from dphg.de

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from dphg.de

Frankfurt/Main
September 24 – 26, 2014
at Goethe University
www.2014.dphg.de
ISBN 978-3-9816225-1-5
DPhG Annual Meeting 2014 – Conference Book
Annual Meeting of the
German Pharmaceutical Society – DPhG
Annual Meeting of the German
Pharmaceutical Society – DPhG
Trends and Perspectives in
Pharmaceutical Sciences
Conference Book
Frankfurt/Main, September 24 – 26, 2014
www.2014.dphg.de
Annual Meeting of the German
Pharmaceutical Society DPhG
Trends and Perspectives in
Pharmaceutical Sciences
Conference Book
Frankfurt/Main, September 24 26, 2014
www.2014.dphg.de
Sponsors of the DPhG Annual Meeting 2014
MEDIEN FÜR DIE APOTHEKE
Institutional Sponsors
Förderer der DPhG-Jahrestagung 2014
CONFERENCE COMMITTEES
Scientific Committee
Prof. Dr. Thomas Efferth
Prof. Dr. Christoph Friedrich
Prof. Dr. Peter Gmeiner
Prof. Dr. Ulrike Holzgrabe
Prof. Dr. Ulrich Jaehde
Prof. Dr. Jochen Klein
Prof. Dr. Heyo Kroemer
Prof. Dr. Peter Langguth
Prof. Dr. Stefan Laufer
Prof. Dr. Kristina Friedland
Prof. Dr. Andreas Link
Prof. Dr. Irmgard Merfort
Prof. Dr. Klaus Mohr
Dr. Olaf Queckenberg
Prof. Dr. Peter Ruth
Prof. Dr. Andrea Sinz
Prof. Dr. Angelika Vollmar
Prof. Dr. Hermann Wätzig
Prof. Dr. Werner Weitschies
Prof. Dr. Gerhard Winter
Organisation Committee
Seniorprof. Dr. Theodor Dingermann
Prof. Dr. Jennifer Dressman
PD. Dr. Gunter Eckert
Prof. Dr. Robert Fürst
Dr. Ann-Kathrin Häfner
Dr. Bettina Hofmann
Prof. Dr. Michael Karas
PD. Dr. Thorsten Maier
Prof. Dr. Rolf Marschalek
Jun.-Prof. Dr. Eugen Proschak
Dr. Bernd Sorg
Dr. Michael Stein
Dr. Mario Wurglics
DPhG Annual Meeting 2014
3
ADDRESS OF WELCOME
Dear colleagues,
as President of the German Pharmaceutical Society (DPhG) and congress chairman it is a pleasure for me to welcome you at Frankfurt to attend our Annual
Prof. Dr. Dieter Steinhilber
DPhG President
4
place for such a meeting which is part of the GU100 celebrations on the occath
sion of the 100 birthday of Goethe University. Furthermore, the meeting is
cosponsored by one of our most important partners, the Pharmaceutical Society
of Japan (PSJ).
We can present you an interesting scientific programme which focuses on
recent developments in all pharmaceutical disciplines. The meeting program
covers topics such as antiinflammatory drugs, anticancer drugs and epigenetics,
biotechnology, clinical pharmacy, drug design/medicinal chemistry/analytics,
natural compounds, biopharmaceutics as well as many aspects of pharmaceutical technology and drug delivery.
I thank the scientific and the organisation committee as well as the chair
persons of the sessions for creating an exciting programme. I would like to
thank all participants of the meeting for sharing their recent results and for
their contributions. The abstract book provides you with an overview about the
scientific posters and presentations and we hope, it will contribute to a successful meeting and to scientific exchange and discussions.
TABLE OF CONTENTS
General Information.................................................................................................................................................................................6
Conference Program Overview .............................................................................................................................................................7
Plenary lectures ...................................................................................................................................................................................... 15
Scientific Sessions .................................................................................................................................................................................. 23
Antiinflammatory Drugs ................................................................................................................................................................................. 24
Neurodegeneration ........................................................................................................................................................................................... 29
Pharmaceutical Technology and Drug Delivery....................................................................................................................................... 33
Medicinal Chemistry (PSJ) .............................................................................................................................................................................. 38
Biomarker and Modeling ................................................................................................................................................................................ 42
Ligand Binding Assays ..................................................................................................................................................................................... 45
Computational Chemistry and Molecular Design ................................................................................................................................... 49
Natural Compounds ......................................................................................................................................................................................... 54
Analytics ............................................................................................................................................................................................................... 59
Case studies from Pharmaceutical Research and Development ........................................................................................................ 64
GPCR Medicinal Chemistry............................................................................................................................................................................. 69
Industrial Pharmacy ......................................................................................................................................................................................... 73
Biopharmaceutics and Pharmaceutical Technology .............................................................................................................................. 77
Anticancer and Epigenetic Drugs ................................................................................................................................................................. 82
Evidence-based Medication Management ................................................................................................................................................ 86
Optimizing Oral Drug Performance ............................................................................................................................................................. 90
Multitarget Drugs ............................................................................................................................................................................................. 94
Non-canonical GPCR-Signaling ................................................................................................................................................................... 99
Biopharmaceuticals/Biotechnology ........................................................................................................................................................... 103
Short lectures ........................................................................................................................................................................................ 108
Posters ..................................................................................................................................................................................................... 121
Antiinflammatory Drugs (AD01-AD19) .................................................................................................................................................... 122
Anticancer Drugs and Epigenetics (ACE01-ACE33) .............................................................................................................................. 128
Biotechnology (BT01-BT16) ......................................................................................................................................................................... 140
Clinical Pharmacy (CP01-CP15).................................................................................................................................................................. 147
Drug Design/Medicinal Chemistry/Analytics (MC01-MC62) ............................................................................................................. 153
GPCR (G01-G14) .............................................................................................................................................................................................. 176
Natural Compounds (NC01-NC21) ............................................................................................................................................................ 182
Neuroactive Drugs (ND01-ND13) .............................................................................................................................................................. 189
Biopharmaceutics (BP01-BP09).................................................................................................................................................................. 194
Pharmaceutical Technology and Drug Delivery (PT01-PT34) ............................................................................................................ 198
Authors Index ........................................................................................................................................................................................ 212
5
GENERAL INFORMATION
INSTRUCTIONS FOR USING CONFERENCE WLAN
If you are a member of an institution (e.g. a university), which is a member of the "eduroam" community, you should use
the wireless network "eduroam". In most cases you can use the wireless network eduroam in the same way you are used
to connect to the wireless network at your home institution. Please use your account and the domain of your institution
(e.g. [email protected]).
If your institution (e.g. a university) is not a member of the "eduroam" community, you have to obtain a guest-account
sword. After authenticating successfully, you will have access to the Internet.
ABSTRACT AND POSTER NUMBERS
Each abstract has a unique identifier, a letter-number combination. Letters refer to the conference topic a contribution
was assigned to. For example, a poster presentation in the area of Antiinflammatory Drugs might have the number
AD.01. Short lectures
SL
Please note that in the case of poster presentations the abstract number is identical with the poster number.
Please refer to the authors index on page 212 for direct access to specific abstracts.
CONFERENCE OFFICE
The Conference office is located at the Conference building (Otto-Stern-Zentrum (OSZ), S1 / S2).
Opening hours:
Wednesday, September 24th, 2014: 10.00 am 6.00 pm;
Thursday, September 25th, 2014: 8.00 am 6.00 pm;
Friday, September 26th, 2014: 8.00 am 12.00 am.
POSTER SESSIONS
There will be two poster sessions:
Topics
Antiinflammatory Drugs, Anticancer Drugs /
Epigenetics and Drug Design / Medicinal Chemistry / Analytics
Biotechnology, Clinical Pharmacy, GPCR, Natural
Compounds, Neuroactive Drugs, Biopharmaceutics and Pharmaceutical Technology/Drug Delivery
Session
Wednesday, Sept. 24th, 6 pm until 10 pm
Thursday, Sept. 25th, 12 o' clock noon until 1.30
pm
Set-up
Wednesday, Sept. 24th, before 6 pm
Thursday, Sept. 25th, before 10 am
Dismantling
Wednesday, Sept. 24th, after 10 pm
Thursday, Sept. 25th, after 6 pm
CONFERENCE DINNER
Separate registration necessary (special fee). Please refer to the Conference Office for registration and details.
The Conference dinner will take place at the Casino on Campus Westend (no public parking available, please use subway
U8 from station Uni Campus Riedberg to station Holzhausenstraße). For detailed maps of Campus Riedberg and Campus
Westend see page 224.
6
CONFERENCE PROGRAM OVERVIEW
Pre-symposia and public talk
Tuesday, 23.9.2014
Bürgersymposium:
Frankfurter Pharmaziegeschichte Von Goethe bis Hoechst
 Ort: Campus Westend (Foyer PA
Gebäude)
15.00-15.15: Begrüßung,
Christoph Friedrich, Marburg
15.15-16.00: Zur Entwicklung der
Pharmazie
an
der
JohannWolfgang-Goethe-Universität
Frankfurt,
Prof. Dr. Axel Helmstädter
16.00-16.45: Zur Geschichte des
Frankfurter Apothekenwesens,
Dr. Caroline Seyfang
Advanced Course in Pharmacology
(DGPT)
'Durchflusszytometrie:
Anwendungen in pharmakologischer
und
pharmazeutischer
Forschung
Separate Anmeldung bei der DGPT
erforderlich
 Ort: OSZ (HS 3) 15.00-18.30 Uhr
15.00-15.10: Einführung,
Prof. Dr. Detlef Neumann
Workshop Drittmittelförderung
 Ort: OSZ (HS 4)
14.00-16.00: Dos und Don'ts beim
Antragschreiben,
T. Hotopp, DFG
 Ort: OSZ (HS 4)
15.10-15.55: Einführung in die 16.00-17.00: Horizon 2020,
M. Ackermann, Nationale KontaktDurchflusszytometrie,
stelle Lebenswissenschaften
Dr. Stefan Schnell
Fluoreszenz-  Ort: OSZ (HS 4)
aktiviertes Zellsortieren mithilfe
der Mikrochip-Technologie,
Dr. Martin Büscher
15.55-16.40: Durchflusszytometrie 15.00-16.30: Beiratssitzung des
für die pharmakologische Charak- VdPPhI
terisierung von GPCR Liganden,
 Ort: OSZ (HS 5)
Prof. Dr. Erich Schneider
16.45-17.15: Kaffeepause
16.40-17.00: Kaffeepause
17.15-18.00: Die Entwicklung der
Firma Hoechst unter besonderer
Berücksichtigung ihrer Geschichte
im Dritten Reich,
Prof. Dr. Stephan H. Lindner
18.00-18.45: Eine Tradition der
besonderen Art: Goethe und sein
Verhältnis zur Pharmazie und zu
Pharmazeuten,
Prof. Dr. Christoph Friedrich
17.00-17.45: Chipzytometrie für
die
omarker-Analyse: Technologie und
Anwendung,
Dr. Christian Hennig
17.45-18.30:
Standardisierung
und
Automatisierung
durchflusszytometrischer Assays in der
pharmakologischen Forschung,
Dr. Peter Engel
17.00-19.30: Mitgliederversammlung des VdPPhI
 Ort: OSZ (HS 5)
anschl. Empfang, Campus Westend, Foyer PA Gebäude
7
Wednesday, 24.9.2014
Fachgruppen-Meetings
9.00-10.30
10.30-12.00
OSZ H2
Pharmazie 2020,
D. Steinhilber,
S. Laufer
Fachgruppe
Pharm./Med.
Chemie,
P. Gmeiner
OSZ H3
Fachgruppe
Pharmakologie,
J. Klein
OSZ H4
OSZ H5
OSZ H6
Fachgruppe Klin.
Pharmazie,
K. Friedland
Fachgruppe
Pharm. Biologie,
A. Vollmar
Fachgruppe
Pharm. Technologie,
P. Langguth
Wednesday, 24.9.2014
Main Symposium (Congress language English)
13.00-13.30
13.30-14.15
14.15-15.00
15.00-15.30
15.30-17.00
8
Opening of the Annual DPhG Meeting 2014,
Trends and Perspectives in Pharmaceutical Sciences
OSZ H1+H2
PL 1, Peter Ruth, New disease relevant functions of Ca2+-activated potassium channels
OSZ H1+H2
PL 2, Shinji Yamashita, Streamlining the development of oral drug product: Role of researchers in
academia
OSZ H1+H2
Coffee break
OSZ
Short talks, parallel sessions I
OSZ H3
OSZ H4
OSZ H5
Antiinflammatory Drugs
Neurodegeneration
Pharmaceutical Technology
Chair:
Chair:
and Drug Delivery
S. Laufer, D. Steinhilber
C. Culmsee, J. Klein
Chair:
L. Meinel, W. Weitschies
15.30 Jan Schwab,
Resolvins,
protectins
and
maresins as candidates to
propagate resolution of inflammation in lesions of the
central nervous system (CNS)
15.50 Andreas Köberle,
Functional lipidomics reveals
phosphatidylcholine-bound
arachidonic acid as regulator
of protein kinase B
15.30 Carsten Culmsee,
New insights into Bidmediated
mitochondrial
demise in neuronal cell death
15.30 Markus Thommes,
Formulation strategies for
poorly water soluble drugs
15.55 Jochen Klein,
Experimental stroke research:
problems and opportunities
15.50 Stephan Reichl,
Valid cell culture models of
the human cornea for drug
transport investigations where are we?
16.10 Thorsten Maier,
Nitro lipids as novel regulators
of leukotriene biosynthesis
16.20 Carina Hohmann,
Emerging options for pharmaceutical care in stroke patients
16.10 Tessa Charlotte Lühmann,
Protein engineering of fibroblast growth factor 2 (FGF-2)
for bioresponsive protein
delivery
OSZ H3
16.30 Olivia Merkel,
Ex vivo and in vivo siRNA
delivery to activated T cells as
novel
anti-inflammatory
asthma therapy
16.45 Christoph Schmidt,
Rational protein-engineering
yields a minimised innate
immune inhibitor with unique
targeting properties
17.00-18.00
OSZ H4
16.40 Amalia Dolga,
SK channel modulation attenuates mitochondrial dysfunction, neuroinflammation, and
neuronal cell death
OSZ H5
16.30 Anne Seidlitz,
In vitro estimation of drug
transfer
from
paclitaxelcoated balloon catheters
16.45 Miriam Pein,
Self-developed sensor membranes for etongue sensors
Short lectures (5 min)
OSZ H1+H2
Hayato Fukuda, Design, synthesis and biological evaluation of a stabilized resolvin E2 analogue
Yudai Matsuda, Biosynthetic studies on fungal meroterpenoids and their fascinating chemistry
Masahito Yoshida, Destruxin E, a potent negative regulator of osteoclast morphology: Solidphase library synthesis and biological evaluation
Tsuyoshi Saitoh, Design and synthesis of NF-κB inhibitors carring epoxyquinol moiety
Marlene Barho, Structure-activity relationship studies on small molecule Bid-inhibitors
Anna Junker, Synthesis and structure affinity relationships of dual chemokine receptor 2 and
chemokine receptor 5 antagonists and development of a selective, fluorinated CCR2 ligand for PET
studies
Ann-Kathrin Schoenfeld, Testing of potential inhibitors of human heparanase in a fluorescence
activity assay
Rico Schwarz, Monitoring conformational changes in PPARβ/δ by cross-linking and mass spectrometry
Wenjin Li, A dynamic pH junction method for monitoring the catalytic activity of cerebroside
sulfotransferase
Dominique Lunter, Confocal Raman microscopic (CRM) methodology for the analysis of the
penetration of pharmaceutical actives into the skin
Julian Schichtel, Determination of the dissolution behaviour of celecoxib-Eudragit E 100nanoparticles using cross-flow filtration
Verena Gotta, Sensitivity of concentration-effect versus dose-effect analysis to detect small
magnitudes of QTc prolongation in preclinical cardiovascular safety setting
18.00-22.00 Poster session I and welcome
reception, OSZ
18.00-18.30 Außerordentliche
sammlung der DPhG, OSZ H3
Hauptver-
9
Thursday, 25.9.2014
8.30-9.15
9.15-10.00
PL 3, Rolf Hartmann, Interference with bacterial quorum sensing: a new antivirulence strategy
OSZ H1+H2
PL 4, Nagayoshi Nagai Lecture, Masakatsu Shibasaki (PSJ), Recent progress in cooperative
asymmetric catalysis
OSZ H1+H2
10.00-10.30
Coffee break
OSZ
Short talks, parallel sessions II
10.30-12.00
OSZ H3
Medicinal Chemistry (PSJ)
Chair:
N. Miyata, K. Tomioka
10.30 Kiyoshi Tomioka,
Paradigm re-shift of medicinal
chemistry in Japan
10.55 Naoki Miyata,
Design, synthesis and biological activity of lysine-specific
demethylase (KDM) inhibitors
11.20 Hiroshi Nagase,
Synthesis of a novel opioid
receptor agonist, SYK-146
with 1,3,5-trioxazatriquinane
skeleton and its pharmacologies
11.45 Hiroaki Ohno,
Gold-catalyzed
annulations
and their medicinal applications
12.00-13.30
OSZ H4
Biomarker and Modeling
Chair:
C. Kloft, T. Lehr
10.30 Markus Joerger,
Implementation of dosing
algorithms of anticancer drugs
based on pharmacological
biomarkers
11.00 Thorsten Lehr,
Mathematical modeling of
amyloid beta for the diagnosis
and treatment of A
disease
OSZ H5
Ligand Binding Assays
Chair:
C. Müller, H. Wätzig
10.30 Frank M. Boeckler,
Biophysical techniques in
fragment hit identification
and lead optimization - A
change of perspective?
10.50 Christian Kramer,
The impact of experimental
uncertainty on decision making in drug design
11.30 Rolf Burghaus,
Understanding
coagulation
biomarkers
and
deriving
clinically relevant surrogates
by use of an in-silico coagulation model
11.20 Dominique Bonnet,
Fluorescent probes to track
GPCR binding and dimerization
11.40 Yosuke Taniguchi,
Development
of
triplexforming
oligonucleotide
having artificial nucleoside
analogues to inhibit the gene
expression as an antigene
strategy
Poster Session II and lunch break
OSZ
Short talks, parallel sessions III
13.30-15.00
10
OSZ H3
Computational
Chemistry and Molecular Design.
Chair:
F. Böckler, O. Koch
OSZ H4
Natural Compounds
Chair:
R. Fürst, A. Vollmar
OSZ H5
Analytics
Chair:
M. Karas, A. Sinz
OSZ H6
Case studies from
Pharmaceutical
Research and Development
Chair:
B. Cezanne, M. Weigandt
OSZ H3
13.30 Andreas Bender,
Integrating chemical
and biological data
for drug design and
mode-of-action
analysis
OSZ H4
13.30 Verena Dirsch,
Neolignans:
from
PPARγ
RXRα
13.45 Steve Maginn,
A
knowledge-based
approach to assessing
propensity for polymorphism
in
the
pharmaceutical
crystalline solid form
14.00 Thomas Exner,
Direct integration of
ligand-based
NMR
data into proteinligand docking
13.50
Andreas
Bechthold,
Waking up biosynthetic gene clusters in
a row
14.15
Alexander
Dömling,
Screening
reaction
pathway-driven very
large chemical space:
Discovery of potent
mdm2-p53 antagonist
14.30 Holger Gohlke,
Identification of a
mechanism-of-action
target
exploiting
similarities of chemotypes and signalling
events, and biophysical simulations
14.45 Discussion
14.10 Jennifer Hermann and Florian
Förster,
Chondramides: setting
the stage for actin
binding compounds in
cancer therapy
14.30 Johanna Liebl,
Cdk5
inhibition
potentiates imatinib
responsiveness
of
Philadelphia chromosome positive chronic
myeloid leukemia cells
14.45 Finn Hansen,
Plasmodium falciparum histone deacetylases (PfHDACs) as
epigenetic
drug
targets
15.00-15.30
OSZ H5
13.30
Dietrich
Volmer,
Analysis of vitamin D
metabolic markers by
mass
spectrometry:
Advantages
and
limitations of the gold
standard method
13.50
Andreas
Römpp,
High resolution MALDI
imaging:
Reliable
molecular identification at cellular resolution
14.10 Kai Scheffler,
Mass
spectrometric
characterization
of
biopharmaceuticals possibilities, challenges and limitations
OSZ H6
13.30 Andrea Hanefeld,
Dendritic
celltargeting
cancer
vaccine formulations
for pulmonary and
peroral delivery
14.30 Ganna Kalayda,
Fluorescent oxaliplatin
analogue as a model
for the anticancer
drug oxaliplatin for
the investigation of its
cellular trafficking
14.30 Sonja Skopp,
Fighting schistosoiasis
in young children: The
Pediatric Praziquantel
Consortium
14.45
Christian
Wischke,
A polymeric multifunctional glaucoma
implant
14.50 Steffen Lüdeke,
Chirality in polyketide
antibiotics: substratedependent inversion
of stereoselectivity in
Tyl-KR1-catalyzed
reductions
13.50 Christoph Saal,
Selection of solid
state forms for new
chemical
entities:
Challenges, opportunities, adventures and
lessons learned
14.10
Matthias
Winzer,
Fast track formulation
development
for
biotherapeutics
Coffee break
OSZ
Short talks, parallel sessions IV
15.30-17.00
OSZ H3
GPCR Medicinal Chemistry
Chair:
P. Gmeiner, U. Holzgrabe
OSZ H4
Industrial Pharmacy
Chair:
A. Link, S. Schmidt
15.30 Armin Buschauer,
Toward selective molecular
tools for histamine receptor
subtypes:
conformational
constraints, bioisosteric and
bivalent approaches
15.30 Norbert Nagel,
Biophysical characterization
of pharmaceutical peptides
OSZ H5
Biopharmaceutics and Pharmaceutical Technology
Chair:
P. Langguth, S. Yamashita
15.30 Heinrich Haas,
Novel injectable RNA formulations for tumor immunotherapy
11
OSZ H3
16.00 Gerhard Wolber,
Modulation of GPCR signaling:
Understanding ligand binding
effects
16.20 Michael Decker,
Molecular combination of
GPCR ligands: bivalent, hybrid
and dualsteric compounds
16.40 Nuska Tschammer,
Boronic acids as probes for
exploration
of
allosteric
regulation of the chemokine
receptor CXCR3
OSZ H4
15.50 Carsten Olbrich,
Subvisible particles in protein
formulations
16.10 Harry F. Abts,
Dissecting pharmacodynamic
action of compound mixtures
by use of in vitro models
16.30 Uwe Muenster,
Current
biopharmaceutics
prediction tools - an overview
OSZ H5
15.50 Herbert Wachtel,
Automated testing of inhalation devices in early development phase
16.10 Peter Serno,
Orodispersible dosage forms
16.30 Mai Anh Nguyen,
Pharmacokinetic
drug
neutraceutical interactions: A
particular type of food - drug
interaction
16.45 Thomas Nawroth,
Gastro-intestinal simulator for
in-vitro drug and nanoparticle
tracing in oral drug development
Short talks, parallel sessions V
17.00-18.30
OSZ H3
Anticancer and Epigenetic
Drugs
Chair:
C. Brandts, R. Marschalek
17.00 Tom Milne,
Unraveling
the
aberrant
epigenetic programming of
MLL leukemias
17.30 Stefan Fröhling,
Identifying therapeutic targets
in MLL fusion-driven leukemia
using functional genomics
18.00 Rolf Marschalek,
MLL leukemias and future
treatment strategies
18.15 Manfred Jung,
Selective Sirt2-inhibition by
ligand induced rearrangement
of the active site
19.30
12
OSZ H4
Evidence-based Medication
Management
Chair:
K. Friedland, U. Jaehde
17.00
Welcome and short introduction
OSZ H5
Optimizing Oral Drug Performance
Chair:
M. Brewster, J. Dressman
17.00 Marcus Brewster,
O
improving oral bioavailability
17.10
Isabel
Waltering,
Susanne
Koling,
Georg
Hempel,
Evaluation of medication
management in community
pharmacies
17.30 Kristina Friedland,
Evidence-based
medication
management in psychiatric
patients
17.30 Mathew Leigh,
Assessing the gastrointestinal
the media
17.50 André Wilmer, Ulrich
Jaehde,
Evidence-based
medication
management in cancer patients
18.10 Anna Laven,
PHARMAGRIPS:
Structured
pharmaceutical counseling in
the self-medication of the
common cold. A randomised
controlled study (RCT)
18.20 Discussion
Conference dinner
17.50 Edmund Kostewicz,
Will the parachute crash?
transfer models for assessing
performance of optimized
formulations
18.10 Rodrigo Cristofoletti,
Connecting oral formulation
performance to therapeutic
effect
Friday, 26.9.2014
8.30-9.15
9.15-10.00
10.00-10.30
PL 5, Charlotte Kloft, Pharmacometrics for better therapies?
OSZ H1+H2
PL 6, Ernst Wagner, Sequence-defined carriers for targeted intracellular drug and nucleic acid
delivery OSZ H1+H2
Coffee break
OSZ
Short talks, parallel sessions VI
10.30-12.00
OSZ H3
Multitarget Drugs
Chair:
T. Efferth, E. Proschak
10.30 Jens-Uwe Peters,
An introduction to polypharmacology in drug discovery
11.00 Thomas Efferth,
Multifactorial activity of the
naphthoquinone
shikonin
against cancer cells
11.15 Eugen Proschak,
Polypharmacology: In silico
recognition vs. rational design
11.30 Samuel N. Okpanyi,
Study of the anxiolytic actions of Valeriana officinalis
L., Melissa officinalis L., Passiflora incarnata L. and their
combination STW 32 in experimental models of anxiety
11.45 Olaf Kelber,
Motility modulation beyond
MCP: Mechanisms of action of
a clinically proven herbal
medicinal product, STW 5, in
functional GI diseases
12.00-13.00
13.00-13.45
14.00-15.00
OSZ H4
Non-canonical
GPCRSignaling
Chair:
C. Hoffmann, K. Mohr
10.30 Andreas Bock,
Dynamic ligand binding
10.55 Andreas Rinne,
Voltage-dependent
GPCRactivation
11.20 Michael Mederos y
Schnitzler,
Mechanosensitivity of histamine H1 receptor
11.40 Annette Kaiser,
Signaling in malaria parasites:
A non-canonical G-protein
from plasmodium falciparum
OSZ H5
Biopharmaceuticals/ Biotechnology
Chair:
O. Germershaus, E. Wagner
10.30 Michael Adler,
Major trends and challenges in
biotherapeutic product development: "polysorbate degradation" and "drug-device
combination product development"
10.50 Eva-Maria Ruberg,
Finding the right candidate
integrated lead ID of nextgeneration molecules
11.10 Carsten Rudolph,
Non-immunogenic messenger
RNA therapeutics
11.30 Leonard Kaysser,
Divergent pathways for the
biosynthesis of merochlorins,
cyclic meroterpenoid antibiotics from a marine streptomycete
11.45 Arnold Grünweller,
Combination strategies for
targeting the oncogenic Pim1
kinase
Short Lunch break
OSZ
PL 7, Zoltan Takats, London, Direct mass spectrometric characterization of biological tissues from automated histology to DMPK studies
OSZ H1+H2
Closing ceremony
OSZ H1+H2
Gründungsversammlung der DPhG-Arbeitsgemeinschaft Notfall- und
Katastrophenpharmazie = AG KatPharm (OSZ H4)
13
Post-symposium
Saturday, 27.9.2014
Tag der Offizinpharmazie, OSZ H2
Organizing Committee: Kathrin Müller, Annegret Birr, Erika Fink, Michael Hannig, Juliane Kresser
Moderation: Prof. Dr. Dieter Steinhilber
Sprecher der Akademie für Pharmazeutische Fortbildung der LAK Hessen
Personalisierte Pharmakotherapie
14.30-15.30
Interaktionen Welche sind häufig und relevant,
Dr. Nina Griese-Mammen
Zentrum für Arzneimittelinformation und Pharmazeutische Praxis (ZAPP)
der ABDA, Berlin
15.30-16.00
Kaffeepause
16.00-17.00
Patientenorientierte Arzneimitteltherapie: Ein Starter
Prof. Theo Dingermann
Institut für Pharmazeutische Biologie, Frankfurt am Main
Einfluss genetischer Variabilität auf die Wirkung von Arzneimitteln
Prof. Dr. Manfred Schubert-Zsilavecz
Institut für Pharmazeutische Chemie, Frankfurt am Main
17.00-18.00
Ab 18.00
14
Mitgliederversammlung der Fachgruppe Allgemeinpharmazie
PLENARY LECTURES
15
New Disease relevant Functions of Ca2+‐activated Potassium Channels Title
PL.01
Ruth, P.
Department of Pharmacology, Toxicology & Clinical Pharmacy, Institute of Pharmacy, University of Tübingen
The big conductance, Ca2+-activated K+ (BK) channel represents an important pathway for the outward flux of K+ ions
from the intracellular compartment in responses to membrane depolarization and elevation in cytosolic free [Ca2+],
which serves to drive cell membrane potential in the negative direction. BK channels are functionally expressed in a
range of mammalian tissues (e.g., neurons, neurosensory cells, smooth muscles, tumor cells), where they can either
enhance or dampen membrane excitability and thus Ca2+ influx into cells. BK channels can be regulated by several
stimuli that allow them to work as integrators of cell signaling, excitability and metabolism. Genetic ablation of the BK
channel in mice leads to several disease states (e.g., elevated blood pressure, hyperaldosteronism due to impaired K+
excretion, overactive bladder syndrome and cerebellar ataxia) but has also beneficial effects like suppressing breast
cancer and preventing dietary obesity. Accordingly, BK channels are potential targets for therapeutic approaches.
In the auditory and visual system they play a crucial role in the maintenance of neurosensory function. In the inner ear
BK channels have an important role in the signal transduction process of cochlear inner and outer hair cells. Its genetic
ablation leads to progressive hearing loss due to activation of apoptotic pathways. In addition, BK channel deletion led to
a higher vulnerability towards noise-induced hearing loss, a global health hazard with considerable social consequences,
suggesting that BK currents are involved in survival mechanisms of cochlear hair cells. Moreover, BK channels in inner
hair cells are also crucial for central coding of the temporal fine structure of sound and for detection of signals in a noisy
environment. In the visual system, BK channels modulate adequate visual responses at the bipolar cell level at dim light
conditions as deduced from BK channel knockout mice. In pain sensing neurons, BK channels exert inhibitory control on
sensory input in inflammatory states.
These results suggest that the overall analysis of a protein´s function in a mammalian organism by gene knockout is able
to predict adverse as well as desired effects of modulatory drugs acting on this protein target.
16
Streamlining the development of oral drug product: Role of researchers in academia
PL.02
Yamashita, S.
Setsunan University, College of Pharmacy
45-1 Nagaotoge-cho, Hirakata,Osaka 573-0101, Japan
Japanese pharmaceutical industries have developed many of innovative medicines so far such as pravastatin, tacrolimus, that made Japan as one of the leading countries in the pharma-business. However, with the advent of paradigm
shift in drug development, activity of pharmaceutical industry in Japan decreased gradually. Although pharmaceutical
science and technology in Japan are still in the high level, rather small size of companies (compared with megacompanies in USA and EU) makes a new drug development difficult due to the smaller size of compound library, database and budget. As one of the possible ways to keep the high activity of drug development, Japanese companies
should share the knowledge and technologies in some parts.
“Consortium of Oral Drug Absorption Screening” with 25 Japanese pharmaceutical companies was started at 2001. Final
goal of the consortium is to establish the effective strategy for development of oral drug product. Also, it aims to give a
platform for mutual interaction of researchers working in different companies. During past 10 years, various projects
were performed in the consortium relating to oral drug absorption and outcomes were shared with all member companies. In the lecture, as an organizer, activity of the consortium will be presented with showing outcomes of 2 main projects for “assessment of oral absorption of poorly-soluble drugs” and “BCS Classification to identify drug developability
as oral products”.
Governmental support is also essential to activate the industry. As a new strategy of drug development, the early phase
clinical study (exploratory IND (eIND) study) including Microdose (MD) study was proposed to increase the success
probability of clinical trials. In Japan, in order to promote the eIND study, the national project on MD clinical study was
started at 2008 (supported by NEDO: New Energy and Industrial Technology Development Organization), entitled as
“Establishment of Evolutional Drug Development with the Use of Microdosing Clinical Trial”. In the project, we performed
more than 30 MD clinical studies with marketed drugs to validate the usefulness of this new strategy and to construct the
system (both soft and hard) for conducting MD clinical study in Japan. Furthermore, we performed the molecular imaging
study with PET to investigate the process of GI absorption and subsequent bio-distribution of orally administered drugs
[1-4]. This is also a collaborative work with governmental research center “Riken”. As a second issue of the lecture,
results of this innovative work will be highlighted with emphasizing an importance of integration of new technologies in
drug development such as a molecular imaging technology.
Acknowledgments:
I would like to thank to the members of “Consortium of Oral Drug Absorption Screening” for their enthusiastic help to accomplish the projects in the
consortium. Also, the study of molecular imaging was done as a part of “Research Project for Establishment of Evolutional Drug Development with
the Use of Microdose Clinical Trial”, sponsored by the New Energy and Industrial Technology Development Organization (NEDO).
References:
1. Yamashita, S. et al.: J Nucl Med. 2011, 52(2): 249-256.
2. Kataoka, M. et al.: Pharm Res. 2012, 29(9): 2419-2431.
3. Shingaki, T. et al.: Clin Pharmacol Ther. 2012, 91(4): 653-659.
4. Takashima, T. et al.: Mol Pharm. 2013, 10(6): 2261-2269.
17
Interference with bacterial quorum sensing - a novel
anti-virulence strategy
PL.03
Hartmann, R.W.1,2
1
2
Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123 Saarbrücken, Germany
Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany
The emergence and spread of bacteria resistant to current antibiotics are a serious and growing health problem worldwide. In pursuing our objective to develop antibacterials with novel modes of action we have focused our interest on the
bacterial cell-to-cell communication. Among other infections, Pseudomonas aeruginosa causes severe pneumonia in
patients suffering from cystic fibrosis. It is difficult to be eradicated, especially when present in biofilms. Biofilm formation
and virulence factor production are regulated by intercellular signal molecules. A selective blockade of this so called
quorum sensing system is a novel therapeutic strategy to limit pathogenicity and is considered to delay resistance
development. Two proteins are targeted, PqsD, a key enzyme in the biosynthesis of the signal molecules PQS and HHQ
and their receptor PqsR.
Following different approaches, structure-based1 and ligand-based2-4 via transition states analogs of the enzymatic
reaction3-4, the first PqsD inhibitors described so far were obtained. The nitrophenylmethanols were able to permeate the
gram-negative bacterial cell wall and inhibited signal molecule production and biofilm formation.
Regarding PqsR, SPR biosensor experiments led to the discovery of highly efficient binders with low molecular weights
including antagonists5. Site-directed mutagenesis combined with isothermal titration calorimetry led to insights into the
binding mode. The first highly potent antagonists have been developed by structural modification of the natural ligand6.
After an unexpected functional inversion of these antagonists in P. aeruginosa had been revealed, second generation
PqsR antagonists were rationally developed and able to efficiently reduce virulence factor formation 7. One of these
antagonists showed a strong reduction of P. aeruginosa pathogenicity in vivo.
References:
1. Sahner et al.: J. Med.Chem. 2013: 8656-8664.
2. Weidel et al.: J. Med. Chem. 2013: 6146−6155.
3. Storz et al.: J. Am. Chem. Soc. 2012: 16143-16146.
4. Storz et al.: ACS Chem. Biol. 2013: 2794-2801.
5. Zender et al.: J. Med. Chem. 2013: 6761−6774.
6. Lu et al.: Chem. Biol. 2012: 381-390.
7. Lu et al.: Angewandte 2014: 1109 –1112.
18
PL.04
Recent Progress in Cooperative Asymmetric Catalysis
Shibasaki, M.
Institute of Microbial Chemistry, Tokyo (BIKAKEN), 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Our research focuses on the development of catalytic asymmetric C-C bond-forming reactions with particular emphasis
on high atom economy and their application to the synthesis of biologically significant compounds. Thus, the concept of
asymmetric cooperative catalysis such as Lewis acid-Brønsted base catalysis [1,2] and Lewis acid-Lewis base catalysis
[3] plays the key role in our research paradigm. In this lecture, we report our recent progress in asymmetric Lewis acidBrønsted base cooperative catalysis. In 1995, we developed the first example of a syn-selective catalytic asymmetric
nitroaldol reaction using LLB (La-Li-BONOL) as catalyst. At this time the anti-selective reaction remained a longstanding
problem. Finally, by changing the catalyst design to a Nd/Na heterobimetallic catalyst possessing a chiral amide ligand,
we succeeded in developing an efficient and practical anti-selective catalytic asymmetric nitroaldol.
In addition, the development of direct catalytic asymmetric aldol-type reaction of thioamides with aldehydes such as
RCH2CHO was also considered to be impossible due to the low acidity of the a-proton. Recently we could overcome this
inherent problem by identifying an asymmetric soft Lewis acid- hard Brønsted base cooperative catalyst. How to overcome this problem as well as application to an efficient and practical catalytic asymmetric synthesis of atorvastatin will
also be discussed.
References:
1. Shibasaki, M. et al.: Acc. Chem. Res., 2009, 42: 1117-1127.
2. Kumagai, N. and Shibasaki, M.: Angew. Chem. Int. Ed., 2011, 50: 4760-4772.
3. Shibasaki, M. et al.: Synlett, 2005, 10: 1491-1508.
19
Pharmacometrics for better therapies?
PL.05
Kloft, C.
Freie Universitaet Berlin, Institute of Pharmacy, Dept. of Clinical Pharmacy & Biochemistry, Kelchstr. 31, 12169 Berlin, Germany
Pharmacometrics, a young but emerging computer-based science1, aims to understand and elucidate qualitatively,
quantitatively and over time the relationships between drug intake – drug concentrations, preferentially at the site of
action (pharmacokinetics) – resulting desired und undesired drug effects measured by e.g. biomarkers (pharmacodynamics) – and therapeutic outcome by influencing disease progression and exerting toxicity (benefit/ risk) considering
knowledge of patient characteristics and their disease.
The developed in silico models, pharmacometric models, attempt to incorporate data of multiple levels, namely on the
molecular, cellular, tissue, organ and patient level and thus provide an increased quantitative understanding of dynamic
complexity in time, space, and population diversity. Ultimately, a thorough understanding of the underlying mechanisms
of drug disposition, target binding, drug-target complex signal transduction, e.g. inside the cell in various sub-cellular
compartments and target dynamics, as well as the impact of patient, treatment and study characteristics shall be covered in a coherent modelling framework. Hence, pharmacometrics creates a paradigm2 for enabling an integrated and
higher level of understanding of drugs, (diseased) systems characteristics, and their interactions through mathematical
models throughout the entire drug development process (model-based or model-integrated drug development, MBDD or
MIDD)3 and for therapeutic usage (model-based or model-integrated patient care, MBPC or MIPC).
Pharmacometrics is based on a transdisciplinary approach bridging concepts of biology, physiology, pharmacology,
pharmacotherapy, clinical pharmacy and clinical pharmacology, medicine, mathematics and statistics. The advancements and success of pharmacometrics substantially increased the demand for scientists educated and trained to
understand the science and underlying framework as well as to develop pharmacometric concepts for drugs and diseases. To this end, the Structured Graduate Research Training program PharMetrX (www.PharMetrX.de) has been
launched to implement this field also in the academic environment.
Pharmacometrics has already demonstrated an added value on, e.g., predictive (translational) pharmacokinetic/pharmacodynamics models and proved instrumental in decision-making in the drug development process and will
hopefully in future contribute to better bridge the gap toward improving patient care.
References:
1. Barrett, J.S. et al.: J. Clin. Pharmacol. 2008, 48: 632-649.
2. Vlasakakis, G. et al.: Clin. Pharmacol. Ther.: Pharmacomet. Syst. Pharmacol. 2013, 2: e40.
3. Milligan, P.A. et al.: Clin. Pharmacol. Ther.. 2013, 93(6): 502-514.
20
Sequence-defined carriers for targeted intracellular drug and nucleic acid delivery
PL.06
Wagner, E.1,2
1 Pharmaceutical
2 Nanosystems
Biotechnology, Department of Pharmacy, Ludwig–Maximilians-University, Butenandtstrasse 5-13, 81377 Munich,
Initiative Munich, Schellingstraße 4, 80799 Munich, Germany
Already five decades ago cationic polymers were used for transfection of nucleic acids [1]. Since then our understanding
of polymers and their rational use in gene delivery has gradually increased. Biological challenges in extra- and intracellular delivery without toxicity were recognized and resulted in breakthrough developments, including surface-shielded
polyplexes, polymers with endosomal escape properties, and biodegradable polymers. Despite this, significant challenges remain and medical translation still has to be demonstrated.
The therapeutic perspectives have widened from pDNA based gene therapy to application of various novel therapeutic
nucleic acids including mRNA, siRNA and microRNA. High resolution microscopy strongly supported our insight on
involved biological delivery mechanisms. Improved precision in macromolecular chemistry enables better design of
polymeric carriers. In our recent research solid phase assisted synthesis was applied in the design of >700 precise
oligomers [2-3] for targeted transfer of therapeutic nucleic acids. Such oligomers self-assemble with nucleic acids or are
conjugated, which results in nanostructures resembling “synthetic viruses”. Like natural viruses, they contain functional
subdomains for facilitating the various delivery steps, including improved packaging [4], facilitated intracellular release
[5], and targeted cell uptake [5-7].
Recently we realized that such carriers can be quite useful for intracellular delivery of other therapeutic agents including
recombinant proteins [8] or low-molecular weight drugs [9] as will be presented in this talk.
References:
1. Vaheri, A.; Pagano, J.S.: Virology 1965, 27: 434-436.
2. Schaffert, D. et al.: Angew. Chem. Int. Ed. Engl. 2011, 50: 8986-8989.
3. Scholz, C. et al.: Chem. Med. Chem. 2014, online.
4. Troiber, C. et al.: Biomaterials 2013, 34: 1624-1633.
5. Lächelt, U. et al.: Nanomedicine NBM 2014, 10: 35–44.
6. Dohmen, C. et al.: ACS Nano 2012, 6: 5198–5208.
7. Zhang, C.Y. et al.: J. Control. Release 2014, 180: 42–50.
8. Maier, K.; Wagner, E.: J. Am. Chem. Soc. 2012, 134: 10169-10173.
9. Lächelt, U. et al.: Mol. Pharmaceutics 2014, 11: 2631–2639.
21
Direct mass spectrometric characterization of biological tissues - from automated
histology to DMPK studies
PL.07
Takats, Z.
Direct mass spectrometric analysis of tissues has been developed in a parallel fashion with the development of desorption ionization methods from the 1970’s. In spite of the success of early studies, the technology remained at the proof-ofconcept stage until the advent of mass spectrometric imaging (MSI) by Matrix-assisted laser desorption ionization
(MALDI) in the late 1990’s.
MSI is a unique assay capable of providing spatial distribution information on hundreds or thousands of chemical species, which information is particularly valuable in case of biological tissues. Prior to the advent of MSI, chemical analysis
of tissues started with an obligatory homogenization step, where any spatial resolution information was immediately lost.
In order to obtain such information, histological approaches (e.g. immunohistochemistry) or autoradiography were
employed, targeted at one or another chemical species. In contrast, imaging mass spectrometry provides semiquantitative information on all detectable species in an untargeted fashion. This information can be interpreted in two,
markedly different ways. On one hand, one can use the intensity distribution of individual species to gain concentration
distribution-type information, which is particularly important in case of DMPK studies. Thanks to the untargeted nature of
MSI, information on the distribution of drugs and their metabolites is obtained in a single experiment without radioactive
(or fluorescent) labelling. On the other hand, the full spectral information (i.e. the entire mass spectrum belonging to
single pixel) can also be used for the histological or histopathological classification of tissues using multivariate statistical
tools. This type of data analysis itself has the potential of substituting classical morphology-based histological examinations and it can also facilitate the co-registration of histological and drug distribution information.
The capabilities of MSI are demonstrated using data obtained by the more recently developed Desorption Electrospray
Ionization (DESI) MSI technique. DESI – in contrast to MALDI – focuses solely on low molecular weight species (metabolites, lipids), however the technique does not require any chemical modification of the sample, which makes it particularly useful for pharmacological studies. In case of DESI MSI the structural lipid composition of the tissue is used to
establish the histological identity of the individual pixels, while the abundance of the molecular ions of drugs is associated with their concentration distribution. The combined results give unique information on the histologically localised
metabolism and accumulation of drugs and their metabolites.
22
SCIENTIFIC SESSIONS
23
ANTIINFLAMMATORY DRUGS
Resolvins, protectins and maresins as candidates to propagate resolution of inflammation in lesions
of the central nervous system (CNS)
Schwab, J.1,2
1 Department
of Neurology and Experimental Neurology, Clinical and Experimental Spinal Cord Injury Research (Neuroparaplegiology), Charite Universitatsmedizin Berlin, D-10117 Berlin, Germany
2 Department of Neurology & Center for Brain and Spinal Cord Repair, Department of Neuroscience, The Ohio State University Medical Center,
Columbus, OH 43210, USA
Inflammatory resolution is an active, highly regulated process already encoded at the onset of inflammation and required
to prevent the transition into chronic inflammation associated with spreading of tissue injury, exacerbated scarring and
pain. We introduce the concept of resolution of inflammation in CNS lesions and summarize emerging evidence for the
bioactivity of specialized pro-resolving mediators (SPM) such as the resolvins, protectins and maresins to attenuate
inflammation-associated neuropathogy. Leukocyte composition in the acute inflammatory milieu in CNS lesion differs
from that after peripheral lesions as being an immune privileged site. PMN infiltration is not a conserved prominent
feature of the CNS inflammatory milieu. The defective resolution of acute inflammation is a major hallmark of a dysfunctional immune response after experimental and human CNS injury. The persisting, chronified inflammation is composed
mostly of microglia/macrophages and likely to participate in late degeneration and non-resolving inflammation. Traditional anti-inflammatory treatment strategies only might be insufficient since impaired resolution of inflammation is part of the
underlying pathophysiology. Modelling neuroinflammatory phenotypes targeting impaired resolution remain underecognized to date. Given that the acute inflammatory response in central nervous system (CNS) lesions is late or nonselflimiting per se SPMs qualify as causal candidates to shape a maladaptive immune response. SPMs constitute a
group of drug targets validated to excert robust bioactivity in CNS neuropathology.
24
Functional lipidomics reveals phosphatidylcholine‐bound arachidonic acid as regulator of protein
kinase B
Koeberle, A.1; Shindou, H.2; Koeberle, S.3; Laufer, S.4; Shimizu, T.2,5; Werz, O.1
Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena, Philosophenweg 14, 07743 Jena, Germany
National Center for Global Health and Medicine, 162-8655 Tokyo, Japan
3 Leibniz Institute of Age Research - Fritz-Lipmann-Institute, 07745 Jena, Germany
4 Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Tübingen, 72076 Tübingen, Germany
5 Department of Lipidomics, Faculty of Medicine, The University of Tokyo, 113-0033 Tokyo, Japan
1
2
Dysregulation of membrane lipids has been associated with disease (e.g., inflammation and cancer), though the underlying mechanisms are poorly defined. Functional lipidomics combines comprehensive lipid profiling with mechanistic and
cell-biological studies to unravel the signalling mechanism of bioactive lipid mediators. We applied this approach to study
the role of membrane lipids for the cell cycle-dependent regulation of protein kinase B (Akt) - a major kinase for cell
proliferation, survival and innate immunity. Since Akt is recruited to membranes for activation, we speculated that its
activity might be regulated by an oscillating membrane lipid component. By monitoring the lipid profile of synchronized
mouse fibroblasts during the cell cycle by UPLC-MS/MS, we found an inverse correlation between the proportion of
arachidonic acid-containing phosphatidylcholine (20:4-PC) and Akt activity (1). Increasing the ratio of 20:4-PC inhibited
Akt membrane binding, Akt (S473) phosphorylation, Akt downstream signalling, S-phase transition and cell proliferation.
In a lipidome-wide screen, the small molecule indirubin-3’-monoxime was identified to reprogram cells towards an
accumulation of 20:4-PC thereby blocking Akt signaling and cell proliferation. The direct influence of 20:4-PC on Akt
membrane binding together with the specificity by which 20:4-PC inhibits Akt activation is surprising. Akt activity is
usually regulated through the level of phosphatidylinositol-3,4,5-trisphosphate (PIP3) - which anchors Akt to membranes
– and not through the affinity of Akt for binding PIP3, and biological effects of 20:4-PC are ascribed in most studies to the
release of arachidonic acid and biosynthesis of eicosanoids instead of to the phospholipid itself. We speculate that
targeting the metabolism of polyunsaturated phospholipids might be a promising approach for the treatment of hyperproliferative and inflammatory diseases.
References:
1. Koeberle, A. et al.: PNAS 2013, 110: 2546.
25
Nitro lipids as novel regulators of leukotriene biosynthesis
Awwad, K.1,2; Steinbrink, S.D.3; Frömel, T.1,2; Lill, N.1,2; Isaak, J.1,2; Häfner, A.-K.3; Roos, J.3; Hofmann, B.3; Heide, H.4;
Geisslinger, G.5; Steinhilber, D.3; Freeman, B.A. 6, Fleming, I.1; Maier, T.J. 3,7
1 Institute
for Vascular Signaling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany.
(German Centre for Cardiovascular Research) partner site Rhine-Main, Frankfurt, Germany.
3 Institute of Pharmaceutical Chemistry, Goethe-University, Frankfurt, Germany.
4 Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Goethe-University, Frankfurt am Main, Germany.
5 Pharmazentrum Frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University, Frankfurt, Germany.
6 Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
7 Institute of Biomedicine, Pharmacology, Aarhus University, DK-8000 Aarhus C, Denmark
2 DZHK
The reaction of nitric oxide generated during inflammatory processes with polyunsaturated fatty acids yields electrophilic
nitro fatty acids (NO2-FA), which display anti-inflammatory properties [1]. Because the 5-lipoxygenase enzyme contains
critical nucleophilic amino acids potentially sensitive to electrophilic attack [2], we investigated whether NO2-FA suppress 5-LO enzyme activity in vitro and 5-LO-dependent inflammatory reactions in vivo. We were able to show that
treatment of human polymorphonuclear leukocytes (PMNL) with nitro-oleic (NO2-OA) or nitro-linoleic acid (NO2-LA) (but
not the parent lipids) led to a concentration dependent and irreversible inhibition of 5-LO product formation. Suppressive
effects were also observed in cell lysates and using the recombinant human 5-LO protein, indicating a direct reaction
with 5-LO. Activity of the related enzymes 12-LO or 15-LO-1 as well as cellular prostaglandin E2 synthesis were not
affected by NO2-FAs. Mechanistically, NO2-FA-induced inhibition of 5-LO was due to nitroalkylation of a specific cysteine residue (Cys418) via a Michael reaction, and the exchange of Cys418 to serine by mutation rendered 5-LO insensitive to NO2-FA. Notably, structurally related derivatives of NO2-OA containing a Michael-acceptor also caused direct and
potent suppression of 5-LO enzyme activity. Systemic administration of NO2-OA to mice decreased neutrophil and
monocyte mobilization in response to lipopolysaccharide (LPS), attenuated the formation of the 5-LO product 5hydroxyeicosatetraenoic acid (5-HETE), and inhibited lung injury. Administration of NO2-OA to 5-LO knockout mice had
no effect on LPS-induced neutrophil or monocyte mobilization as well pulmonary inflammation. NO2-FAs are a novel
type of endogenous 5-LO inhibitor directly and irreversibly suppressing 5-LO and contributing to resolution of inflammation in vivo. We hypothesize that synthetic homologues of the NO2-FAs may represent an innovative strategy to treat
inflammatory diseases and may represent a novel potential pharmacological option for 5-LO inhibition, which might have
therapeutic implications for asthma.
The authors are indebted to Marie von Reutern, Isabella Schlöffel, and Sven George for their excellent technical support. This study was supported
by the LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF) and the Deutsche Forschungsgemeinschaft (Exzellenzcluster 147 ‘‘CardioPulmonary Systems’’ and SFB 815/Z1). T.J.M. is the recipient of a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft.
References:
1. Baker, P.R., Schopfer, F.J., Freeman, B.A.: Free Radic Biol Med 2009, 46(8): 989-1003.
2. Hornig, M. et al.: Biochim Biophys Acta 2011, 1821(2): 279-286.
26
Ex vivo and in vivo siRNA delivery to activated T cells as novel anti-inflammatory asthma therapy
Xie, Y.1; Kim, N.H.1; Nadithe, V.1; Thakur, A.1,2; Lum, L.G.1,2; Bassett, D.J.P.1; Merkel, O.M.1,2
1 Wayne
State University, DETROIT, MI, United States of America
Cancer Institute, DETROIT, MI, United States of America
2 Karmanos
Local, targeted, cell-specific RNA interference (RNAi)-based therapies could improve patients’ ability to control asthma.
Allergen-induced airway dysfunction was shown to be prevented by downregulating the secretion of Th2 cytokines.
However, T cells are hard to transfect cells and not easily accessible for RNAi-based therapies. We recently reported
that activated T cells (ATCs) overexpress the transferrin receptor (TfR) which is an internalizing transmembrane receptor
that mediates endocytosis of transferrin-bound iron and which is broadly exploited for targeted nucleic acid delivery.1, 2
Here we aim to therapeutically downregulate the Th2 transcription factor GATA-3 which is known to drive IL-4, IL-5, and
IL-13 secretion in asthma to silence all its downstream inflammatory cascades.
T cells are isolated from full blood or by magnetic bead isolation from mouse spleens. TfR overexpression is measured
by flow cytometry upon activation with anti-CD3. TfR-targeted nanocarriers are designed by optimizing the coupling
chemistry of Tf and low molecular weight polyethylenimine (PEI) and are purified by FPLC and ultrafiltration. Fluorescently labeled siRNA is delivered to ATCs, and the uptake is evaluated by flow cytometry. Gene knockdown in primary
ATCs after siRNA delivery is quantified by qRT-PCR. Balb/c mice are sensitized and challenged with ovalbumin (OVA)
or with NaCl as negative control. Mice are treated intratracheally with free fluorescent siRNA, targeted, or non-targeted
nanocarriers. Their lung function is measured as changes in resistance to metacholine challenge.
A: Coupling of Tf to PEI, B: siRNA uptake in ATCs, C: Asthma model, D: siRNA uptake in CD4+ cells in OVA-challenged mice.
The coupling of Tf to PEI was optimized and the nanocarriers were shown to be smaller than 100 nm in size. ATCs
selectively took up targeted nanocarriers, and 70% gene knockdown was achieved in primary ATCs. OVA-challenged
mice showed specific uptake of fluorescently labeled siRNA in pulmonary T cells mediated by targeted nanocarriers. No
other cell types (macrophages, epithelial cells, endothelial cells, eosinophils, B cells) took up significant amounts of
siRNA. The lung function was not negatively affected by the Tf-shielded nanocarriers.
Wayne State Start-Up Grant, FRAP, BOOST and NanoIncubator grants to OMM are gratefully acknowledged.
References:
1. Kim, N.H. et al.: J Aerosol Med Pul Drug Del 2013, 26 (2): A46-A47.
2. Kim, N.H. et al.: J Drug Del Sci Tech 2013, 23 (1):17-21.
27
Rational protein-engineering yields a minimised innate immune inhibitor with unique targeting properties
Harder, M.; Simmet, T.; Schmidt, C.Q.
Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany
Introduction: The complement system is an integral part of the human immune system and is well recognized
for its contributions to host defense and tissue homeostasis [1]. While complement activation is necessary to protect and
maintain self-tissue, prolonged and unrestricted activation is causative or associated with several illnesses [2]. The
increasing evidence of complement involvement in many frequent chronic conditions has revived the surge for intervening options, but several challenges specific to the complement system have prevented the availability of an efficient and
cost effective complement therapeutic. Three complement activation pathways exist. Especially the so called “alternative
pathway” of complement activation is the underlying factor in several human disease conditions (e.g. age-related macular degeneration). However, treatment options that specifically block the alternative pathway (AP), leaving other complement pathway uninhibited, are not available in the clinic [3].
Objectives: This study describes the rational engineering of the innate immune modulator “mini-FH”, which is
based on the template glycoprotein “Factor H”, a natural complement regulator in human serum. The study also evaluates the biological functions and pharmacokinetic behaviour of mini-FH and mini-FH-derived second generation products.
Results: Based on resent insights into the structure and function of the crucial complement AP-regulator Factor
H [4-6], we rationally engineered a protein therapeutic (mini-FH) by directly joining selected N- and C-terminal domains
of FH through a rationally optimized linker in such way that Factor H functionality is preserved. Importantly, the chosen
design also attributes a novel function: mini-FH shows a strong preference over Factor H in binding complement inactivation products. This furnishes mini-FH with a unique triple targeting mechanism to facilitate simultaneous targeting to (i)
complement activation/inactivation products and (ii) polyanionic host surface markers and (iii) to markers of oxidative
stress. The engineered mini-FH protein was expressed in the methylotrophic host Pichia pastors, purified to homogeneity and submitted to several in vitro assays including an AP-mediated disease assay on patient-derived cells: mini-FH
conferred efficient complement regulation at an IC50 of 60 nM. In a next step novel mini-FH derivatives with extended
structures were generated to optimise efficacy and pharmacokinetic profiles. The resulting 2nd generation mini-FH class
of proteins were tested in a panel of established interaction, functional and inhibition studies and exhibited improved
activity when compared to the first generation. Finally, a first pharmacokinetic profile and the in-vivo activity of a selected
mini-FH candidate-protein were assessed after in vivo administration into a wildtype mouse and transgenic mice mimicking a complement-mediated disorder, respectively.
Conclusions: By employing a rational engineering approach of the natural complement alternative pathway regulator FH, we gained a set of very efficient complement regulators that largely exceed (about ten- to twenty-fold) the
biological activity of the template molecule Factor H. Our data show that the rationally engineered mini-FH proteins are
able to prevent disease-related complement activation by targeting sites of ongoing complement amplification on host
cells; thus, the mini-FH class of engineered proteins has high therapeutic potential for a variety of complement-mediated
diseases.
Acknowledgments: Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA, John D. Lambris
& Daniel Ricklin.
References:
1. Ricklin, D. et al.: Nat Immunol. 2010, 11: 785-797.
2. Ricklin, D.; Lambris, J.D.: Nat Biotechnol. 2007, 25: 1265-1275.
3. Ricklin, D.; Lambris, J.D.: J Immunol. 2013, 190: 3839-3847.
4. Schmidt, C.Q. et al.: Clin Exp Immunol. 2008, 151: 14-24.
5. Schmidt, C.Q. et al.: J Immunol. 2008, 181: 2610-2619.
6. Morgan, H.P. et al.: Nat Struct Mol Biol. 2011, 18: 463-470.
28
NEURODEGENERATION
New insights into Bid-mediated mitochondrial demise in neuronal cell death
Culmsee, C.
Institute of Pharmacology and Clinical Pharmacy, University of Marburg, 35032, Germany
Mitochondria are highly dynamic organelles with essential functions in the physiology of energy metabolism, controlled
reactive oxygen species (ROS) formation and the regulation of intracellular Ca2+ homeostasis. In the nervous system,
mitochondrial integrity is crucial for the maintenance and function of neurons. In fact, mitochondrial damage is a major
feature of many neurological and neurodegenerative diseases, and cellular stress and death signaling pathways converge at the level of mitochondria. In particular, disturbed intracellular Ca2+ homeostasis, increased ROS formation,
dysbalanced fusion and fission of the organelles, loss of the mitochondrial membrane potential, and the release of
mitochondrial proteins such as apoptosis inducing factor (AIF) are prominent in many different paradigms of neuronal
dysfunction and death.
The lecture will highlight novel insights into the molecular regulation of increased mitochondrial fission and associated
damage in models of neuronal cell death induced by oxidative stress, glutamate toxicity and oxygen glucose deprivation.
In particular, the role of Bid will be discussed as potential targets for therapeutic approaches in models of neuronal death
in vitro and in vivo. The data demonstrate that Bid interacts with dynamin related protein-1 (Drp-1) and the voltagedependent anion channel (VDAC1) in mediating mitochondrial damage and intrinsic pathways of cell death, while pharmacological inhibition or genetic deletion of each factor preserves mitochondrial functions, thereby providing neuroprotective effects. In addition, further approaches of mitoprotection are discussed, including strategies of mitochondrial
preconditioning mediated by inhibition of complex I, AIF depletion or inhibition of Cyclophilin A (CypA).
Overall, several lines of evidence expose mitochondria as key regulators in pathways of cellular stress with relevance to
progressive neuronal dysfunction and death which is prominent in many neurodegenerative diseases and conditions of
acute brain damage. Thus, novel concepts aiming at preserved mitochondrial integrity and function may provide effective
neuroprotection.
29
Experimental stroke research: problems and opportunities
Klein, J.
Department of Pharmacology, FB 14, Goethe University, Frankfurt, Germany
A stroke is an acute incidence in humans and can hardly be foreseen. Accordingly, clinical stroke research is largely
restricted to patients that have sustained a stroke hours or days earlier. In experimental stroke research, brain ischemia
can be induced in rodents in a controlled manner, parameters such as cerebral blood flow and brain damage can be
monitored in situ, and neurologic outcome can be determined for weeks afterwards. Nevertheless, many compounds that
were identified as therapeutically effective in rodents did not work when tested in clinical studies. To improve the translation of drugs from bench to bedside, we have therefore attempted to get a clearer picture of the metabolic status of
ischemic brain tissue in rodents, using microdialysis and analytical approaches to quantify energy metabolites in ischemic and healthy tissue. In our experimental work, we presently focus on preventive measures which could be used to
reduce ischemic damage in patients at risk.
In one approach, we administered bilobalide, a neuroprotective compound isolated from Ginkgo biloba extracts which
are known to be well tolerated by humans. At 1-10 mg/kg, bilobalide reduces ischemic brain damage up to three hours
after stroke and also improves motor function in mice. Microdialysis studies showed that the dramatic, more than 50-fold
increase of glutamate which is observed in untreated animals is reduced by 80% in treated mice. At the same time,
mitochondrial function is preserved, especially in complex I of the electron transport chain (ETC). Protection of mitochondria leads to higher energy (ATP) levels, reduced glutamate release and protects neurons from excitotoxicity and
cell death.
In a second approach, we used an anaplerotic diet in mice. Anaplerosis, the mechanism of refilling lost substrates in the
citric acid cycle (CAC), is a new approach for neuroprotection. Using triheptanoin (glycerol-triheptanoate) as a dietary
supplement in mice, we expected the formation of odd-chain fatty acids which can be metabolically transformed into
succinate, a metabolite of the CAC which also fuels directly into complex II of the ETC. After 14 days of diet, stroke was
induced and neurological deficits were determined by behavioral testing. Triheptanoin-fed mice showed a significant
improvement in three behavioral tests whencompared to mice on control diet containing soybean oil. In microdialysis
studies, blood flow and glucose consumption were similar between the two dietary groups, but glutamate release was
reduced by 66% in triheptanoin-fed mice. What is more, the activities of complexes II and IV of the ETC were higher in
the triheptanoin group, and ATP levels and mitochondrial membrane potentials were also better preserved. This data
shows that the triheptanoin-rich diet produced a neuroprotective effect in ischemic stroke in mice. It should therefore be
tested as a prophylactic treatment against brain ischemia in humans.
In summary, experimental stroke research helps to identify potential prophylactic treatments that could be tested in
clinical trials. It may be more promising to prevent ischemia-induce damage than to treat it after the damage has been
done. Alternatively, these treatments could be used to improve regeneration after stroke. Experiments to that effect are
currently in the planning stage.
30
Emerging options for pharmaceutical care in stroke patients
Hohmann, C.1,2; Neumann-Haefelin, T.2; Radziwill, R.1
1 Klinikum
2 Klinikum
Fulda gAG, Department of Pharmacy, Pacelliallee 4, 36043 Fulda, Germany
Fulda gAG, Department of Neurology, Pacelliallee 4, 36043 Fulda, Germany
Stroke is one of the leading causes of death in Europe and the major cause of disability in the elderly. Ischaemic stroke
accounts for 85–90 % of all strokes and is characterized by the sudden occlusion of a cerebral artery resulting in reduced blood flow and loss of neurological function. Ischaemic stroke is mainly caused by large-artery atherosclerosis,
cardio embolism (e.g. atrial fibrillation), or small-vessel occlusion. Determing the cause of ischaemic stroke is very
important for further therapy management, especially the secondary stroke prevention. The main risk factors for stroke
are hypertension, atrial fibrillation, diabetes mellitus, high cholesterol and smoking. A consistent, safe, and effective
secondary prevention after ischemic stroke is very important and contains the intake of acetylsalicylic acid in atherosclerotic stroke or an oral anticoagulant for the prevention of cardioembolic stroke as well as the treatment of the cardiovascular risk factors.
The main issues involved with clinical pharmacy practice are (i) medication reconciliation, (ii) drug therapy optimization, and detection, resolution and prevention of drug-related problems (DRPs), (iii) consultation with the patient regarding new drugs including advice about indication, dosage, adverse events, and drug-interactions, (iv) giving detailed
information on medication upon hospital discharge in the discharge letter by the clinical pharmacist.
These different issues of the clinical pharmacy service in stroke patients will be presented [1-5].
In conclusion, clinical pharmacists can provide a valuable contribution in the multidisciplinary team by detecting and
resolving DRPs that lead to an optimized and safe pharmacotherapy, especially regarding to antihypertensive medication, secondary prevention, and statin therapy. Furthermore, they support the information transfer through medication
review and medication reconciliation on hospital admission and hospital discharge that lead to an optimized transition
care process.
References:
1. Hohmann, C. et al.: Pharm World Sci. 2009, 31(5): 550-558.
2. Hohmann, C. et al.: Health Qual Life Outcomes. 2010, 8: 59.
3. Hohmann, C. et al.: Int J Clin Pharm 2012, 34(6): 828-831.
4. Hohmann, C. et al.: Stroke 2013, 44(2): 522-524.
5. Hohmann, C. et al.: J Clin Pharm Ther. 2014, 39(3): 286-291.
31
SK channel modulation attenuates mitochondrial dysfunction, neuroinflammation, and neuronal cell
death
Dolga, A.M. 1; Terpolilli, N.2; Netter, M.3; Richter, M.4; Decher, N.3; Plesnila, N.2; Culmsee, C.1
1 Institute
of Pharmacology and Clinical Pharmacy, University of Marburg, 35032, Germany
of Neurosurgery, Ludwig-Maximilians-Universität München, München, 81377, Germany
3 Institute of Physiology and Pathophysiology, Vegetative Physiologie, University of Marburg, 35037, Germany
4 Department of Neurology, University of Marburg, 35043, Germany
2 Department
Potassium channels are a family of highly diverse transmembrane proteins with multiple functions in the physiology of
excitable cells, and according dysfunctions have been linked to degeneration and death of neurons in various neurological diseases. According to current knowledge, small-conductance calcium-activated potassium (SK/KCa2) channels are
located in close vicinity to synaptic NMDA receptors and control excitability and Ca 2+ influx by reducing the amplitude of
synaptic potentials. Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in
intracellular calcium ([Ca2+]i), mitochondrial dysfunction, ER stress and inflammation are established hallmarks of neuronal cell death. Recently, we showed that pathological [Ca2+]i deregulation occurring after glutamate receptor stimulation is effectively modulated by SK channels. Activation of SK channels preserved SK expression and significantly
reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo in a model of middle cerebral artery occlusion. In addition, SK channel opening restores microglial activation, cytokine production and nitric oxide
release.
Besides their plasma membrane localization we have demonstrated that functional SK2 channels are also expressed in
the mitochondrial inner membrane and prevent glutamate-induced neuronal oxidative stress and mitochondrial dysfunction by 80-90%. Activation of SK channels inhibits mitochondrial fragmentation, loss of mitochondrial membrane potential, and translocation of apoptosis inducing factor (AIF) to the nucleus. Using a neuronal cell line devoid of NMDA
receptors, we demonstrated that the neuroprotective effects were independent of direct interaction of SK channels with
NMDA receptors and calcium influx, further supporting the functional activity of SK channels in mitochondria. Furthermore, overexpression of SK channels in mitochondria using mitochondrial-targeted SK channels demonstrated substantial neuroprotective effects in a model of oxidative stress (induced by glutamate) and ER stress (induced by brefeldin A).
SK channel activation altered the level of unfolded protein response proteins, i.e. further increased CHOP levels and
reduced PERK levels compared to brefeldin A-treated cells. Moreover, activation of SK channels resulted in slight ATP
depletion and reduced mitochondrial metabolic activity, as assessed by the Seahorse Bioscience XFe cell metabolism
analyzer. These results expose a pre-conditioning effect as a mechanism for neuroprotection mediated by SK channel
activation.
Our findings show a critical role for SK channels in excitotoxic neuronal cell death, mitochondrial dysfunction, ER stressassociated cell death and neuroinflammation, proposing their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.
32
PHARMACEUTICAL TECHNOLOGY AND DRUG DELIVERY
Formulation Strategies for Poorly Water Soluble Drugs
Thommes, M.
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Duesseldorf, Germany
The low aqueous solubility of novel active pharmaceutical ingredients is one of the major challenges pharmacists have
been facing for more than 35 years. Since then several formulation strategies have been developed, which all have
advantages but also limitations.
The presentation covers the physical reasons as there are particularly low crystal lattice energy as well as high lipophilicity as main reasons for low aqueous solubility. This requires different formulation strategies like “emulsifying systems”,
“solid dispersions”, “nano particles” as well as “co-crystals” in order to increase the oral bioavailability. These concepts
are discussed in detail while market products are mentioned and actual research trends are debated.
33
Valid cell culture models of the human cornea for drug transport investigations - where are we?
Reichl, S.; Hahne, M.; Verstraelen, J.; Kölln, C.
Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany
Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treatment. Since the cornea is the main barrier for most topically applied drugs, such preclinical investigations on transcorneal drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell
culture models. Cell culture models of the human cornea can avoid several of the disadvantages of widely used animal
experimental models, including ethical concerns and poor standardisation [1]. However, for widespread use, the contemporary validation of existent systems is required.
Based on SV40 immortalized human corneal epithelial cells and keratocytes we established a serum-free cultivated
human hemicornea construct (HC) that exhibited high degree of equivalence to ex vivo tissue regarding histological
characteristics and barrier properties [2]. In a next step, our standard operating procedures were transferred and the HC
model was independently cultured in three different laboratories, and the intralaboratory and interlaboratory reproducibility was analyzed and compared with animal corneas. This analysis showed that the HC has a barrier in the same range
as excised animal corneas, although with a higher reproducibility and lower variability [3].
While transcorneal passive drug transport processes are well understood, only few is known about the expression of
transporter proteins, in particular ABC transporters, and drug-metabolizing enzymes in both human and animal corneal
tissue as well as human cornea cell culture models. A comparison of the expression of ABC transporters (MDR1, MRP
1-5 and BCRP) and phase I and II enzymes, in particular, cytochrome P450 enzymes (CYP) and glutathione transferases (GST), between HC and the most commonly used ex vivo models, namely, rabbit and porcine corneas, was conducted. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and
bidirectional permeation studies using specific substrates and inhibitors or activity assays in the case of GST and CYP.
The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the
expression and activity of MDR1 transporter was confirmed, whereas HC and porcine corneas did not show MDR1
expression. However, HC possessed MRP1, 3-5 and BCRP expression, whereas no functional expression of MRP 1-3, 5
and BCRP was found in porcine corneas and MRP 3, 4 and BCRP in rabbit corneas. Therefore, the transfer of data
obtained from animal experiments to an in vivo situation in humans should be performed with caution.
Even though the expression levels of drug-metabolizing enzymes in corneal tissue are low in comparison to liver or
kidney, the expression of GSTO1, GSTP1 and CYP2D6 isoenzymes was detectable in HC, human and animal corneas
on mRNA, protein and functionality level. GST activity was found to be higher in rabbit and porcine cornea compared to
HC. In contrast, total CYP450 and CYP2D6 activity were detected to be similar in HC and excised corneas.
The HC represents a promising in vitro alternative to the use of ex vivo tissue and offers a well-defined and standardized
system for drug absorption studies.
Acknowledgments: We are grateful to the German Federal Ministry of Education and Research (BMBF) and German Federal Institute for Risk
Assessment (BfR), which funded this work under grant nos. 0315504E, 0315504B and 3-1328-30652054369.
References:
1. Pepić, I. et al.: Drug Discov Today 2014, 19: 31-44.
2. Hahne, M., Reichl S: Int J Pharm. 2011, 416: 268-279.
3. Hahne, M. et al.: J Pharm Sci. 2012, 101: 2976-2988.
34
Protein engineering of fibroblast growth factor 2 (FGF-2) for bioresponsive protein delivery
Lühmann, T.1; Jones, G.1; Memmel, E.2; Seibel, J.2; Meinel, L.1
1
2
Institute for Pharmacy and Food Chemistry, University of Würzburg, Germany
Institute for Organic Chemistry, University of Würzburg, Germany
Current needs in regenerative medicine include the design and development of sophisticated materials that integrate
tissue specific growth factors. Immobilisation of growth factors to implant materials has been generally performed by (i)
non-covalent physiochemical adsorption of the protein (ii) covalent random immobilization by e.g. primary amino-groups
of the protein or (iii) enzymatic coupling approaches. Methods, which covalently link proteins non-specifically to surfaces,
have certain limitations as the presentation and bioactivity of the protein can be severely diminished during the modification process. Although the bioactivity of growth factors can be retained by non-covalent physiochemical adsorption onto
implant materials, desorption of the protein occurs uncontrolled and in a rapid manner, a bottleneck when e.g. a longterm release of the protein into the environment is desired. An alternative strategy deploys the genetic mechanisms in
archaebacteria, which use a stop codon to encode for the 22nd amino acid pyrrolysine during translation [1]. By replacing
pyrrolysine with its analogue propargyl-L-lysine (plk), genetic engineered proteins can be recombinantly expressed and
modified in a site-directed fashion thereafter [2, 3].
This study was designed to elucidate the impact of site-directed immobilisation by copper catalyzed azide alkyne cycloaddition (CuAAC) of genetic engineered plk-FGF2 on cellular responses in vitro.
Wild-type murine FGF2 and murine plk-FGF2 were expressed in E.coli BL21 (DE3) and were purified using heparin
based affinity chromatography as previously described [4]. Azide groups (azide-mannose) and control groups (mannose)
were introduced on model surfaces and plk-FGF2 was linked via click reaction in comparison to wild-type FGF2 and to
plk-FGF2 in the absence of copper (I). The potency of both soluble and immobilized FGF2 was determined by the
proliferation of human MG-63 cells and by the investigation of ERK signalling, respectively.
Plk-FGF2 was successfully expressed in the presence of 3 mM plk and was purified by heparin chromatography in an
analogue manner to wild-type FGF2. The soluble plk-FGF2 analogue was found to induce proliferation of MG-63 cells in
the same magnitude compared to the wild-type protein. The correct incorporation of plk at position 8 at the N-terminus of
FGF2 was confirmed by ESI-MS analysis and peptide mapping after trypsin digest. Functionality of the introduced
alkyne-group of plk-FGF2 was demonstrated by Cy3-azide conjugation in the presence of copper (I) by monitoring
fluorescence signals by SDS-PAGE and subsequent Coomassie protein staining. Plk-FGF2 was immobilized to azidemannose decorated surfaces by CuAAC and immobilized FGF-2 on the surface was visualized by immunosttaining.
Moreover, MG-63 cells were successfully stimulated by immobilized FGF2.
Site-directed immobilisation of growth factors might be a powerful tool to decorate implant surfaces and opens the
possibility to tailor bioresponsive release mechanism in the future.
References:
1. Gaston, M.A. et al.: Curr Opin Microbiol 2011, 14: 342-349.
2. Eger, S. et al.: Methods Mol Biol 2012, 832: 589-596.
3. Nguyen, D.P. et al.: J Am Chem Socl 2009, 131: 8720-8721.
4. Zhao, H. et al.: Journal of structural biology 2014, 186(3): 420-430.
35
In vitro estimation of drug transfer from paclitaxel-coated balloon catheters
Seidlitz, A.1; Kempin, W.1; Reske, T.2; Kaule, S.2; Grabow, N.2; Petersen, S.2; Nagel, S.1; Weitschies, W.1
1 Ernst-Moritz-Arndt
2 University
University of Greifswald, Institute of Pharmacy, C_DAT, Felix-Hausdorff-Straße 3, 17487 Greifswald, Germany
of Rostock, Institute for Biomedical Engineering, Friedrich-Barnewitz-Straße 4, 18119 Rostock, Germany
Drug-coated balloons (DCB) are an innovative approach to locally treat stenosis of coronary arteries for lesions in which
a therapy with drug-eluting stents is considered impossible (for example due to small vessel size or length of lesion) or
unpromising (e.g. after multiple stent implantations in the concerned vessel segment). In 2011 at least 5 different types
of such medical devices had received the CE-mark in Europe [1]. The current drug of choice for this application is
paclitaxel, a mitotic inhibitor which is intended to supress hyperproliferation and migration of smooth muscle cells which
may cause a re-narrowing of the treated vessel portion. For this treatment, the drug-coated balloon is advanced through
the vascular system to the generally previously angioplastically re-opened lesion and is expanded. During this expansion
which typically allows for contact times of up to 60 s the drug is delivered to the vessel wall. Due to this short time frame
available for drug transfer in combination with a challenging application through the vascular system, the coating morphology and the mechanical stability of the coating are of greatest importance.
To systematically evaluate the drug transfer from DCB in vitro, a previously established perfused model of the implantation pathway including a guiding catheter and a simulated arterial passage was combined with a hydrogel cylinder [2].
Using this system drug loss during the passage to the site of application and drug transfer to the gel representing the
vessel wall in this test setup were evaluated. The model coronary artery pathway (fig. 1, left) was perfused with PBS pH
7.4 at a flow rate of 35 mL/min for 60 s. During this time the DCB was rapidly advanced through the system until reaching the resting position (indicated by asterisk). After the perfusion time the balloon was further advanced until exiting the
model. The DCB was inserted into the gel cylinder and expanded (inflation pressure 8 atm, contact time 60 s). After refolding and removal of the balloon the drug content of the gel, the perfusion liquid, the residual amount on the balloon
surface and potential residual drug in the pathway model were determined via hplc. Balloons coated with Paclitaxel in
combination with different excipients (fabricated via micro-pipetting) and commercially available SeQuent® Please
balloons were tested using this method.
The results of the drug transfer testing are depicted in figure 1 (right). It becomes evident, that the simulation of the
implantation process had a great influence on drug transfer due to the occurrence of great losses of the coated drug.
The best drug transfer rates were obtained for the coatings containing iopromide (SeQuent® Please) or PVP as an
additive with 18 % and 6 % of the initial drug load. The coatings fabricated without additives except for the solvents
showed very low drug transfer rates from 0.8 % to 2.6 %. Opposed to the other two purely solvent containing formulations which lost almost the entire drug load during the passage, the ethyl acetate coating possessed a very smooth
glassy structure and 15 % of the drug remained on the balloon after the transfer experiment. These findings emphasize
the necessity to design coating strategies for DCB which produce mechanically stable coatings that are able to withstand
the implantation procedure while at the same time allowing for fast drug transfer at the site of application. The use of
certain additives in the coating seems to be favourable over purely solvent-containing coating liquids. In addition, the
solvent also seems to greatly influence coating morphology and adhesion to the balloon surface.
Acknowledgement: Financial Support by the German Federal Ministry of Education and Research (BMBF) within “REMEDIS” and by the European
Social Fund (ESF) and European Regional Development Fund (ERDF) is gratefully acknowledged.
References:
1. Scheller, B. et al.: Kardiologe 2011, 5(6): 411-435.
2. Seidlitz, A. et al.: PLOS ONE 2013, 8(12): e83992.
36
Self-developed sensor membranes for etongue sensors
Pein, M.; Schneider, K.
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University Düsseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany, email: [email protected], phone: +49 2118114225
Purpose: Sensor membranes for etongue sensors should be developed by applying solvent-casting. Their performance
should be assessed regarding taste-masked oral formulations and the results compared with those of commercially
available sensors.
Materials & Methods: Sensor membranes were prepared based on high molecular weight polyvinylchloride (PVC,
Sigma-Aldrich), acetone (VWR), tetrahydrofuran (THF, AppliChem), isopropyl myristate (IPM, Cognis), tetradodecylammonium bromide (TB, Sigma-Aldrich), trioctylmethyl-ammonium chloride (TC, Alfa Aesar), bis(2-ethylhexyl) phosphate (BP, Sigma-Aldrich) and hydroxypropyl-ß-cyclodextrin (HP, Roquette). Solutions were casted, dried and adhered
to PVC based sensor heads [1]. The sensors were filled with KClaq (3.33 M) in saturated AgClaq and equipped with an
Ag/AgCl electrode. Sensor performance was compared to the commercially available astringency (SB2AAE) and bitter
sensors (SB2AC0, SB2AN0, SB2BT0) utilizing the Insent taste sensing system TS-5000Z (Insent, Inc.) according to
Woertz et al. [2], but with a stability criterion of 2 mV. Evaluated samples were A: oral films containing dimenhydrinate
(DMH), B: oral films containing DMH and sweeteners, C: placebo oral films, D: placebo oral films containing sweetener,
E: pure DMH, F: DMH + sweetener, G: pure sweetener [3]. 20 films of sample A-D were dissolved over 3 min in 100.0 ml
of purified water at 37 °C and immediately filtered.
Results & Discussions: Principle component analyses (PCA) were performed based on the responses of the commercially available and of the self-developed sensors. The information of the DMH containing samples is influenced by the
responses of the commercial bitter sensors SB2AC0, SB2AN0 and SB2BT0 (Figure a: Loading Scatter Plot) and thus
displayed on the right side of the according PCA map (Figure A). A comparable result is seen for the self-developed
sensors with the major influence of sensor TBHP (Figure B and b). In both cases, the information is mainly separated
along principle component 1 (PC1: 98.8 % and 94.1 %). Regarding the similar location, DMH containing samples A, F
and E are detected comparably. Data points of sample B are located in the direction of the (sweet) placebo samples C,
D and G, indicating a slight taste-masking effect. Minor differences between Figure 1A and 1B can be seen regarding the
location of the placebo film sample C, indicating that the self-developed sensors enable a discrimination between film
based samples (A-D) and the pure substances (E-F), resulting in a slight separation along the y-axes.
Conclusion: Sensor membranes with comparable performance to commercially available ones were developed. Both
sensor sets registered a taste-masking effect of sample B. The indicated benefit of the self-developed sensors – to
discriminate between film based samples and pure substances – will be further investigated.
References:
1. Schneider, K.: Diploma Thesis: 2014, Ernst-Moritz-Arndt-Universitaet Greifswald.
2. Woertz, K. et al.: J. Pharm. Biomed. Anal. 2010, 51: 497-506.
3. Pein, M. et al.: Int. J. Pharm. 2014, 469: 228–237.
37
MEDICINAL CHEMISTRY (PSJ)
Paradigm Re-shift of Medicinal Chemistry in Japan
Tomioka, K.
Faculty of Pharmaceutical Sciences, Doshisha Women’s College of Liberal Arts, Kodo Kyotanabe 610-0395 Japan
Drug discovery of small molecules is the main and only one aim of medicinal chemistry. Approach to this excellent goal
had been influenced by the great progress of biological sciences. Especially many and distinct proteins provide us the
targets for small molecules called as, for example, genom-based drug discovery. This trends expanded the criteria of
drugs to antibody drugs. Recent trend on this line is the development of antibody-drug conjugate, again a revival of small
molecules. These paradigm shifts in drug discovery of medicinal chemistry and recent progress of catalytic bond forming
reactions which enable the efficient synthesis of target small molecules1 would be the subject of this lecture.
Reference:
1. Xinyu, H. et al.: Catalysis Science & Technology, 2011, 1(1): 62-64.
38
Design, synthesis and biological activity of lysine-specific demethylase (KDM) inhibitors
Miyata, N.1; Suzuki, T2; Nakagawa, H.1
1
2
Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi 467-8603, Japan
Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 603-8334, Japan
Methylation of histone lysine residues is reversibly controlled by histone lysine methyl transferases (KMTs) and histone
lysine demethylases (KDMs) and plays an important role in the regulation of gene expression. Two classes of KDMs
have been identified. One is lysine-specific demethylases (LSDs), which are flavin-dependent amine oxidase domaincontaining enzymes, and the other is Jumonji domain-containing demethylases (JMJDs), which are Fe(II) and αketoglutarate-dependent enzymes. KDMs are associated with various disease states, and have emerged as attractive
targets for the development of new therapeutic drugs. We designed and synthesized several selective KDM inhibitors
(Figs. 1 & 2) and will discuss their potential as cancer therapeutic agents.
References:
1. Ueda, R. et al.: J. Am. Chem. Soc., 2009, 131(48): 17536-13537.
2. Ogasawara, D. et al.: Angew. Chem. Int. Ed., 2013, 52(33): 8620-8624.
3. Hamada, S. et al.: Bioorg. Med. Chem. Lett., 2009, 19(10): 2852-2855.
4. Hamada, S. et al.: J. Med. Chem., 2010, 53(15): 5629-5638.
5. Suzuki T. et al.: J. Med. Chem., 2013, 56(18): 7222-7231.
39
Synthesis of a novel opioid receptor agonist, SYK-146 with 1,3,5-trioxazatriquinane skeleton and its
pharmacologies
Nagase, H.2; Hirayama, S.1; Wada, N.1; Kuroda, N.1; Iwai, T.1; Fujii, H.1
1
2
School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
International Institute for Integrative Sleep Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan
We designed and synthesized of 1,3,5-trioxazatriquinanes with m-hydroxyphenyl groups. The designed 1,3,5trioxazatriquinanes include the phenethylamine structure within them, which is a common structure observed in
morphinan derivatives like morphine. Among the synthesized compounds, SYK-146 (1) with two m-hydroxyphenyl
groups selectively bound and exerted full agonist activity toward the κ opioid receptor (KOR).[1] Subcutaneously administered (1) exhibited significant antinociceptive effects via the KOR in a dose dependent manner. These results suggest
the emergence of a novel class KOR agonist.
We also report the pharmacological activities of the optically active SYK-146, and the o- and p-hydroxyphenyl derivatives.[2]
References:
1. Hirayama, S.; Nagase H. et al.: ACS Med. Chem. Lett. 2014. in press.
2. Hirayama, S.; Nagase H. et al.: Bioorg. Med. Chem. Lett. 2014. in press.
40
Gold-catalyzed annulations and their medicinal applications
Ohno, H.; Suzuki, Y.; Hou, Z.; Tokimizu, Y.; Oishi, S.; Fujii, N.
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
The development of cascade reactions is an area of considerable interest in modern organic chemistry. Efficient cascade
reactions realize the economical synthesis of complex target molecules through multiple bond formations in a single
operation. Elementary reactions that form fewer waste products are desirable in terms of atom economy and to suppress
side product formation in sequential processes. Recent advances in homogeneous gold catalysis have opened up
further possibilities for cascade reactions. We are involved in development of gold-catalyzed atom-economical cascade
reactions and their medicinal applications.
(1) Synthesis of dihydropyrazoles by gold-catalyzed three-component annulation
(1-1) Efficient synthesis of highly functionalized dihydropyrazoles1
A transition metal-catalyzed Mannich-type three-component coupling of alkynes, aldehydes and amines (A3 coupling) is
an attractive reaction not only for facile preparation of propargylamines but also as an elementary reaction for a cascade
process. We developed a gold-catalyzed A3 coupling to produce polysubstituted dihydropyrazoles 5, in which all the
reaction components are incorporated in the newly-formed ring.
R1
+
R2
H
O
1
R
+
2
3
N
H
H
N
R4
R
cat. [Au+ ]
4
H
N
N
R3
R2
R
1
3
[Au +]
4
R4
R3
N N
R1
R2
5
(1-2) Fused pyrazole syntheses and identification of novel CK2 inhibitors2,3
Using the gold-catalyzed three-component annulation, various benzo[g]indazole derivatives 8 and pyrazolo[4,3-b]indole
derivatives 9 were prepared. Evaluation of their CK2 inhibitory activities revealed that benzo[g]indazole and pyrazolo[4,3b]indole are appropriate scaffolds for potent CK2 inhibitors.
or
RO 2C
6
R1
RO2 C
N3
R
HO2C
7
H
N N
HN N
cat. [Au+ ]
R
1
2
or
HO2C
8
9
R2
N
H
indoloquinolines4
(2) Gold-catalyzed cascade cyclization of (azido)ynamides for the construction of
(Azido)ynamides 10 were efficiently converted into indoloquinolines 12/13 by the use of gold catalyst. While ynamides
bearing an allylsilane gave terminal alkenes 12, ynamides bearing a simple alkene gave cyclopropanes 13. These
reactions proceed through the formation of an α-amidino gold-carbenoid 11.
Ts
N
R
R
+
cat. [Au ]
[Au+]
R
H
or
N
N
N
N
H
Ts
Ts
N3
10
12 (R = CH2TMS)
13 (R = Ph or n-Bu)
11
Acknowledgments: The authors wish to thank Prof. Tsujimoto, G. and Prof. Hirasawa, A. (Graduate School of Pharmaceutical Sciences, Kyoto
University) for the CK2 inhibition assay; Professor Nakanishi, I. (Department of Pharmaceutical Sciences, Kinki University) for the docking simulation and valuable comments for the molecular design. This work was supported by a Grant-in-Aid for the Encouragement of Young Scientists (A),
as well as Platform for Drug Design, Discovery, and Development from the MEXT, Japan.
N
N
Ts
References:
1. Suzuki, Y. et al.: Org. Lett. 2012, 14(1): 326.
2. Suzuki, Y. et al.: Org. Biomol. Chem. 2012, 10(25): 4907.
3. Hou, Z. et al.: Org. Biomol. Chem. 2013, 11(20): 3288.
4. Tokimizu, Y. et al.: Org. Lett. 2014, 16(11): 3138.
41
BIOMARKER AND MODELING
Implementation of dosing algorithms of anticancer drugs based on pharmacological biomarkers
Joerger, M.
Kantonsspital St.Gallen, Switzerland
Anticancer drugs are typically given either as a flat-dose or based on the patient’s body-surface area (BSA). Both
methods do not account for the wide interindividual variability in drug pharmacokinetics and pharmacodynamics. This
may not be a substantial issue in drugs with a broad therapeutic window, but is a major concern in oncology. Modern
technology such as drug immunoassays or population PKPD-modeling has enabled to elaborate more sophisticated
dosing algorithms that implement therapeutic drug monitoring (TDM) with the aim to reduce interindividual PK variability,
and improve the benefit-risk ratio of common anticancer drugs. Still the only anticancer drug were standard TDM is done
in adult oncology is high-dose methotrexate (MTX), with the aim to avoid severe MTX-related toxicity by guiding
leucovorin-rescue. At present, there is evidence from one randomized study on TDM-based administration of continousinfusional 5-fluorouracil (5FU), and there is mainly retrospective evidence for TDM-based administration of paclitaxel,
docetaxel, busulfan and imatinib. Limited retrospective evidence is also available for other oral tyrosine kinase inhibitors.
Pharmacogenetic markers of increased risk for toxicity are available for selected anticancer drugs, chiefly DYPD for the
fluoropyrimidines, UGT1A1 for irinotecan and TPMT for 6-mercaptopurine. The clinical significance of CYP2D6 (2C19)associated metabolism for tamoxifen is still controversial and results from prospective clinical studies are awaited. In the
future, individualized dosing algorithms will become more ‘mainstream’ in oncology as an inherent part of personalized
treatment.
42
Mathematical modeling of amyloid beta for the diagnosis and treatment of Alzheimer’s disease
Lehr, T.
Saarland University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany
Accumulation of amyloid-β (Aβ) peptide in the central nervous system (CNS) is believed to play a crucial role in the
pathogenesis of Alzheimer’s disease (AD) [1]. In order to gain more insight in Aβ metabolism in healthy subjects as well
as in AD patients Bateman et al. developed a method to quantify Aβ turnover in the cerebrospinal fluid (CSF) [2]. Therefore, the stable isotope labelled amino acid leucine is infused into the bloodstream, wherefrom it can be transported to
the brain and incorporated into newly synthesized proteins such as the amyloid precursor protein (APP). Labelled APP is
processed by the enzymes β- and γ-secretase to produce labelled Aβ. Both labelled and unlabelled Aβ are cleared
through the CSF, where sampling and quantification occurs at several time points. This technique of stable isotope
labelling kinetics (SILK) has been successfully used to measure endogenous Aβ production and degradation rates in
human CSF [3]. An additional study comparing the fractional production and clearance rates between control and AD
patients revealed 30% impairment in the clearance of both Aβ, while average production rates did not differ between the
two groups [4]. Thus, altered clearance mechanisms may contribute to the Aβ accumulation in the CNS.
Based on the exciting data from various SILK studies several mathematical models were generated in order to describe
Aβ biosynthesis and degradation in humans [5] and to link these processes to the Alzheimer disease status. Aim of this
presentation is to outline the future use of these mathematical models to diagnose Alzheimer’s disease in humans and to
potentially guide the treatment of Alzheimer’s disease.
References:
1. Karran, E. et al.: Nat Rev Drug Discov. 2011, 10(9): 698-712.
2. Bateman, R.J. et al.: J Am Soc Mass Spectrom. 2007, 8(6): 997-1006.
3. Bateman, R.J. et al.: Nat Med. 2006, 12(7): 856-861.
4. Mawuenyega K.G. et al.: Science 2010, 330(6012): 1774.
5. Haug, K.G. et al.: Journal of Clinical Pharma 2012, 7(53): 691–698.
43
Understanding Coagulation Biomarkers and Deriving Clinically-Relevant Surrogates by Use of an InSilico Coagulation Model
Burghaus, R.1; Willmann, S.1; Siegmund, H.-U.2; Lippert, J.1
1 Bayer
2 Bayer
HealthCare Pharmaceuticals, Clinical Pharmacometrics, 42113 Wuppertal, Germany
Technology Services, Technology Development, 51368 Leverkusen, Germany
Objectives: Mechanisms of coagulation are being successfully investigated and characterized in the scientific community for decades. As a consequence, there is a large body of related knowledge, information and data in the public as well
as proprietary R&D space. This evidence can be used to understand coagulation biomarkers in mechanistic detail and
generate clinically relevant InSilico surrogates to assess expected clinical performance of novel pro- and anticoagulant
drugs and treatment options.
Methods: A mechanistically detailed systems pharmacology simulator called “Blood Coagulation Simulator” (BCS) was
established integrating and summarizing community and proprietary information about blood coagulation. This tool is
used to simulate conditions typical for standard blood coagulation assays as well as physiological conditions representing clinically relevant scenarios.
Results: The BCS is able to integrate biological structural knowledge and quantitative information about the dynamics of
actual protein activation processes as well as processes on a cellular and blood vessel scale. Systemic properties of the
simulator – e.g. platelet activation responses - are in line with experimental observations. The impact of anti-thrombotic
agents on standard coagulation assays is simulated and found to be in accordance with ex-vivo data. Simulations of anticoagulant treatments in physiologically motivated settings identify doses which show clinically favorable risk/benefit
profiles.
Discussion/Conclusions: Systems Pharmacology methods have been successfully applied to blood coagulation. Such
an approach allows to understand systemic properties of coagulation assays as well as pharmacological interventions.
Translatability into a clinical framework has been demonstrated by simulation work being in line with confirmatory clinical
trials. Application of the technology allows for evaluating innovative treatment paradigms in terms of their expected
clinical profile and streamlining development programs by early identification of reasonable dosing schemes.
The application described can be seen as a prototype for applying Systems Pharmacology in combination with biomarker data to rationalize pharmaceutical research and streamline development.
References:
1. Burghaus, R. et al.: PLoS ONE 2011, 6(4): e17626.
2. Küpfer, L.; Lippert, J.; Eissing, Th.: Adv Exp Med Biol. 2012, 736: 543-561.
44
LIGAND BINDING ASSAYS
Biophysical techniques in fragment hit identification and lead optimization ‐
A change of perspective?
Boeckler, F.M.1; Wilcken, R.1,2; Bauer, M.R.1,2; Cieslik, M.B.1; Rutherford, T.J.2; Fersht, A.R.2; Joerger, A.C.2
Laboratory for Molecular Design and Pharmaceutical Biophysics, Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy,
Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
2 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
1
In recent years we have worked on various projects involving fragment hit identification and optimization toward lead
structures using different biophysical approaches. We have identified lead structures for rescuing the p53-mutant Y220C
[1-4] and identified lithocholic acid as an inhibitor of MDMX and MDM2 [5]. Experiences with the advantages and disadvantages of using methods including NMR-based techniques, fluorescence-based techniques and calorimetric techniques for the different stages of the projects will be discussed. During the initial stages of the project there is a strong
preference for techniques that help to avoid false positives and false negatives. However, the perspective can change
significantly when the project evolves.
References:
1. Boeckler, F.M. et al.: Proc. Natl. Acad. Sci. U. S. A. 2008, 105(30): 10360-10365.
2. Wilcken, R. et al.: J. Am. Chem. Soc. 2012, 134(15): 6810–6818.
3. Wilcken, R. et al.: Proc. Natl. Acad. Sci. U. S. A. 2012, 109(34): 13584-13589.
4. Wang, G.; Fersht, A.R.: Proc. Natl. Acad. Sci. U. S. A. 2012, 109(34): 13590-13595.
5. Vogel, S. et al.: Proc. Natl. Acad. Sci. U. S. A. 2012, 109(42): 16906-16910.
45
The Impact of Experimental Uncertainty on Decision Making in Drug Design
Kramer, C.
Center for Molecular Biosciences; Institute for General, Inorganic, and Theoretical Chemistry; Leopold-Franzens University Innsbruck; Innrain 82;
6020 Innsbruck; Austria
All experimental results contain some experimental uncertainty. This is well appreciated in almost all engineering disciplines, and good strategies for coping with the uncertainty have been developed there. In contrast, the analysis of
experimental uncertainty in physicochemical and biochemical measurements is often worked out less stringent, and the
impact of the amount of experimental uncertainty on SAR analysis and drug design decisions are not rigorously taken
into account. Even more, the experimental uncertainty of the individual measurements itself is often not really known.
Figure 1: Independently measured pKi values for the same protein-ligand system from CHEMBL14 (8524 pairs). The
diagonals indicate the 1:1 agreement and the thresholds where two measurements disagree by 2.5 log units.
Upon comparison of a large number of independent affinity measurements of pairs of protein-ligand systems, we found
that the average experimental uncertainty of heterogeneous public Ki values is 0.54 log units (see Figure 1 below).[1]
This is in sharp contrast to repeated measurements of the same assay in the same laboratory, where we found an
experimental uncertainty of 0.2 log units.[2]
The amount of experimental uncertainty has a limiting effect on all rational techniques that help guiding drug design. For
three different standard data analyses and affinity prediction techniques that are in daily use of drug designers, we will
show how the ignorance of experimental uncertainty can lead to wrong conclusions: Without taking into account experimental uncertainty, pseudo activity cliffs will be detected in SAR analysis, the true predictive power of QSAR models will
be underestimated, and Matched Molecular Pair Analysis will seem more fuzzy than it really is.[3] We thus need to either
reduce the experimental uncertainty in the data, or develop a far better understanding of its magnitude and the impact on
the daily drug design decisions.
References:
1. Kramer, C. et al.: J. Med. Chem. 2012, 55(11): 5165-5173.
2. Kalliokoski, T. et al.: PLoS One 2013, 8(4): e61007.
3. Kramer, C. et al.: J. Med. Chem. 2014, 57(9), 3786-3802.
46
Fluorescent probes to track GPCR binding and dimerization
Bonnet, D.; Hibert, M.
Laboratoire d’Innovation Thérapeutique, UMR7200 CNRS/Université de Strasbourg, Labex Médalis, Faculté de Pharmacie, 74 route du Rhin,
67401 Illkirch, France
G-protein-coupled receptors (GPCRs) represent the largest family of cell surface membrane proteins encoded by the
human genome and more than 40% of all marketed therapeutics act on them. However, these drugs target only few
members of the family (15%). So, there is an enormous potential to exploit the remaining family members, including the
orphan receptors for which no ligands have so far been identified. Besides, in the last decade, homo- and heterooligomerization of GPCRs have been described as a new way to modulate receptor pharmacology and functional activity. Thus, heteromer-targetted drug discovery opens new perspectives both in Academic pursuits and for the Pharmaceutical industry.
In this context, we have set up innovative fluorescent-based assays in order to gain a better understanding of GPCR
functional architecture but also to set up new receptor-selective high-throughput screening (HTS) assays for classical,
orphan and heterodimeric GPCRs. Owing to their high sensitivity and to their reduced environmental safety risk, fluorescent technologies represent a powerful molecular tool to study ligand-GPCR interactions [1]. However, the prerequisite
to develop such methods is to design and to synthesize high affinity and selective fluorescent probes.
As we will illustrate, synthetic methods have been set up to facilitate the access to original fluorescent GPCR probes with
potential applications in drug discovery. For instance, the first environment sensitive (“Turn-on”) probe was developed to
detect and to monitor oxytocin GPCR at the surface of living cells [2]. We have also designed and synthesized fluorescent compound-based libraries allowing the discovery by FRET of the first non-peptidic agonist of the apelin receptor [3].
Finally, selective fluorescent ligands were developed to detect vasopressin V1a-V2 heterodimers at the cell surface and to
set up a novel TR-FRET assay to screen for heterodimers [4].
Acknowledgments: Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université de
Strasbourg
References:
1. (a) Durroux, T. et al.: Nat. Chem. Biol. 2010, 6(8): 587-594; (b) Ilien, B. et al. : J. Med. Chem. 2012, 55(5): 2125–2143.
2. Karpenko, I. et al.:: ChemBiochem 2014, 3(15): 359-363.
3. (a) Bonnet, D. et al.: Chem. Eur. J. 2008, 14(20): 6247-6254; (b) Iturrioz, X. et al.: FASEB J. 2010, 24(5): 1506-1517; (c) Bonnet, D. et al.: J.
Med. Chem. 2014, 57(7): 2908−291.
4. Bonnet, D. et al.: J. Med. Chem. 2012, 55(20): 8588−8602; Patent PCT/EP2013/070837.
47
Development of triplex-forming oligonucleotide having artificial nucleoside analogues to inhibit the
gene expression as an antigene strategy
Taniguchi, Y.; Okamura, H.; Sasaki, S.
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, JAPAN
The sequence specific formed triplex DNA is one of the most important method for the inhibition of gene expression as
an antigene strategy in living cell. Basically, the triplex DNA is formed by the interaction of triplex forming oligonucleotides (TFOs) with duplex DNA, which is stabilized by the hydrogen bonding between dG and GC or between dA and AT
base pair. Thus, the TA and CG base pair within target duplex DNA hampered the stable triplex DNA. We have designed
and synthesized W-shaped nucleoside analogues (WNA) for the formation of stable triplex DNA, and the WNA-T
having thymine as a recognition base and WNA-C having cytosine as a recognition base showed a high selectivity for
the TA and CG interrupting site, respectively (Table 1 and Figure 1).1 However, the recognition ability of WNA was
dependent on the sequence context of TFOs. In order to overcome this limitation, we developed the several WNA
analogues and evaluated the ability of triplex formation by the gel-shift assay. Consequently, the combination use of
WNA-T derivatives showed the recognition of the TA base pair at any different sequences.2 Furthermore, we applied
the WNA-T for the antigene strategy for targeting the survivin oncogene. The antigene TFOs having WNA-T showed
the effective anti-proliferative effect for the living cell by the inhibition of the survivin expression (Figure 2).3
Recently, we have found the novel nucleoside unit, isocytidine skeleton, for the selective recognition of CG interrupting
site. It was revealed that TFOs containing 5-methylisocytidine with guanidinoethyl group or aminopyridine methyl group
showed the selective and stable triplex DNA including the CG site without sequence dependency (Table2, Figure 3).4,5
In conclusion, we have developed the artificial nucleoside analogues to recognize the TA or CG interrupting sites for the
formation of stable triplex DNA.
And we have demonstrated that
the antigene TFOs containing WNA
analogues showed the effective
anti-proliferative effect in comparison to the effect of the corresponding natural antigene TFOs.
References:
1. Sasaki, S. et al.: J. Am. Chem. Soc. 2004, 126: 516-528.
2. Taniguchi, Y. et al.: J. Org. Chem. 2006, 71: 2115-2122.
3. Taniguchi, Y.; Sasaki, S.: Org. Biomol. Chem.: 2012, 10: 8336-8341.
4. Okamura, H.; Taniguchi, Y.; Sasaki, S.: Org. Biomol. Chem.: 2013, 11: 3918-3924.
5. Okamura, H.; Taniguchi, Y.; Sasaki, S.: ChemBioChem: 2014, in press.
48
COMPUTATIONAL CHEMISTRY AND MOLECULAR DESIGN
Integrating Chemical and Biological Data for Drug Design and Mode-of-Action Analysis
Bender, A.
Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
More and more chemical and biological information is becoming available, both in public databases as well as in company repositories. However, how to make use of this information in chemical biology and drug discovery settings is much
less clear. In this work, we will discuss how chemical and biological information from different domains – such as compound bioactivity data, pathway annotations from the bioinformatics domain, and gene expression data – can be used
for a variety of purposes, such as the mode-of-action analysis from phenotypic readouts,[1,2] anticipating compound
toxicities in early discovery, and for designing and selecting compound with the desired bioactivities.[3,4] We will show
that cheminformatics algorithms trained on large chemogenomics databases can be employed to support target deconvolution in high-content screening as well as organism-based screens using e.g. Xenopus laevis[2] as well as phenotypic
data obtained from rat models. When anticipating compound adverse compound properties early on, we will show than
gene expression data can be used for this purpose; however, how to generate and analyze data is very much casedependent. Relating to compound design and selection, we can employ both bioactivity-driven approaches as well as
gene expression based resources, and examples of both will be presented. Hence, overall, while the chemical and
biological data available currently is very diverse, we are able to show that it can already be used successfully for understanding the mode of action of compounds, anticipating their toxicities early on in discovery, and designing and selecting
novel chemical matter to modulate biology.
References:
1. Koutsoukas, A. et al.:J. Proteomics 2011, 74(12): 2554-2574.
2. Liggi, S. et al.: Mol. Inf. 2013, 32(11-12): 1009-1025.
3. Van Westen, G.J.P. et al.: MedChemComm 2011, 2(16): 16-30.
4. Van Westen, G.J.P. et al.: PLoS Comp. Biol. 2013, 9(2): e1002899.
49
A Knowledge-Based Approach to Assessing Propensity for Polymorphism in the Pharmaceutical
Crystalline Solid Form
Maginn, S.J.; Feeder, N.
Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, United Kingdom
Uncontrolled crystal form polymorphism can have a critical impact on pharmaceutical or agrochemical product robustness. This has been particularly exemplified by the pharmaceutical cases of Norvir™ [1] and Neupro™ [2], which were
withdrawn from the market after the unexpected appearance of a more stable polymorph. The Norvir™ example illustrates how such polymorphism can be driven by a stronger set of hydrogen bonds in the stable form.
At the CCDC we are developing structural informatics approaches that can mitigate solid form risk and move us towards
the notion of solid form by design. Here the vast knowledge base of 700,000+ crystal structures of the CSD and the
millions of discrete data points on the geometry of intermolecular interactions contained therein are mined to reveal the
underlying rules that control crystal packing. Such an approach compliments more brute force ab-initio energy calculations yet offers the advantage of being applicable across all solid form types and accessible to solid state scientists
rather than just computational specialists. For example, we have developed a CSD based Hydrogen-Bond Propensity
tool which would have clearly predicted the likely existence of the more stable polymorph of ritonavir (Norvir™)[3].
The principles behind these approaches and their practice will be described, and a view to the future will be provided.
References:
1. Bauer, J. et al.: Pharm. Res. 2001, 18: 859-866.
2. Cajigal, S.: Neurology Today 2008, 8: 1 & 8.
3. Galek, P.T.A. et al.: CrystEngComm 2009, 11: 2634-2639.
50
Direct Integration of Ligand-Based NMR Data into Protein-Ligand Docking
Exner, T.E.1,2; Onila, I.2; Fredriksson, K.2; Codutti, L.3; Mazur, A.4; Möller, H.M.2; Carlomagno, T.3; Griesinger, C.4
1 Institute
of Pharmacy, Eberhard Karls University of Tübingen, Tübingen, Germany
of Chemistry, University of Konstanz, Konstanz, Germany
3 European Molecular Biology Laboratory, Heidelberg, Germany
4 Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
2 Department
In structure-based drug design, the experimental elucidation of protein-ligand complexes plays a central role in the
design of high-affinity drug candidates from weakly bound lead compounds. As an alternative to X-ray crystallography,
NMR techniques can be applied to obtain information on the bound ligand. STD [1] (saturation transfer difference) is
often used to find the epitope in the ligand by determining the distance between a specific part of the ligand to the
surface of the protein. In contrast, the relative new INPHARMA [2,3] (Internuclear NOEs for Pharmacophore Mapping)
approach, determines the relative orientation of two competitive ligands in the receptor binding pocket. It is based on the
observation of interligand transferred NOEs mediated by spin diffusion through protons of the protein and is, therefore,
sensitive to the specific interactions of each of the two ligands with the protein.
The docking program PLANTS [4,5] (Protein-Ligand ANT System) developed in our group was extended to directly
include the experimental information. The standard scoring function ChemPLP [5] is augmented with STD and/or
INPHARMA scores, which describes the agreement between the experimental spectrum and a back-calculated spectrum
by defining distance constraints or using the full relaxation matrix approach. We will show that this integration is more
beneficial and efficient than a two-step procedure generating first trial poses with a docking program and then identification of the correct pose by a rescoring with the experimental data as used in [6]. We will also demonstrate that, besides
this improved scoring function, the careful preparation of the input structures and the docking setup including protonation
states is essential.
References:
1. Mayer, M., Meyer, B.: J. Am. Chem. Soc. 2001, 123: 6108 – 6117.
2. Sanchez-Pedregal, V.M. et al.: Angew. Chem. Int. Edit. 2005, 44(27): 4172 – 4175.
3. Orts, J. et al.: Angew. Chem. Int. Edit. 2008, 47(40): 7736 – 7740.
4. Korb, O.; Stützle, T.; Exner, T.E.: Swarm Intell. 2007, 1: 115 – 134.
5. Korb, O.; Stützle, T.; Exner, T.E.: J. Chem. Inf. Model. 2009, 49: 84 – 96.
6. Skjaerven, L. et al.: J. Am. Chem. Soc. 2013, 135(15), 5819-27.
51
Screening reaction pathway-driven very large chemical space: Discovery of potent mdm2-p53 antagonist
Dömling, A.1; Camacho, C.2; Holak, T.3; Koes, D.2; Neochoritis, C.1; Khoury, K.4
Dept. Drug Design, RUG, Netherlands,
University of Pittsburgh, USA,
3 Jagiellonian University, Krakow, Poland,
4 Carmolex Inc, Pittsburgh, USA
1
2
Current industrial screening paradigm is HTS: Most medicinal chemistry program start with hits from high throughput
screening. Virtual screening of very large chemistry space combined with structure-based drug discovery offers a valuable alternative. However, until recently, most virtual screening exercises used rather small libraries of limited chemical
diversity. And, although, identified compounds are often commercially available for validation, such as in ZINC, PUBCHEM or ChEMBL databases, follow-up SAR is often challenging and expensive. For instance, GB-11 (what is GB11) is
very large and hard to test due to the lack of efficient synthetic access to hit compounds for validation.
We have introduced ANCHOR.QUERY, a web-based and google-like technology for the structure-based mining of
billions of small molecules (anchorquery.csb.pitt.edu/).1 The compound database is based on >20 diverse scaffolds with
a defined reaction pathway based on efficient and rapid multicomponent reaction chemistry (MCR).2 The virtual compound library is design for a high level of confidence in synthetic feasibility and speed: Every hit compound can be
rapidly accessed from commercial starting materials in less than four chemical steps. Protocols for the synthesis are
provided online. To increase virtual screening hit rates the compound library is biased towards deeply buried anchor
residues which play a key role in molecular recognition, e.g. deeply buried amino acid side chains in PPIs.
We have validated ANCHOR.QUERY with the discovery of more than 10 different compound classes able to antagonize
the protein protein interaction p53-Mdm2-Mdm4.3-6 Several of the predicted compounds could be validated by cocrystal
structure analysis and compared with the predicted binding poses (Figure). Some scaffolds were optimized towards low
nM compounds highly active in cancer cells and a xenograft.
3LB
K
3TJ
2
3LB
J
4MD
N
Acknowledgments: The Dömling laboratory is generously funded by the University of Groningen, the Innovative Medicines Initiative (grant agreement n° 115489), Qatar National Science Foundation (NPRP 6 - 065 - 3 - 012), the National Institute of Health (1R01GM097082-01) and Carmolex
Inc.
References:
1. Koes, D. et al.. PLoS One 2012, 7: e32839.
2. Dömling, A.; Wang, K.; Wang, K. : Chem. Rev. 2012, 112: 3083-3135.
3. Czarna, A. et al.. Angew. Chem. Intl. Ed. 2010, 48: 5352-5356.
4. Bista, M. et al.. Structure 2013, 21: 2143-2151.
5. Huang, Y. et al. ACS Chem. Biol. 2014.
52
Identification of a mechanism-of-action target exploiting similarities of chemotypes and signalling
events, and biophysical simulations
Gohlke, H.1; Schmitz, B.1; Bonus, M.1; Sommerfeld, A.2; Reinehr, R.2; Häussinger, D.2
1 Institute
for Pharmaceutical and Medicinal Chemistry, Department of Mathematics and Natural Sciences, Heinrich-Heine-Universität, Düsseldorf,
Germany
2 Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-Universität, Düsseldorf, Germany
Recent studies reported that for about 7% of approved drugs, a primary target is unknown.[1] One such drug has been
ursodeoxycholic acid, which is in vivo converted to tauroursodeoxycholic acid (TUDC). This bile acid is a mainstay for
the treatment of cholestatic liver disease. Earlier work showed that TUDC exerts its choleretic properties in an 51
integrin-mediated way.[2, 3] However, an intracellular receptor/sensor specific for TUDC that initiates the signaling has
remained unknown.
Exploiting that I) TUDC-induced signaling events strongly resemble those when mechano/swelling-sensitive 51 integrin become activated, II) TUDC-induced signaling is inhibited in the presence of an 5 1 integrin inhibitory peptide, and
III) TUDC bears structural similarity with tirofiban, an IIb3 integrin inhibitor, both in terms of molecular shape and
molecular recognition properties, we hypothesized that 5 1 integrins act as sensors for TUDC in hepatocytes.
The hypothesis was verified by immunofluorescence staining experiments showing direct integrin activation by TUDC, by
Western blot analyses showing the initiation of integrin signaling involving downstream kinases, and by integrin knockdown abolishing TUDC signaling. TUDC-induced integrin activation occurs predominantly inside the hepatocyte and
requires TUDC uptake via the Na+/taurocholate cotransporting peptide. Furthermore, molecular dynamics simulations of
a model of the 5 1 integrin ectodomain with TUDC revealed that TUDC induces pronounced allosteric conformational
changes known to be associated with integrin activation.
This finding of a long-sought intracellular sensor of TUDC is key for understanding the choleretic and cytoprotective
effects as well as the hepatocyte-specificity of TUDC at a molecular level.[4] Moreover, our results yield the first structural model of a bile acid binding to 5 1 integrin, which is expected to foster the development of novel small-molecule
integrin agonists. Finally, our results suggest that exploiting similarities of signaling events and biophysical simulations,
in addition to chemical similarity as done previously,[5] can lead to highly accurate predictions of drug-target associations, which is of high interest currently.
Acknowledgments: We acknowledge support by the Deutsche Forschungsgemeinschaft through the Collaborative Research Centers SFB 575 and
SFB 974 and the Clinical Research Group KFO 217, and by the initiative ‘‘Fit for Excellence’’ at the Heinrich-Heine-Universität.
References:
1. Drews, J.: Science 2000, 287: 1960-1964.
2. Häussinger, D. et al.: Gastroenterology 2003, 124: 1476-1487.
3. Schliess, F. et al.: Gastroenterology 1997, 113: 1306-1314.
4. Gohlke, H. et al.: Hepatology 2013, 57: 1117–1129.
5. Gregori-Puigjane, E. et al.: Proc. Natl. Acad. Sci. 2012, 109: 11178-11183.
53
NATURAL COMPOUNDS
Neolignans: from PPAR to RXR
Dirsch, V.M.
Department of Pharmacognosy, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria
Peroxisome proliferator-activated receptor gamma (PPAR) agonists have been and are still used to treat type 2 diabetes and the metabolic syndrome due to their ability to increase “safe” free fatty acid (FFA) storage in adipocytes thereby
lowering FFA-mediated lipotoxicity and increasing insulin sensitivity, among others [1]. Most full PPAR agonists, however, display serious side effects, which led to great interest in novel ligands with more favorable properties. Thus, one
aim of our group is to identify new PPAR agonists. Using pharmacophore-based virtual screening of 3D natural product
libraries we discovered several neolignans (dieugenol, tetrahydrodieugenol, and magnolol) as partial PPAR agonists
[2]: These neolignans bound to the PPAR ligand binding domain with Ki values in the nano- to low micomolar range. In
intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPAR-mediated luciferase reporter gene
expression with EC50 values comparable to pioglitazone (pioglitazone: 0.26 µM; dieugenol: 0.63 µM; tetrahydrodieugenol: 0.33 µM) but with a considerable lower maximal response suggesting partial agonism. All three compounds promoted 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPAR expression [2].
Interestingly, the structurally closely related neolignan, honokiol did not induce adipogenesis under the same conditions
[3]. Nevertheless, as pioglitazone it led to an increase in glucose uptake in adipocytes. In diabetic KKAy mice oral
application of honokiol prevented hyperglycemia and suppressed weight gain [3]. Additional studies by other groups
characterized honokiol as specific partial retinoid X receptor alpha (RXR agonist [4], and magnolol as dual agonist of
RXR and PPAR [5]. Based on these findings we screened a library of 53 (semi)synthetic neolignan derivatives for
their activity against PPAR and RXR in respective luciferase reporter models. As a result, we identified several new
RXR agonists with EC50 levels below 1 µM. Further studies will characterize these compounds in functional models for
adipogenicity, glucose uptake in adipocytes, cholesterol efflux from human THP-1 macrophages and lipid accumulation
in HepG2 hepatocytes.
Acknowledgements: This collaborative study was conducted by groups from the Universities of Vienna, Innsbruck and Graz, as well as by the
China Academy of Chinese Medical Sciences. The study was mainly funded by the Austrian Science Fund (FWF): S107 (NFN: Drugs from Nature
Targeting Inflammation)
References:
1. Ahmadian, M. et al.: Nat Med. 2013, 19(5): 557-566.
2. Fakhrudin, N. et al.: Mol. Pharmacol. 2010, 77(4): 559-566.
3. Atanasov, A.G. et al.: Biochim. Biophys. Acta 2013, 1830(10): 4813-4819.
4. Kotani, H. et al.: J. Nat. Prod. 2010, 73(8): 1332-1336.
5. Zhang, H. et al.: PLOS one 2011, 6(11): e28253.

54

Waking up biosynthetic gene clusters in a row
Bechthold, A.
Department of Pharmaceutical Biology and Biotechnology, Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104
Freiburg, Germany
Natural products are a rich source of commercial products for the pharmaceutical and other industries. An enormous
number of such compounds have derived from microorganisms colonizing various habitats. However, industrial interest
in metabolites research from microorganisms has declined significantly in the past years because of the rediscovery of
the same bioactive compounds and redundancy of the sample strains.
In the last few years, microbial natural product research has been revolutionized by genomic technologies. Complete
sequences of microbial genomes revealed a remarkable number of gene clusters encoding enzymes involved in the
production of undetected and unknown secondary metabolites.
Different strategies have been pursued to wake up and express these ‘cryptic’ gene clusters including culture manipulation, the genetic manipulation of pathway specific regulatory genes and the use of heterologous expression techniques.
In my talk I will give examples for each of these strategies and I will introduce an unusual way of activating “cryptic” gene
clusters resulting in the isolation of novel undiscovered natural products.
55
Chondramides: setting the stage for actin binding compounds in cancer therapy
Herrmann, J.1; Förster, F.2; Hüttel, S.1; Müller, R.1; Vollmar, A.M.2
1 Helmholtz
Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Campus C2.3, Saarland University, 66123
Saarbrücken, Germany
2 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, 81377 Munich, Germany
Myxobacteria have been established as a rich source of bioactive compounds with unique structures.[1,2] We routinely
screen crude extracts of novel myxobacterial isolates by LC/hrMS and Chondromyces sp., strain MSr9030, was found to
produce the already described chondramides A-D. These cyclodepsipetides were originally isolated from Chondromyces
crocatus due to their nanomolar cytotoxic activity on cancer cell lines, which originates from a stabilizing effect on actin
filaments[3].
Of note the mode of cytotoxicity and moreover the molecular mechanism of action linked to actin have not been examined yet. We report here that chondramide A disrupts the actin cytoskeleton and forms bundles of amorphous actin
aggregates (FRAP analysis and confocal microscopy). Chondramide induces apoptosis (Annexin-V/PI-costaining, Parp
cleavage, Caspase 9 activation) via a collapse of the mitochondrial membrane potential (decrease of mitochondrial
membrane potential and Cytochrome C release) and affecting the mitochondrial permeability transition pore (mPTP)
(translocation of Hexokinase II to the cytosol and Bad dephosphorylation). In search of a link between effects of chondramide on actin cytoskeleton and apoptosis induction the oncogene PKCε gained attention as this kinase posseses an
actin-binding site and is involved in the control of the mPTP. In fact, a colocalization of PKCε and chondramide A induced actin bundles was shown by confocal microscopy and Westernblot analysis of cytoskeletal fractions. Furthermore,
PKCε membrane translocation after phorbol ester stimulation was decreased upon chondramide A treatment indicating
reduced activity of PKCε. Finally, PKCε overexpression was able to significantly reduce cell death induced by chondramide A proving our hypothesis. Of special importance the mechanism of trapping PKCε in actin bundles could also be
found in in vivo tumor samples, which showed a decreased tumor volume compared to control tumors. These results set
the stage for actin binding compounds as innovative leads for future tumor therapies.
Along this line the MSr9030 crude extract was analyzed in more detail as the chondramides A-D might mask the presence of further interesting bioactive metabolites. For bioactivity-guided isolation of new natural products we applied highcontent-screening (HCS) and could finally detect more than 30 novel chondramide derivatives, some of which were
present in minute amounts only and which would have not been detected in conventional cytotoxicity assays. Initial
biological profiling of 11 new natural derivatives in comparison to the chondramides A-C showed that brominated variants were the most active (GI50 in the low nanomolar range) on a panel of human cancer cell lines. Given the fact that
these analogs were also by factor 2-4 less potent on non-cancerous human cells in comparison to average values on
cancer cell lines, our results aid the further SAR-guided development of chondramides via chemical syntheses.[4]
Financial support by the Deutsche Forschungsgemeinschaft (DFG; FOR1406) is gratefully acknowledged.
References:
1. Weissman, K.J., Müller, R.: Nat. Prod. Rep. 2010, 27(9): 1276-1295.
2. Krug, D., Müller, R.: Nat. Prod. Rep. 2014, 31(6): 768-783.
3. Sasse, F. et al.: J. Natl. Cancer Inst. 1998, 90(20): 1559-1563.
4. Herrmann, J., Hüttel, S., Müller, R.: Chembiochem 2013, 14(13): 1573-1580.
56
Cdk5 inhibition potentiates Imatinib responsiveness of Philadelphia chromosome positive chronic
myeloid leukemia cells
Mandl, M.; Vollmar, A.M.; Liebl, J.
Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, Munich, Germany
Chronic myeloid leukemia (CML) is a malignancy that arises from transformation of the hematopoietic stem cell. The
hallmark of CML is the Philadelphia chromosome, a reciprocal translocation of chromosomes 9 and 22, which generates
the BCR/ABL fusion gene encoding a constitutively active tyrosine kinase. BCR/ABL tyrosine kinase exerts oncogenic
function by activating various intracellular signaling pathways leading to increased cell survival and proliferation as well
as abrogated dependency on growth factors. The ATP-competitive BCR/ABL tyrosine kinase inhibitor (TKI) Imatinib has
revolutionized CML therapy. However, resistance to imatinib treatment due to mutations in BCR/ABL that impair the
ability of imatinib to interact with the tyrosine kinase occurred as major problem. Moreover, Philadelphia chromosome
positive CML stem cells can be intrinsically insensitive to imatinib. Second generation inhibitors targeting also imatinib
insensitive BCR/ABL mutants show improved effectiveness. Nevertheless, resistance to these second generation TKIs
has been described. Therefore, new therapeutic strategies to improve CML treatment are required. Novel approaches
addressing alternative targets in combination with TKIs might be more effective to treat resistant CML cells.
Our present study provides evidence for Cyclin dependent kinase 5 (Cdk5) as drugable target to improve the therapeutic
response of Philadelphia chromosome positive CML cells to TKI treatment.
Cyclin dependent kinase 5 (Cdk5) is a serine/threonine kinase with essential functions in neuronal development, function, and disease. Regulation and downstream signalling of neuronal Cdk5 is well-established whereas knowledge about
Cdk5 in peripheral tissues - particularly in cancer - is limited. We recently demonstrated that Cdk5 exerts important
functions in the endothelium and angiogenesis and elucidated Cdk5 as drugable target for treatment of hepatocellular
carcinoma (HCC).
In neurons, Cdk5 gets phosphorylated and activated by the Abelson tyrosine kinase (c-Abl). Along this line, we hypothesized that Cdk5 is a downstream target of BCR/ABL in CML. In fact, Cdk5 phosphorylation and activity was increased in
the Philadelphia chromosome positive CML cell line K562 in comparison to normal CML and T-ALL cells. Moreover,
imatinib decreased Cdk5 phosphorylation and activity. Importantly, combined Cdk5 inhibition and imatinib treatment
resulted in increased apoptosis and reduced proliferation of BCR/ABL CML cells.
Therefore, the present study provides first evidence for the combination of Cdk5 inhibition and TKI as promising novel
alternative approach for Philadelphia chromosome positive CML therapy.
57
Plasmodium falciparum histone deacetylases (PfHDACs) as epigenetic drug targets
Hansen, F.K.1; Sumanadasa, S.D.M.2; Stenzel, K.1; Duffy, S.2; Marek, L.1; Meister, S.3; Skinner-Adams, T.S.2; Held, J.4;
Schmetter, R.1; Mordmüller, B.4; Hamacher, A.1; Kassack, M.U.1; Winzeler, E.A.3; Avery, V.M.2; Andrews, K.T.2; Kurz, T.1
Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
Eskitis Institute for Drug Discovery, Don Young Road, Nathan Campus, Griffith University, QLD 4111, Australia
3 Department of Pediatrics, University of California, San Diego, School of Medicine, 9500 Gilman Drive 0741, La Jolla, CA 92093, USA
4 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany
1
2
Histone deacetylases (HDACs) play a key role in the epigenetic modulation of gene expression by altering chromatin
structure and HDAC inhibitors (HDACi) are widely studied and developed as effective treatments for human cancers.
Interestingly, HDAC gene homologues have been discovered in all Plasmodium species that can infect humans and five
HDAC encoding genes have been identified in the P. falciparum genome.[1] Three of these Plasmodium falciparum
histone deacetylases (PfHDACs) show homology to human class I (PfHDAC1) or class II HDACs (PfHDAC2,3), whereas
two genes are class III homologues (PfSir2A and B). The enzyme PfHDAC1 is considered as an emerging target for
malaria intervention strategies.[1]
Recently, we demonstrated for the first time that HDACi cause death and histone hyperacetylation in Plasmodium
falciparum gametocytes.[2] Moreover, the transcription of all five PfHDACs in early gametocytes (stage III) and late stage
gametocytes (stage V) was confirmed by diagnostic RT-PCR.[2] Based on these results, it was our aim to discover novel
HDACi with activity against multiple malaria life cycle stages using LMK235, a HDACi with a unique preference for
human HDAC4 and HDAC5,[3] as starting point. Thus, we synthesized two series of HDACi containing novel connectionunit linker regions.[4,5] An extensive biological evaluation disclosed that some compounds showed nanomolar activity
against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages), while other compounds
revealed increased parasite selectivity in combination with at least dual-stage activity.[5] Mode of action studies with
representative compounds showed that our HDACi caused hyperacetylation of P. falciparum histones and inhibited
deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts.[4,5]
In summary, our data identify HDACi as being among a limited number of compounds that target asexual, exoerythrocytic and gametocyte stage Plasmodium parasites, making them a potential new starting point for future development of antimalarial drug leads with multistage activity.
References:
1. Andrews, K.T.; Tran, T.N.; Fairlie, D.P.: Curr. Pharm. Des. 2012, 18(24): 3467–3479.
2. Trenholme, K. et al.: Antimicrob. Agents Chemother. 2014, 58(7): 3666–3678.
3. Marek, L.; Hamacher, A. et al.: J. Med. Chem. 2013, 56(2): 427–436.
4. Hansen, F.K. et al.: ChemMedChem 2014, 9(3): 665–670.
5. Hansen, F.K. et al.: Eur. J. Med. Chem. 2014, 82: 204–213.
58
ANALYTICS
Analysis of vitamin D metabolic markers by mass spectrometry: advantages and limitations of the gold
standard method
Volmer, D.A.
Institute of Bioanalytical Chemistry, Saarland University, 66123 Saarbrücken, Germany
Vitamin D compounds are secosteroids, which are found naturally as vitamin D3 in mammals and D2 in plants. Vitamin D
is essential for bone health; recent studies, however, have shown involvement of vitamin D in the pathologies of a much
wider range of diseases such as cancer, diabetes, autoimmune, neurodegenerative, mental and cardiovascular diseases. Vitamin D is synthesized in the human skin under the influence of UVB radiation, and subsequent hepatic and renal
metabolism generates a number of transformation products over a large dynamic range from picomolar to nanomolar
levels.
D3
25-hydroxylase
1-hydroxylase
liver
kidney
25(OH)D3
1,25(OH)2D3
Vitamin D3 and most significant metabolites, 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3).
Capturing vitamin D metabolite levels requires sensitive and selective analytical methods that permit quantitative determination of the low concentration levels of vitamin D compounds in relevant tissues such as blood. Ideally, vitamin D
assessment would be performed by a standardized reference method, available to nutritional and clinical laboratories,
that provides reliable, precise and accurate quantitative results for all relevant vitamin D metabolites with sufficiently high
throughput. Unfortunately, no such method exists at the present time. Currently, LC-MS/MS assays are the most promising assay techniques for vitamin D. This presentation focuses on developments in recent mass spectrometry methodologies for vitamin D and its metabolites, and the experimental approaches chosen in our laboratory for determining fingerprints (“chemotypes”) of relevant vitamin D metabolites. It will highlight detrimental influences of the biological matrix,
epimer contributions, problems with specific mass spectrometry data acquisition routines (in particular, multiple reaction
monitoring, MRM), ionization efficiency, chemical derivatization reactions to improve detectability, gas-phase separation
techniques (ion mobility spectrometry) for removing chemical noise, and accuracy issues and inter-laboratory comparisons.
59
High Resolution MALDI Imaging: Reliable Molecular Identification at Cellular Resolution
Römpp, A.; Bhandari, D.; Schober, Y.; Guenther, S.; Spengler, B.
Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Schubertstrasse 60, D-35392 Giessen, Germany
Mass spectrometry imaging (MS imaging) is the method of scanning a sample of interest and generating an image of the
intensity distribution of a specific analyte ion. In contrast to most histochemical techniques, mass spectrometry imaging
can differentiate (amino acid) modifications and does not require labelling of compounds. Our work is focused on obtaining reliable chemical information and on increasing the spatial resolution in order to detect (sub)cellular features. Here
we present a number of improvements in instrumentation, sample preparation, measurement parameters and data
processing.
MS imaging experiments were performed with a high resolution atmospheric-pressure imaging source (AP-SMALDI10,
TransMIT GmbH, Giessen) attached to ‘LTQ Orbitrap’, ‘Exactive Orbitrap’ or ‘Q Exactive’ mass spectrometers (Thermo
Scientific GmbH, Bremen) [1,2]. Pixel size was between 2 and 10 µm. Mass accuracy was better than 2 ppm (root mean
square error) under imaging conditions. Tentative identification based on accurate mass was confirmed by on-tissue
MS/MS experiments.
Phospholipids were analysed in a wide range of tissue types in order to characterize and differentiate cell types. This
includes investigation of intra-tumor heterogeneity in a human biopsy of gastric cancer at 10 µm pixel size and mouse
model tissue at 2 µm pixel size.
The spatial distribution of the tyrosine-kinase inhibitor Imatinib was imaged in mouse kidney at 10 mm pixel size [3].
These measurements revealed the detailed distribution of the compound within the mouse organ. At the same time our
method provides detailed information on histological features based on the distribution of phospholipids and other
endogenous compounds. Correlation of these different images allows for fast and easy interpretation of the drug compound distribution and areas of accumulation can be directly linked to certain tissue types. Additional examples of drug
compound include the analysis of whole-body rat sections.
In an effort to investigate metabolites in cell cultures, a dedicated sample preparation protocol was established for the
analysis of single cells. A range of metabolites including nucleic acids, cholesterol and phospholipids were imaged in
single cells at 7 µm pixel size [4].
MS image analysis for all these experiments showed excellent agreement with histological staining evaluation. In addition it provided highly specific molecular information. In many cases signals with very similar mass (∆m/z<0.1) showed
distinctly different distributions, which demonstrates the need for high mass resolution in order to obtain reliable information from MS imaging experiments of complex biological samples.
General trends and developments in the field of mass spectrometry will be briefly discussed. This includes strategies for
flexible data analysis on the basis of the data format imzML (www.imzml.org) and activities in the framework of COST
action (European Cooperation in Science and Technology) „Mass Spectrometry Imaging: New Tools for Healthcare
Research” (BM1104).
References:
1. Römpp, A. et al.: Angew. Chem. Int. Ed. 2010, 49(22):3834-3838.
2. Römpp, A.; Spengler, B.: Histochem. Cell Biol. 2013, 139(6): 759-783.
3. Römpp, A. et al.,: Anal Bioanal Chem 2011, 401(1):65-73.
4. Schober, Y. et al.: Anal Chem 2012, 84(15):6293-6297.
60
Mass Spectrometric Characterization of Biopharmaceuticals - Possibilities, Challenges and Limitations
Scheffler, K.
Thermo Fisher Scientific, Dreieich, Germany
Biopharmaceuticals are in most cases challenging molecules that require a variety of different techniques to perform full
characterization including all qualitative and quantitative aspects and the analysis of modifications on the protein and
peptide level. Monoclonal antibodies (mAbs) play a major role in the treatment of a variety of conditions such as cancer,
infectious diseases, allergies, inflammation, and auto-immune diseases. Because mAbs can exhibit significant heterogeneity, extensive analytical characterization is required to obtain approval for a new mAb as a therapeutic product. Mass
spectrometry has become an essential tool in the characterization of mAbs, providing molecular weight determinations of
intact proteins as well as separated light and heavy chains, elucidation of glycosylation and glycan structures, confirmation of correct amino acid sequences, and identification of impurities such as host cell proteins (HCP) inherent to the
production process.
The analysis of intact proteins and especially large proteins such as intact antibodies and protein complexes on the
OrbitrapTM platform have steadily advanced on a technical level ever since the Orbitrap mass analyzer became commercially available in the year 2005, only been made possible due to several technological advancements we implemented
in newer generations instruments. One of the newer generation instrument platforms is the Thermo Scientific Q Exactive
benchtop Orbitrap mass spectrometer, an instrument we introduced into the market in the year 2011.
This presentation is focused on qualitative and quantitative LC-MS workflows applicable to the Orbitrap platform aiming
at the full characterization of biopharmaceuticals. During the course of the presentation many examples of application
data obtained from these workflows will be discussed.
61
Fluorescent oxaliplatin analogue as a model for the anticancer drug oxaliplatin for the investigation of
its cellular trafficking
Kalayda, G.V.1; Gollos, S.2; Kullmann, M.1; Metzger, S.3; Jaehde, U.1
1 Department
of Clinical Pharmacy, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
of Pharmaceutical Chemistry, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
3 Cologne Biocenter, University of Cologne, Züplicher Str. 47b, 50674 Cologne, Germany
2 Department
Oxaliplatin (Eloxatin®) is a platinum-based anticancer drug with a better safety profile and the lack of cross-resistance
with cisplatin. It is used as a standard treatment of advanced colorectal cancer. Nevertheless, tumour cells can develop
resistance to oxaliplatin as well [1]. In contrast to the mechanisms of cisplatin resistance, resistance to oxaliplatin is less
well investigated. As in the case of the other platinum drugs, cytotoxic action of oxaliplatin is assumed to result from the
binding of the drug to genomic DNA, which subsequently triggers apoptosis in cancer cells. However, only a small
fraction of oxaliplatin, which enters the cell, reaches genomic DNA. The fate of the rest of intracellular oxaliplatin and its
relevance for oxaliplatin sensitivity of tumour cells and for resistance to the drug remains unclear.
Investigation of the intracellular trafficking of oxaliplatin represents a challenge, as the number of methods allowing
detection of platinum drugs inside the cell is very limited. Moreover, many procedures require tiresome cell fractionation,
which often do not provide entirely pure cellular organelles.
For these reasons, there is a need for model compounds, which are easy to detect in the cells on one hand, and still
reflect biological properties of the parent drug on the other hand. Several fluorescent cisplatin analogues were developed
and one of them was shown to mimic biological behaviour of cisplatin with respect to cisplatin resistance [2,3]. Fluorescence allows monitoring the processing of the compound in living cells. Furthermore, it enables detection of binding
partners of platinum drugs.
The aim of our work was to develop a fluorescent oxaliplatin analogue and to evaluate it as a model compound for the
studies of cellular trafficking of oxaliplatin. An oxaliplatin derivative labelled with carboxyfluorescein diacetate (CFDAoxPt, the structure is shown below) has been synthesized and fully characterized. The label is introduced into the cyclohexane ring, as the oxalate ligand is readily exchanged upon interaction of oxaliplatin with (cellular) nucleophiles. The
cytotoxicity of CFDA-oxPt in HCT-8 human ileocecal colorectal adenocarcinoma cell line and its oxaliplatin-resistant
derivative HCT-8ox was studied using a MTT-based assay. Cellular processing of the model complex in both cell lines
was investigated by fluorescence microscopy. Based on the results, the suitability of CFDA-oxPt as a model for investigation of the cellular trafficking and intracellular interactions of oxaliplatin is discussed.
O
O
O
O
H2
N
O
O
O
O
O
Pt
N
H2
O
O
O
HN
H2
N
oxaliplatin
O
O
Pt
N
H2
O
O
CFDA-oxPt
The authors acknowledge the financial support by the Deutsche Forschungsgemeinschaft (grant JA 817/4-1). The authors are grateful to Prof.
Christa E. Müller (Institute of Pharmacy, University of Bonn) for providing laboratory facilities for synthetic chemistry.
References:
1. Mishima, M. et al.: Eur. J. Cancer 2002, 38(10): 1405-1412.
2. Kalayda, G.V. et al.: BMC Cancer 2008, 8: 175.
3. Kalayda, G.V.; Wagner, C.H.; Jaehde, U.: J. Inorg. Biochem. 2012, 116: 1-10.
62
A polymeric multifunctional glaucoma implant
Wischke, C.1; Löbler, M.2; Neffe, A.T.1; Hanh, B.D.1; Sternberg, K.2; Stachs, O.3; Guthoff, R.3; Lendlein, A.1
Institute of Biomaterial Science and Berlin-Brandenburg Center of Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Kantstr. 55, 14513
Teltow, Germany
2 Institute for Biomedical Engineering, University of Rostock, F.-Barnewitz-Str. 4, 18119 Rostock, Germany
3 Department of Ophthalmology, University of Rostock, Doberaner Str. 140, 18057 Rostock, Germany
1
Glaucomas are eye diseases that can lead to blindness due to ocular and neuronal tissue damage by elevated intraocular pressure. If topical medication fails, surgical intervention such as implantation of aqueous drainage devices is advised
in open angle glaucoma. Still, due to deficiencies of existing systems that can lead to hypotony, fibrosis, long-term
failure, and damage of adjacent tissues, alternative polymer-based implants should be developed.
This study presents a degradable copolyester blend with remarkable mechanical properties and the development of
multilayered, drug releasing glaucoma microstents for suprachoroidal implantation based on these materials [1, 2]. The
degradation of poly(ε-caprolactone) [PCL] could be accelerated in a controlled manner by introduction of 8 wt.% glycolide units leading to poly[(ε-caprolactone)-co-glycolide] (PCG), which, however, exhibited poor mechanical properties.
By blending of PCG with PCL (50/50 w/w), the advantageous individual properties of both components could be combined, i.e. elastic properties comparable to pure PCL and a water uptake during degradation similar to pure PCG [1]. At
the same time, processing by hot melt extrusion to tubes with internal diameters as low as 50 µm was realized. In this
continuous process that did not involve solvents for drug loading, diclofenac sodium could be incorporated as a model
drug. In addition to microstents with a wall from one polymer layer, also bilayer microstents could be obtained. By using
a drug free internal polymer layer, drug diffusion was directed to the outer side of the microstent that would be in contact
with the tissue. The apparent diffusion coefficients in the different polymer layers could be determined in systematic
diffusion experiments. Furthermore, comprehensive degradation and mechanical studies, sterilization experiments, and
analysis of in vitro biocompatibility e.g. with human fibroblasts were conducted. Finally, an in vivo study in rabbits with a
100 day follow-up examination illustrated the general suitability of the microstents for ocular implantation [2].
Scheme of multifunctional microstent and ocular implantation for suprachoroidal drainage, reprinted from [2] with permission.
Overall, this study found that blending of the two (co)polyester components allowed combining interesting elastic properties with a desired accelerated degradation. Ocular single and bilayer microstents have been prepared as thin diameter,
sterilizable candidate devices for aqueous drainage in glaucoma. These multifunctional systems combine i) a drainage
function with adjustable outflow rates by their tailorable diameters in the micrometer range, ii) a mechanical support
function, iii) hydrolytic degradability for a regenerative medicine approach, iv) biocompatibility, and v) spatially directed
controlled drug release.
In the future, the incorporation of antiproliferative drugs and the in vivo functional analysis of long-term performance
should be addressed.
References:
1. Wischke, C. et al.: Macromol. Sympos. 2011, 309: 59-67.
2. Wischke, C. et al.: J. Controlled Release 2013, 172: 1002-1010.
63
CASE STUDIES FROM PHARMACEUTICAL RESEARCH AND DEVELOPMENT
Dendritic cell-targeting cancer vaccine formulations for pulmonary and peroral delivery
Hanefeld, A.1; Schiller, S.1; Wolf, M.1; Weigandt, M.1; Scherließ, R.2; Diedrich, A.2; Janke, J.2; Knolle, P.3, Schröder, M.4;
Walden, P.5; Baleerio, R. 5; Lehr, C.-M.6; Rietscher, R.6; Schneider, M.7
1 Merck
KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany
of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9, 24118 Kiel, Germany
3 Klinikum rechts der Isar, Technische Universität München, Schneckenburgerstr. 8, 81675 München, Germany
4 BioMedX, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany
5 Department of Dermatology, Venerology and Allergy, Charité, Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
6 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Campus,
Geb. A4.1, 66123 Saarbruecken, Germany
7Institute for Pharmaceutics and Biopharmacy, Philipps University Marburg, Ketzerbach 63, 35037 Marburg, Germany
2 Department
Dendritic cells (DCs) play a crucial role in initiating anti-tumor immunity. We designed nanoparticles for passive dendritic
cell-targeting with the aim to trigger potent and specific anti-tumor T cell responses.
Activated cytotoxic T lymphocytes (CTLs) are needed to combat tumor cells.
Cross-presentation of antigen via the MHC Class I pathway is necessary to effectively induce a CTL response. Current
adjuvants are optimized for Th2-biased responses and high antibody serum titers. Ideally, a tumor vaccine formulation
would combine direct CTL activation (MHC Class I) with indirect Th1/2 mediated support. We studied a variety of nanoparticles (NPs) on their ability to do so and ways to formulate those antigen carriers into dosage forms for mucosal
administration.
We could elucidate the routing of different NPs in human dendritic cells. The particle fate seems to be determined by its
composition.
In vitro assays showed that the immunological response to the model antigen ovalbumin (OVA) could be increased by
factor 10 to 30 in comparison to soluble OVA depending on the NP type.
OVA-loaded NPs could be successfully formulated into inhalable or enteric microparticles for pulmonary or peroral
delivery.
Acknowledgments: BMBF (Förderkennzeichen 13N11455)
64
Selection of solid state forms for New Chemical Entities: Challenges, opportunities, adventures and
lessons learned
Saal, C.
Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany
Selection of solid state forms including pharmaceutical salts, polymorphs and co-crystals has drawn increasing interest
since the coincidental discovery of Ritonavir's stable polymorph which represented a major challenge for one of the first
anti-HIV drugs. The so called "Ritonavir case" together with the advent of New Chemical Entities which became more
and more demanding with regards to physico-chemical properties stimulated the systematic assessment and development of solid state forms for clinical development. The presentation will provide an overview on selection of solid state
forms to overcome hurdles for New Chemical entities including experiences made during the last decade and new
technological approaches.
We are greatful to Axel Becker and Michael Lange for fruitful discussion and strong support.
References:
1. Paulekuhn, S.; Dressman, J.; Saal, C.: J. Med. Chem., 2007, 50(26): 6665-6672.
2. Saal, C.; Becker, A.: Eur. J. Pharm. Sci., 2013, 49(4): 614-623.
3. Paulekuhn, S.; Dressmann, J.; Saal, C.: Die Pharmazie, 2013, 68: 555-564.
65
Fast track formulation development for biotherapeutics
Winzer, M.
66
Fighting Schistosomiasis in Young Children: The Pediatric Praziquantel Consortium
Skopp, S.
Merck KGaA, R&D Chemical and Pharmaceutical Development, Frankfurter Str. 250, 64293 Darmstadt, Germany
The current gold standard treatment of schistosomiasis employs annual single oral dose of the drug praziquantel (PZQ)
600 mg tablets, jointly developed by Bayer and Merck in the 1970ies. The tablet is available as an oral immediate
release tablet for adults and school-aged children1.
The available formulation of PZQ is not suitable for pediatric use and cannot be readily administered to children especially to preschool-age children and infants2. The drug product consisting of the two enantiomers, levopraziquantel (L-PZQ)
and dextropraziquantel (D-PZQ), has a severe bitter taste, which can lead to gagging or vomiting if tablets are chewed.
To date traditional methods of taste masking have been ineffective for PZQ. The described age groups of children have
difficulties swallowing the medication due to the large size of the tablet. Important quality and efficacy issues can be
raised3. Morever, the tablets are not registered for pediatric use in pre-school aged children and lack adequate clinical
data in this population.
In order to tackle this important public health problem, a consortium was formed in July 2012 under the leadership of
Merck KGaA with the goal of developing a suitable pediatric praziquantel formulation appropriate for children from the
age of 3 months to 6 years and register its use in schistosomiasis. The new formulation will preferably contain the L-PZQ
enantiopure active pharmaceutical ingredient (API), yet formulation of the racemate PZQ is also considered as a parallel
development. The project has completed the pre-clinical phase and will enter Phase I clinical trials in the last quarter of
2014.
References:
1. Preventive chemotherapy in human helminthiasis: coordinated use of anthelminthic drugs in control interventions: a manual for health professionals and programme managers (Geneva: World Health Organization) 2006.
2. Ekpo, U.F. et al.: Parasitology. 2012, 139: 835-841.
3. Richey, R.H. et. al.: BMC Pediatr. 2013, 13:81.
67
Chirality in polyketide antibiotics: substrate-dependent inversion of stereoselectivity in Tyl-KR1catalyzed reductions
Häckh, M.; Lucas, X.; Müller, M.; Günther, S.; Lüdeke, S.
Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany
Polyketide synthases (PKS) are mega-enzyme clusters that assemble acetate and propionate subunits for the biosynthesis of numerous antibiotics. During this process ketoreductases (KR) inside of the PKS are responsible for the stereoselective introduction of chiral secondary alcohols that are characteristic for polyketide scaffolds. The absolute configuration of their reduction products correlates strongly with conserved amino acid motifs within the involved KR domain. 1
Recently, we found a significant dependence of stereoselectivity and –specificity of recombinant PKS ketoreductase TylKR1 from Streptomyces fradiae on both the chain length and the kind of ester used for different non-natural keto-ester
substrates.2 This enzyme exhibits high (S,S)-selectivity for a number of artificial substrates despite of its physiological
(R,R)-selectivity.
On the basis of modeling studies using the crystal structure of Tyl-KR1,3 and 2-methyl-3-oxovaleric acid Nacetylcysteamine thioester, a surrogate for the physiological substrate,4 we identified molecular features that may be
crucial for (R,R)-selectivity and –specificity. We synthesized different analogs, for which at least one of these interactions
would be interrupted (Scheme 1). The stereochemical outcome of the Tyl-KR1-catalyzed reduction of these analogs was
studied qualitatively and quantitatively by vibrational circular dichroism and chiral phase gas chromatography. This study
does not only provide new insights into the understanding of stereoselectivity in the biosynthesis of polyketide antibiotics
but also provides a basis for the utilization of Tyl-KR1 or related enzymes in the biocatalytic synthesis of valuable chiral
compounds.
O
O
S
Tyl-KR1
NADPH
H
N
OH
O
S
O
O
2R,3R O
84% ee
Tyl-KR1
NADPH
O
S
Scheme 1
References:
1. Caffrey, P.: ChemBioChem 2003, 4(7): 654–657.
2. Häckh, M.; Müller, M.; Lüdeke, S.: Chem. Eur. J. 2013, 19(27): 8922–8928.
3. Keatinge-Clay, A.T.: Chem. Biol. 2007, 14(8): 898–908.
4. Siskos, A.P. et al.: Chem. Biol. 2005, 12(10): 1145–1153.
68
H
N
OH
O
S
2S,3S
18% ee
GPCR MEDICINAL CHEMISTRY
Toward selective molecular tools for histamine receptor subtypes: conformational constraints, bioisosteric and bivalent approaches
Buschauer, A.
Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry II, University of Regensburg, D-93040 Regensburg, Germany
Four histamine receptor subtypes, designated H1 (H1R), H2 (H2R), H3 (H3R) and H4 receptors (H4R), are encoded in the
human (h) genome. Antagonists for hH1R and hH2R are well established drugs for the treatment of allergic conditions
and gastroduodenal diseases, respectively, whereas pitolisant, the first clinically available hH3R antagonist, has obtained
orphan drug status for the treatment of narcolepsy. H4R antagonists are not yet available as approved drugs, but are
considered to be of potential value for the pharmacotherapy of inflammatory diseases and pruritus. With the discovery of
the H3R and, in particular, the H4R, the field became considerably more complex. Numerous compounds developed as
subtype-selective ligands for H1R and H2R decades ago, have been proven to possess much higher affinity to H3R and
H4R [1]. This holds, e. g., for imidazole-type ligands such as the potent H4R agonist 5-methylhistamine, which was
initially described as the first selective H2R agonist, as well as for guanidines derived from impromidine.
Aiming at subtype-selective, radiolabeled and fluorescent hHxR ligands as pharmacological tools, bioisosteric and
bivalent approaches were explored in our laboratory, starting from guanidine-type H2R agonists or piperidinomethylphenoxypropylamine-type H2R antagonists, respectively. Modification of the latter gave tritiated (([3H]UR-DE257) and
fluorescent ligands for the H2R and paved the way to fluorescent H3R antagonists. In the guanidine series, the replacement of the imidazole ring by 2-aminothiazole in combination with an acyl- or carbamoylguanidine moiety resulted in
highly potent and selective H2R agonists, including bivalent agonists [2,3]. With regard to H4R selectivity, the suitability of
guanidine replacements, various heterocycles and conformationally constrained linkers was explored, and the substitution pattern of acylguanidines [4] and cyanoguanidines [5] was varied, resulting, e. g., in the high-affinity H3/4R radioligand [3H]UR-PI294 [6] and the potent H4R agonists UR-PI376 [5] and trans-(+)-(S,S)-UR-RG98.
Selectivity for H4R over H3R is especially challenging. Beyond HR subtype selectivity, activities of many ligands differ
significantly from those at the human H4R, especially at rodent H4Rs, in terms of ligand efficacies, potencies and affinities [7, 8]. Such differences were extremely pronounced in case of proximal readouts ([32P]GTPase, [35S]GTPγS assay).
Moreover, in contrast to the mouse and rat H4R, the hH4R shows a high degree of constitutive activity [9].
In conclusion, agonists, antagonists as well as labeled ligands are required as molecular tools for the investigation of HR
subtypes of various species. With respect to predictivity of in vitro studies and translational animal models, ortholog- and
assay-dependent activity profiles have to be considered, e. g. binding and functional data (GPTase, GTPγS assay,
reporter gene, arrestin and label-free assays) using recombinant human, murine and rat H4Rs including mutants as well
as native cells.
Acknowledgments: This work was supported by the Graduate Training Programmes GRK 760 and GRK 1910 of the DFG and by the European
Cooperation in Science and Technology, COST Action BM0806
References:
1. Seifert, R. et al.: Trends Pharmacol. Sci. 2013, 34: 33-58.
2. Kraus, A. et al.: ChemMedChem 2009, 4: 232-240.
3. Birnkammer, T. et al.: J. Med. Chem. 2012, 55: 1147-1160.
4. Igel, P. et al.: J. Med. Chem. 2009, 52: 2623-2627.
5. Igel, P. et al.: J. Med. Chem. 2009, 52: 6297-6313.
6. Igel, P. et al.: ChemMedChem 2009, 4: 225-231.
7. Schnell, D. et al.: Naunyn. Schmiedebergs Arch. Pharmacol. 2011, 383: 457-470.
8. Nordemann, U. et al.: PLoS ONE 2013, 8: e73961.
9. Wifling, D. et al.: Br. J. Pharmacol. 2014, in press, doi: 10.1111/bph.12801.
69
Modulation of GPCR signaling: Understanding ligand binding effects
Bermudez, M.; Wolber, G.*
Computer-Aided Drug Design, Institute of Pharmacy, Department Pharmaceutical Chemistry, Freie Universität Berlin, 14195 Berlin
E-Mail: [email protected]
G-Protein coupled receptors (GPCRs) have played a key role for drug development over the last decades. Each class of
GPCRs can trigger different functions by adopting specific conformations upon ligand binding. Recent advances in X-ray
protein crystallography now provide us with new fundamental structural data on GPCRs with co-complexed ligands [1].
These new insights give us the opportunity to develop structure-based binding models by computer simulation in order to
rationally develop drug candidates that selectively bind to a specific conformation. Two case studies for such models will
be presented:
The first model, based on the recently published crystal structure of the M2 muscarinic acetylcholine receptor (PDB:
3UON [2]) provides the possibility to rationalize and understand the binding of ligands, explain their subtype preference
and give insights into the flexibility of their binding pockets. Since allosteric, orthosteric and dualsteric ligands are available, we were able to connect multiple signaling roles with conformational states [3-4]. Extensive molecular dynamics
(MD) simulations in combination with molecular docking and 3D-pharmacophore analyses of known ligands and related
structures were used to understand and explain this relation.
The second model describes the binding of two recently discovered ligands (JSM10292 and MEN16132) of the bradykinin B2 receptor. Based on the crystal structure of CXCR3 receptor, a homology model could be developed that explains
specific conformational changes and provides a basis for further rationally designing optimized ligands [5].
Fig. 1.: Conformation of the atropin-based hybrid ligand atr-6-phth complexed with the M2 receptor
References:
1. Venkatakrishnan, A.J. et al., Nature 2013, 494(7436): 185–194.
2. Haga, K., et al.: Nature 2012, 482(7386): 547–551.
3. Antony, J., et al.: Faseb Journal 2009, 23(2): 442-450.
4. Holzgrabe, U.; Mohr, K.: Drug Discov Today 1998, 3(5): 214-222.
5. Schmitz, J. et al.: J. Med. Chem. 2014, 57(15):6739-6750.
5. Leschner, J. et al.: J Pharmacol Exp Ther 2013, 1344(1):85-95.
70
Molecular combination of GPCR ligands: bivalent, hybrid and dualsteric compounds
Decker, M.
Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-Universität Würzburg, Am Hubland, D97074 Würzburg, Germany
The development of GPCR ligands with high affinity and selectivity is one of the major success stories in medicinal
chemistry, pharmacy and medicine. An array of methods has been developed to find such selective compounds starting
from suitable lead structures. Nevertheless, many problems based on receptor function are yet unsolved. It is still unclear how GPCR move spatially and temporally to mediate their intracellular activity. Also the role of receptor aggregation and/or dimer formation for mediation of effects and receptor internalization is not understood. Based solely on the
ligand structure, there is no general knowledge - although there is numerous empirical data – about what determines the
intrinsic activity of a chemical compound. Molecular tools are needed to help and enable pharmacologists to investigate
these effects [1]. Furthermore, pharmaceutical companies face the demanding task to identify lead structures. Improving
a lead’s binding profile can be achieved as mentioned above. Nevertheless, the efforts to find a lead, like screening
compound libraries, are difficult to perform and cost-intensive. Another unmet problem lies in the fact that several diseases seem to be multifactorial in nature, such as neurodegenerative disorders. Even the best (in terms of affinity and
selectivity) GPCR ligand will most of the time only target one aspect of combating the disease, but will never be able to
target a larger spectrum of biological effects.
Out of these reasons, in the last years several approaches have emerged and successfully applied, in which known
GPCR ligands are connected or merged with other biologically active molecules: either combination of identical or
related ligands to bivalent ones [1], combination of orthosteric with allosteric ligands [2], sometimes leading to dualsteric
(or bitopic) ones, and finally merging chemical structures to dual-acting or multifunctional compounds.
In this talk some examples for the above approaches at different GPCRs and their subtypes will be presented showing
the versatility of this approach. This includes the synthesis and characterization of bivalent ligands at opioid (OP) [3] and
both cannabinoid (CB) 1 and CB2 receptors [4] and some aspects to consider when designing bivalent ligands will be
discussed such as an alteration of intrinsic activity. Tasks emerging when designing dualsteric ligands will be presented
with regard to muscarinic acetylcholine 1 (M1) receptor ligands [5,6], here it is of major importance to determine whether
dualsteric ligands are actually formed. Finally, developing a low-molecular-weight dual active compound acting at the
histamine (H) 3 receptor as an antagonist and inhibiting the enzyme and Alzheimer’s disease drug target acetylcholinesterase (AChE) at the same time will be presented [7]. Such a compound might represent an approach to combat multifactorial diseases like Alzheimer’s disease. All these methods offer unique chances for developing novel GPCR ligands,
but also challenge the medicinal chemist when applying them in terms of design, synthetic approaches, and pharmacological characterization.
Financial support by the German Research Foundation is gratefully acknowledged (DFG DE1546/4-1)
References:
1. Hiller, C.; Kühhorn, J.; Gmeiner, .P: J. Med. Chem. 2013, 56(17): 6542-6559.
2. Mohr, K. et al.: Angew. Chem. Int. Edit. 2013, 52(2): 508-516.
3. Decker, M. et al.: J. Med. Chem. 2010, 53(1): 402-418.
4. Nimczick, M. et al.: Bioorg. Med. Chem. 2014, 22(15): 3938-3946.
5. Fang, L.. et al.: J. Med. Chem. 2010, 53(5): 2094-2103.
6. Decker, M., Holzgrabe, U.: Med. Chem. Commun. 2012, 3(7): 752-762.
7. Darras, F.H. et al.: ACS Chem. Neurosci. 2014, 5(3): 225-242.
71
Boronic acids as probes for exploration of allosteric regulation of the chemokine receptor CXCR3
Bernat, V.; Admas, T.H.; Brox, R.; Tschammer, N.
Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Schuhstraße 19, 91052
Erlangen, Germany
The G protein coupled chemokine receptor CXCR3 is associated with numerous pathologies including autoimmune
diseases, cancer, vascular disease and transplant rejection. To facilitate the understanding of complex allosteric regulation of CXCR3, we combined computational modelling with the synthesis of novel chemical tools containing boronic acid
moieties, site-directed mutagenesis and detailed functional characterization followed by the analysis of biased signalling.
The design of boronic acid derivatives was based on the predictions from homology modelling and docking. Boronic
acids derivatives are scarcely used as GPCR ligands. The choice of the boronic acid moiety was dictated by its unique
ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like
boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand
RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different
binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109 2.60
and Lys3007.35 inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation.
Furthermore we report the boronic acid derivative as the first biased negative allosteric modulator of the receptor that
preferentially inhibits the recruitment of β-arrestin 2 over G protein activation. Overall, our data demonstrate that boronic
acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of
ligand-biased signalling.
N.T., T.H.A. and R.B. were financially supported by Graduate Training Schools GRK1910 and GRK1962, and grant TS287/2-1 of German Research Foundation (DFG). N.T. participates in the European COST Action CM1207 (GLISTEN: GPCR-Ligand Interactions, Structures, and Transmembrane Signalling: a European Research Network).
72
INDUSTRIAL PHARMACY
Biophysical Characterization of Pharmaceutical Peptides
Nagel, N.
Sanofi-Aventis Deutschland GmbH, R&D LGCR / Analytical Sciences, D-65926 Frankfurt am Main
Physical characterization of pharmaceutical peptides involves not only secondary, tertiary and quaternary structure
investigations, but also exploring the stability of peptides with respect to non-specific aggregation or amyloid fibril formation. Recent examples from sanofi development Frankfurt illuminate potentials and limitations of up to date analytical
methods.
73
Subvisible Particles in Protein Formulations
Olbrich, C.
Bayer Pharma AG, Building 230, 42117 Wuppertal
Particles are a challenge in parenteral formulations. Specific attention is paid to particle formation in protein formulations,
as they can be immunogenic and can have severe side effects. For a long time, particles could only be monitored in the
range of visible particles and with respect to subvisible particles in the size range of ≥ 10 and ≥ 25 µm (particulate
matter; USP monograph). Besides this, size exclusion chromatography was available for the determination of Dimers
and Oligomers in the lower nm range. Due to the lack of suitable analytical methods, the range of particles between 100
m and 10µm was not accessible to analytical charav´cterisation, especially the quantification of particles.. Recent
developments in analytical equipment opens now the perspective for monitoring protein particles in the size range
mentioned allowing the optimisation of formulation and manufacturing processes to minimise particle formation and
monitor consitencey over manufacturing changes, which is an important aspect in comparability excersises for protein
drugs.
Mikroflow Imaging opens the perspectzive of counting and characterising particles between 1 µm and 10 µm and
resonant mass measurement is available to to so with particles from 50 nm to 5 µm. This allows to cover the interesting
size range for protein particles and to study mechanistically protein particle formation as a consequence of stress.
These new analytical methods allow a more reliable development of protein formulations and to conecutively fulfill the
upcoming requirements of limitations of partaicle formation and occurance in protein therapeutics, e.g. as a
consequence of the FDA Immunogenicity guidline.
Examples are shown of stress factors leading to the formation of protein aggregates, as well as to mitigation strategies to
avoid or minimise these effects.
74
Dissecting pharmacodynamic action of compound mixtures by use of in vitro models
Hengl, T.1; Riegel, K.1; Schlinzig, K.1; Ansari, N.²; Stelzer, E.²; Abts, H.F.1
1 Merz
Pharmaceuticals, 60318 Frankfurt, Germany
² Buchmann Institute for Molecular Life Science (BMLS), Goethe University, 60438 Frankfurt, Germany
Established use of traditional or “grandfather” products are often based mainly on clinical effects while detailed
knowledge of the underlying pharmacodynamics might not be available. In particular information on the detailed mode of
action on the target cell would be of interest in order to further support current use but also allow further development.
For obtaining such information on pharmacodynamics appropriate cell based in vitro assay and molecular identification
of potential functional pathways could be used.
In vitro cultured human keratinocytes are a well-established tool for investigating general aspects of skin and hair physiology. Within the epidermis normal human epidermal keratinocytes (NHEK) generate the multi-layered skin barrier by a
distinct differentiation process. The major part of the hair follicle is build up by hair follicle associated keratinocytes
(HHFK) that form as a result of another differentiation program the hair-shaft. General aspects of keratinocyte physiology
could be investigated in both cells, while examination of hair-specific processes requires the analysis in HHFKs. Cultivation of keratinocytes in a minimal growth medium (MGM) was used as an in vitro model for undersupplied keratinocytes,
mimicking the situation of diminished hair growth for analyzing the effect of a hair growth promoting formulation, Panto(vi)gar. To investigate the genes that are modulated during Panto(vi)gar treatment we performed an Agilent whole
genome array expression analysis using HHFK and NHEK cultivated either in a minimal growth medium (MGM) alone or
supplemented with a Panto(vi)gar in vitro correlate (P-IC). In accordance with the P-IC induced cellular phenotype P-IC
modulated genes were identified that are involved in cell cycle, proliferation and metabolic processes in NHEK and
HHFK. Furthermore P-IC appears to regulate genes associated with cell death, extracellular matrix, stress responses
and hair follicle physiology.
Two-dimensional monolayer cell culture models are thus successfully used for understanding physiological pathways on
a cellular level. However, physiological properties and differentiation processes are in vivo often also the result of threedimensional interactions within organs or mini-organs such as the hair-follicle. Associated processes like differentiation
or morphogenesis can be simulated in vitro by using 3-D culture models.
As second example for the use of in vitro models we thus present a 3-D culture system mimicking more closely the in
vivo situation of the human hair follicle. This 3-D model was used to further investigate the mode of action of hair-growth
promoting formulations. As 3-D hair follicle model heterotypic spheroids were established from human dermal papilla
cells (DP) and hair-follicle-associated keratinocytes (HHFK). The analysis of the initial spheroids formation process was
performed by observing cell type specific migration/distribution patterns by automated live cell fluorescence microscopy.
Spheroid formation process was shown to be fully functional in P-IC whereas cultivation in MGM only resulted in only
reduced spheroid quality and stability. Data indicate that the tested actives could thus deliver a significant contribution to
the formation and preservation of the hair-follicle structure.
In conclusion use of 2-D and 3-D cell based models allow the molecular dissection of the functional aspects of pharmacologic active ingredients. Such knowledge not only uncovers the mode of action but could form also the basis of alternative and improved drugs.
75
Current Biopharmaceutics Prediction Tools - An Overview
Muenster, U.
Bayer Pharma AG, Research Center Aprath, 42096 Wuppertal, Germany
In order to bring innovative drug products to the patient in need in good time, it is key within Pharmaceutical Development to focus on the right formulation strategy early-on. The type of formulation needed (e.g. immediate release (IR) with
or without solubilization technology, or e.g. slow release (SR) tablet) depends on the pharmacokinetic profile as well as
the physicochemical properties of the API. Choosing the right formulation and making an acceptable prediction of its
biopharmaceutics performance has been more and more supported over the last two to three decades by the generation
of various biopharmaceutics prediction tools. These include physiologically based pharmacokinetic (PBPK) in silico
softwares, in vitro experimental set-ups (e.g. mimicking gastrointestinal compartments) and respective in vivo animal
models. The talk will give an overview on the current status and most often used biopharmaceutics prediction tools within
Pharmaceutical Industry.
76
BIOPHARMACEUTICS AND PHARMACEUTICAL TECHNOLOGY
Novel injectable RNA formulations for tumor immunotherapy
Haas, H.1; Fritz, D.1; Meng, M.1; Popa, A.-L.1; Esparza, I.1; Kranz, L.M.3; Reuter, K.C.2; Diken, M.2; Kreiter, S.2; Funari,
S.4; Pawlowska, D.5; Brezesinski, G.5; Sahin, U.1,2,3
1 BioNTech
RNA Pharmaceuticals GmbH, An der Goldgrube 12, 55131 Mainz, Germany
gGmbH, Building 708, Langenbeckstrasse 1, 55131 Mainz, Germany
3 Universität Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany
4 HASYLAB, DESY, Notkestrasse 85, 26607 Hamburg, Germany
5 Max-Planck-Institute for Colloids and Interfaces, Am Mühlenberg 1, 14476 Golm, Germany
2 TRON
Novel nanoparticulate lipoplex formulations to deliver mRNA to Antigen Presenting Cells (APCs) are presented. After
intravenous (i.v.) injection of the lipoplex formulations, expression of mRNA in splenic Antigen Presenting Cells (APCs)
with high selectivity and efficacy can be obtained. For targeting certain physicochemical parameters of the lipoplex
nanoparticles are decisive, and no ligands for specific binding tot the target membrane, such as mannose, are required.
With this type of formulations, tumor immunotherapy based on expression of mRNA coding for tumor antigens in APCs
may be substantially facilitated and provided to high numbers of patients under daily routine conditions.
Similar to the well-known approaches for non-viral gene delivery with DNA, mRNA lipoplexes were formed by incubation
of RNA to cationic (positively charged) liposomes. Ionic conditions, lipid composition, lipid-to-RNA ratio, and further
physicochemical parameters were varied for formation of the lipoplex formulations. Particle size, colloidal stability and
other parameters that are relevant for pharmaceutical products for parenteral administration were investigated. Formulations consisting of positively charged or negatively charged nanoparticles, which were stable in the liquid phase for
several days, and where the RNA was protected with respect to interactions with serum components and degradation
were identified.
After intravenous injection of such lipoplex formulations from mRNA, coding for the reporter gene luciferase into mice,
expression of luciferase was measured in various target organs, dependent on the particle properties. While with positively charged (cationic) particles, such as known from classical gene delivery, preferentially luciferase expression in the
lung was observed, negatively charged lipoplex nanoparticles lead to luciferase expression almost exclusively in Dendritic Cells (DCs) in the spleen. It appears that the charge of the particles was a key factor for the targeting selectivity.
Different mechanisms of cellular uptake in DCs and other cells may play a role for the observed selectivity.
The finding, that high targeting selectivity can be achieved without using specific ligands or complex nanoparticulate
architecture makes formulations assembled by the described approach promising for pharmaceutical development
towards products for application in patients. Robust, straightforward and scalable manufacturing processes can be
realized, using standard lipids which are compliant with the requirements of products for parenteral administration.
This will help to bring novel therapeutic strategies based on selective expression of mRNA in Antigen Presenting Cells
(APCs) readily into clinical practice. Products for tumor immunotherapy by selective expression of tumor antigens in
APCs can be administered systemically (intravenously) on a regular basis.
Acknowledgments: We would like to thank The innovation and technology program by the government of Rhineland Palatinate within the InnoTop
Project “Innovative therapeutic RNA formulations for systemic application of biologicals in humans for financial support. Wealso would like to
acknowledge financial support for the project “Nanopartikuläre Ribopharmaka zur individualisierten Tumortherapie –Ph I im Mammakarzinom,
within the ” CI3 Cluster für Individualisierte ImmunIntervention”.
77
Automated testing of inhalation devices in early development phases
Wachtel, H.; Jung, A.; Richter, F.
Respiratory Drug Delivery Department, Boehringer Ingelheim Pharma GmbH & Co. KG, Binger Str. 173, 55216 Ingelheim, Germany
Advanced testing of quality aspects of the Respimat® Soft Mist™ Inhaler (SMI) is discussed where the required release
tests and one-time studies were complemented by automated tests. The automated testing was established as a voluntary additional effort because it allowed to significantly increase the sample size and thereby to improve the understanding of this device in the early development phase.
Background: The manufacture of inhalers and the applicable testing methods are strictly regulated. According to the
classification of the inhalers they are either medicinal products or medical devices or combination products, depending
on different national legislations. In case of international development programs, the regulations of the importing countries must already be considered during development, where e.g. a design history file is required. The regulations strongly influence the testing strategies applied e.g. for release.
Requirements: During the development phase of a multi-dose inhaler like the Respimat SMI, the reliable dosing is key
and must be checked with high statistical power, increasing the number of test samples in a way that manual testing
according to Pharm. Eur. (0671) is not feasible. In addition, the innovative technology of spray generation motivates
paying special attention to the particle size distribution which by convention previously was checked using cascade
impactors according to Pharm. Eur. 2.9.18. Handling e.g. the torque for preparing the Respimat SMI for use or the force
to press the dose release button should also be recorded thus taking into account a recent initiative towards human
factor engineering which puts emphasis on usability.
Technical solution: Boehringer Ingelheim built a fully automated test system which comprises a six-axis robotic arm for
device transfer between individual stations which are dedicated to the following tasks: 1. Storage of 300 inhalers, 2.
Checking handling parameters, e.g. torque to prepare the inhaler and force to press the release button, 3. Sound detection, 4. Measurement of the particle size distribution (laser diffraction), 5. Detection of the plume geometry, 6. Measurement of spray duration, 7. Check of consistent dosing by determining delivered mass / metered mass. The system was
designed for 1000 actuations within 24h and the large number as well as the high reproducibility (low standard deviation)
help in making rational design decisions. The system is operated in a non-GMP environment because during early
development the flexible adaptation to the changing inhaler features is important. For the same reason there is no full
formal compliance with 21 CFR Part 11 as the complexity of the heterogeneous multi-processor system (see Fig. 1)
prohibits fast revalidation cycles. The accuracy of the measured data however is guaranteed by regular checks of the
individual sensors. All submission data are generated by conventional manually operated tests in the appropriately
regulated environment.
Figure 1: IT structure of the automated system allowing for flexibility on a local level while interfacing to a strictly regulated company-wide IT structure and the Oracle database.
Summary: The Respimat® Soft Mist™ inhaler represents a novel and unique principle for atomizing pharmaceutical
aerosols. Even in early development, the advantage of automated testing of this inhaler is apparent. In early development, the flexibility of an automated system is key and outweighs full compliance (i.e. the system is operated at a nonGMP status). The large number of devices and repeated actuations automatically investigated leads to a better understanding and consequently to high reliability of the finished product.
78
Orodispersible Dosage Forms
Serno, P.
Bayer Pharma AG, Building D303, 51368 Leverkusen, Germany
Orodispersible dosage forms passed through significant steps of evolution during the last two decades and represent a
well accepted formulation option for dysphagic patients, poorly cooperating patients, medication to be taken during
travelling and other instances. Three formulation technologies are being used: Oral lyophilisates, orodispersible tablets
and orodispersible films.
Oral lyophilisates require highly specialized and expensive manufacturing technology. They exhibit limited mechanical
strength but still offer the shortest disintegration time and lead to pleasant mouthfeel upon administration.
Orodispersible tablets have initially been manufactured by highly complex technologies involving spinning of flosses,
compression of fine particular sugars, curing steps and others. Nevertheless, mechanical properties of early
orodispersible tablets had often been poor. Meanwhile a large number of coprocessed excipients has become available
for easy formulation of orodispersible tablets. The “coprocessing” usually comprises spray drying of one or more polyols
and addition of a superdisintegrant like crospovidone. Orodispersible tablets are subsequently manufactured in a direct
compression process with the tablet compression force as a key critical process parameter. Alternatively, orodispersible
tablets may be formulated by addition of silicates and superdisintegrants in high concentrations.
Orodispersible films are manufactured by casting thin layers of organic polymer solutions and subsequent drying in long
drying tunnels.
The biopharmaceutical performance of orodispersible dosage forms ranges from bioequivalence to substantially
enhanced bioavailability in comparison to conventional swallow – tablets. Several case studies suggest that “faster
action”, i.e. faster occurrence of therapeutically relevant plasma concentrations, is usually not achieved.
79
Pharmacokinetic Drug-Nutraceutical Interactions: A Particular Type of Food-Drug Interaction
Nguyen, M.A.; Langguth, P.
Institute of Pharmacy Institute of Pharmacy, Johannes Gutenberg University, Staudingerweg 5, 55099 Mainz, Germany
In addition to the daily diet, components present in food and beverages are increasingly consumed in dietary supplements due to their presumed health promoting effects, for example anti-oxidative and anti-inflammatory activities. These
nutraceuticals represent a relevant source of interaction with drug pharmacokinetics for two main reasons: Specific
components are by several magnitudes higher dosed compared to the normal dietary intake and unlike prescribed drugs,
their consumption is usually not reported to the physician upon visit.
Our data presented here focus on the modulation of drug transport by selected flavonoids in humans. Although quercetin
remarkably inhibits P-glycoprotein (P-gp)-mediated drug efflux in vitro1, the bioavailability of talinolol, a substrate of
intestinal P-gp, was reduced following oral co-administration with the nutraceutical.2 This observation indicates that
inhibition of intestinal uptake by organic anion-transporting polypeptides (OATPs)3 rather than P-gp inhibition dominates
when talinolol and quercetin are co-administered in humans. On the other hand, naringin which had been reported to be
the causative ingredient for the inhibition of intestinal OATPs by grapefruit juice3 did not alter the pharmacokinetics of
talinolol, a substrate of intestinal OATP1A2 and OATP2B1, after 7-day supplementation with naringin capsules. Possible
explanations include the higher dose and the solid state which is different from the common intake of naringin in grapefruit juice. In the proximal parts of the small intestine, the main site of talinolol absorption, the concentration of dissolved
naringin may not be high enough for efficient inhibition of OATP-mediated uptake, whereas in more distal parts, naringin
and its aglycone naringenin may reach higher concentrations provoking P-gp inhibition which counteracts the uptake
inhibition.
Like food, nutraceuticals can interfere with a variety of processes during drug absorption, distribution, metabolism and
elimination. Due to their different dose and dosage forms, however, their effect on drug pharmacokinetics may deviate
from that observed when administered as food components. Better understanding of their pharmacokinetics will help
reducing the risk of uncontrolled modulation of drugs’ efficacy and adverse effects.
The research work discussed here was supported by the German Research Foundation (DFG).
References:
1. Ofer, M. et al.: Eur. J. Pharm. Sci. 2005, 25: 263-271.
2. Nguyen, M.A. et al.: Eur. J. Pharm. Sci. 2014, 61: 54-60.
3. Shirasaka, Y. et al.: J. Pharmacol. Exp. Ther. 2010, 332: 181-189.
4. Bailey, D.G. et al.: Clin. Pharmacol. Ther. 2007, 81: 495-502.
80
Gastro-Intestinal Simulator for in vitro Drug and Nanoparticle Tracing in Oral Drug Development
Nawroth, T.1; Khoshakhlagh, P.1; Kindgen, S.1; Krebs, L.1; Johnson, R.1; Langguth, P.1; Schweins, R.2; Szekely, N.3
Gutenberg-University, Inst. Pharmacy and Biochemistry, Pharmaceutical Technology Department, Staudingerweg 5, D-55099 Mainz, Germany
Institut Laue Langevin ILL, DS / LSS, 71 Avenue des Martyrs, F-38042 Grenoble CEDEX 9, France
3 JCNS-FRM-II outstation at the Mayer-Leibnitz Zentrum MLZ, Lichtenbergstr.1, D-85747 Garching, Germany
1
2
Intestinal modelling and model fluids are tools for the in vitro investigation of drug formulations for oral administration. In
vitro solubility and permeability studies at nearly physiological conditions are the key for an improvement of the bioavailability of drugs, which is difficult for hydrophobic materials, i.e. drugs of the classes II and IV of the Biopharmaceutics
Classification System BCS [1]. These form nanoparticles with amphiphilic materials, e.g. bile and food, which can mediate a drug solubilisation and uptake. The structure of lipidic nanoparticles in solution can be estimated by dynamic light
scattering DLS and small angle scattering, with best contrast using neutrons, i.e. neutron small angle scattering SANS
[2].
We develop a gastro-intestinal simulator system for the in vitro estimation of drug and formulation development in a
modelled human digestion system (fig.1). The flow-through modules for the sections of the GI-system are designed for a
tracing of formulation disintegration, drug dissolution and structure analysis of intermediate nanoparticles, e.g. from biledrug-formulation interaction. The transport can be analyzed by local sampling. The nano-structure is observed in situ
through flat quartz windows with local and time resolution with a computer controlled (CNC) continuous flow (CF) +
stopped flow (SF) system with a projecting DLS device, and for the most important samples with neutron scattering
SANS at large science facilities [2,3].
Fig.1: Concept of the gastro-intestinal simulator for in vitro drug and nanoparticle tracing of oral drug formulations.
Formation and decay of intermediate nanoparticles from bile, drug, fluid and food are analyzed in gut module flow
cells with DLS and SANS with continuous flow (CF) and stopped flow (SF) technology.
This work was contributed to the OrBiTo project (http://www.imi.europa.eu/content/) as sideground.
References:
1. Amidon, G. et al.: Pharm. Res.1995, 12: 413-420.
2. Nawroth, T. et al.: Mol. Pharm. 2011, 8: 2162-2172.
3. Khoshakhlagh, P.: et al. Eur.J.Lipid Sci. Technol 2014 submitted.
4. Johnson, R. et al.: Eur.J.Lipid Sci. Technol 2014 in press.
81
ANTICANCER AND EPIGENETIC DRUGS
Unraveling the aberrant epigenetic programming of MLL leukaemias
Milne, T.
There has been progress in treating human cancers, especially leukaemias, but many remain resistant to treatment. A
potentially exciting approach is the development of small molecule inhibitors that specifically target aberrant processes in
cancer cells but leave normal cells unharmed. In order to be successful, such an approach requires highly detailed
information about normal and aberrant cellular processes on the molecular level. Understanding the function of key
proteins on a molecular level requires the use of model systems that are amenable to experimental manipulation. The
Mixed Lineage Leukaemia protein (MLL) has provided a very useful model system for exploring general mechanisms in
leukaemia. MLL is an important epigenetic regulator normally required for the maintenance of gene activation during
development. Chromosome translocations that fuse the MLL gene to over 75 different partner genes produce novel
fusion proteins that are a major cause of therapy resistant acute leukaemias in children and adults. Relative to other
acute leukaemias, MLL leukaemias have very few cooperating mutations. MLL fusion proteins are thought to promote
leukaemogenesis through the epigenetic activation and maintenance of important gene targets that control cellular
growth and proliferation pathways. Studying MLL leukaemias can aid in the identification of new drug targets that could
also potentially impact a wider range of different cancers.
82
Identifying Therapeutic Targets in MLL Fusion-Driven Leukemia Using Functional Genomics
Fröhling, S.
Department of Translational Oncology, German Cancer Research Center (DKFZ), National Center for Tumor Diseases (NCT), Heidelberg
Chromosomal rearrangements involving the H3K4 methyltransferase MLL trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusionmediated leukemogenesis have not yet translated into better therapies, because MLL is difficult to target directly and the
identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly
annotated. We used a functional genetic approach to uncover that AML cells driven by the MLL-AF9 fusion are exceptionally reliant on the cell cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth
inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also
evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia, and can be
ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering
CDK6 expression are closely phenocopied by palbociclib, a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome
the differentiation block associated with MLL-rearranged AML, and underscore that cell cycle regulators may have
distinct, non-canonical and non-redundant functions in different contexts. Based on this preclinical rationale, we are
currently preparing a phase I/II clinical trial to evaluate the efficacy of palbociclib in patients with newly diagnosed,
relapsed, or refractory MLL-rearranged leukemia who are not candidates for intensive chemotherapy and hematopoietic
stem cell transplantation.
83
MLL leukemias and future treatment strategies
Marschalek, R.
Inst. Pharm Biology, Goethe-University, Biocenter, Max-von-Laue-Str. 9, 60438 Frankfurt/Main
Childhood cancer affects about 1.800 children per year in Germany of which about 620 cases develop acute leukemia
(~35%). This is different in adults, because of the 430.000 newly diagnosed cancer cases per year only about 9.300
cases suffer from acute leukemia (2%). Of all those leukemia cases (childhood and adult), MLL-rearranged leukemia
cases play an important role. Generally, MLL-r leukemia patients are hard-to-treat and the overall survival of these
patients is very poor. Moreover, a large portion of these leukemias is diagnosed in infants after birth, indicating that the
onset of this type of leukemia already occured in utero.
The human MLL protein - encoded by the MLL gene - plays a pivotal role in growth and differentiation processes. The
MLL protein exerts a histone H3 lysine-4 (H3K4) tri-methylation activity. This particular histone modification represents a
key signature of our "epigenetic programming system" which is necessary for developmental process, but also responsible for "transcriptional memory" in differentiated cells.
The MLL gene becomes disrupted in a process termed "chromosomal translocation". Basically, this process describes
how 2 different chromosomes are recombined after a DNA damage situation. During this event, the MLL gene becomes
disrupted and fused reciprocally to one of its many translocation partner genes. So far, 80 different translocation partner
genes have been described. Consequently, all these events result in the expression of MLL fusion proteins, and subsequently, in the development of acute leukemia [ALL (acute lymphoblastic leukemia) or AML (acute myeloid leukemia)].
Interestingly, only 4 out of 80 gene fusions are responsible for the majority of cancer cases (90% of patients in ALL, 50%
of patients in AML). These translocation partner genes are AF4, AF9, ENL and AF10, respectively. Moreover, all 4
encoded proteins are part of a multiprotein complex that steers "transcriptional elongation". Thus, epigenetic imprinting
as well as transcriptional processes are affected in a genome-wide fashion in the leukemic blasts of these leukemia
patients. Consequently, novel drugs have been developed in the past years that affect key functions deriving from these
MLL fusion proteins. This talk will summarize our knowledge about MLL-r leukemia and the different strategies to inhibit
functions of these MLL fusion proteins by using molecules.
This study is supported by grant DKS 2011.09 from the German Children Cancer Aid to RM, and by research grants Ma1876/10-1, Ma1876/11-1
from the DFG to RM. RM is PI within the CEF on Macromolecular Complexes funded by DFG grant EXC 115.
84
Selective Sirt2-inhibition by ligand induced rearrangement of the active site
Jung, M.1; Rumpf, T.1; Schiede, M.l.1; Karaman, B.2; Roessler, C.3; North, N.J.4; Ladwein, K.I.1; Gajer, M.1; Pannek, M.5;
Steegborn, C.5; Sinclair, D.A.4; Gerhardt, S.6; Schutkowski, M.3; Sippl, W.2; Einsle, O.6
1 Albert-Ludwigs-University,
Institute of Pharmaceutical Sciences, Albertstr. 25, 79104 Freiburg, Germany
Martin-Luther-University, Institute for Pharmacy, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
3 Martin-Luther-University Institute for Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany
4 Harvard Medical School, Department of Genetics, 77 Avenue Louis Pasteur, Boston, MA 02115 USA
5 University of Bayreuth, Department of Biochemistry, Universitätsstr. 30, 95445 Bayreuth, Germany
6 Albert-Ludwigs-University, Institute for Biochemistry and BIOSS, Albertstr. 21, 79104 Freiburg, Germany
2
Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegenerative diseases which makes a modulation of Sirt2
activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2inhibitors is lacking so far. Here, we present the first high-resolution structures of human Sirt2 in complex with highly
selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2-selectivity
are based on a ligand-induced structural rearrangement of the active site unveiling a cryptic binding pocket. Application
of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and
induces destabilization of the checkpoint protein BubR1, consistent with Sirt2-inhibition in vivo. Our structural insights
into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective
tools for sirtuin biology.
The studies have been supported by the Deutsche Forschungsgemeinschaft (Inhibitors: Ju295/8-1, Si868/6-1 structural work: SFB992 Medical
Epigenetics, Project Z02) and the EU (cellular studies: SEtTReND, Nr. 241865, FP7 Health). D.A.S. was supported by grants from NIH/NIA (R01
AG028730 and R01 AG019719), the Glenn Foundation for Medical Research, the United Mitochondrial Disease Foundation, The Juvenile Diabetes
foundation, and a gift from the Schulak family. B.J.N was supported by BIDMC/Harvard Translational Research in Aging Training Program (T32
AG023480). We also thank C. Kambach (University of Bayreuth, Department of Biochemistry, Universitätsstr. 30, 95445 Bayreuth, Germany) for
providing Sirt6.
References:
1. Finnin, M.S. et al.: Nat. Struct. Biol. 2001, 8: 621–625.
2. Trapp, J. et al.: J. Med. Chem. 2006, 49: 7307–7316.
3. Outeiro, T.F. et al.: Science 2007, 317: 516–519.
4. Schemies, J. et al.: Cancer Lett. 2009, 280: 222-232.
5. Suzuki, T. et al.: J. Med. Chem. 2012, 55: 5760–5773.
6. Moniot, S. et al.: J. Struct. Biol. 2013,182: 136–143.
7. Yamagata, K. et al.: Structure 2014, 22: 345–352.
8. North, B.J. et al.: EMBO J. 2014: in press.
85
EVIDENCE-BASED MEDICATION MANAGEMENT
Evaluation of medication management in community pharmacies
Waltering, I.; Koling, S.; Hempel, G.
Institute of Pharmaceutical and Medicinal Chemistry, Clinical Pharmacy, University of Muenster, Germany
Patients in community pharmacy – How to improve medication reviews
Medication therapy is a well-known risk. Medication errors, adverse drug events and medication discrepancies are
common, costly and preventable. A possibility to reduce the risk is a medication review conducted in community pharmacies [1-5]. With the Apo-AMTS-Concept a training-program was developed to teach pharmacist and pre-registration
students how to conduct such a review on an intermediate level (PCNE-classification)[6]. After implementation of the
program it was necessary to assess the ability of the participants to correctly and completely identify drug-related problems (DRPs).
This cross-sectional study with 76 patients from a convenience sample was performed within the Apo-AMTS-course.
During the Apo-AMTS course pharmacists and pre-registration students received training in medication reviews consisting of a 4h basic-course and 3-day advanced-course. At the end of the course each participant had to turn in 5 medication reviews conducted in their pharmacy. A checklist to document the detected problems and a documentation template
to record the interventions were provided for this purpose. The results noted in the checklist were reviewed by a clinical
pharmacist with experience in medication reviews and a physician. The number and types of DRPs found by the pharmacists/students were registered in an excel datasheet. Classification of the DRPs was deviated from the MAI Score[7].
The expert reviewers completed the different potential DRPs by reviewing all available information. The ATC-code was
used to assess what drugs caused the main problems and which drugs are responsible for the most overlooked DRPs.
The results showed that pharmacists and students detected interactions, side-effects, problems with time of intake and
adherence problems almost completely. Double medication, duration of intake and dosage intervals were the most
overlooked or not assessed problems. It could also be seen that many issues were connected with PRN (as needed)
medication and with drugs prescribed from the ATC-class “A”.
In conclusion, this study could show that with medication reviews in community pharmacies on an intermediate level,
after training, DRPs that could be detected in the pharmacy. Most of these problems could be solved together with
patients and pharmacists and improve medication and patient safety. DRPs like dosage and double-medication, where
more information and clinical judgment could be necessary or a discussion with the prescriber was necessary were more
often not detected. Here is more support and training necessary.
Because we did not compared results of medication reviews from untrained pharmacists, the correct effect of the training
could not be detected. Also further studies of the clinical importance of the detected and non-detected problems are
necessary.
References:
1. Joint Commission on Accreditation of Healthcare Organizations, U., Using medication reconcilitation to prevent errors. Sentinel Event Alert.,
2006, Jan 23(35): 1-4.
2. Gandhi, T.K. et al.: N Engl J Med, 2003, 348(16): 1556-1564.
3. Bryant, L.J. et al.: Int J Pharm Pract, 2011, 19(2): 94-105.
4. Lenaghan, E.; Holland, R.; and Brooks, A.: Age Ageing, 2007, 36(3): 292-297.
5. Sorensen, L. et al.: Br J Clin Pharmacol, 2004, 58(6): 648-664.
6. van Mil F, G.N., The PCNE guideline for medication review, in PCNE Working Symposium on Medication Review. 2011: Manchester.
7. Hanlon, J.T. and Schmader, K.E.: Drugs Aging, 2013, 30(11): 893-900.
86
Evidence-based medication management in psychiatric patients
Pauly, A.1; Wolf, C.1; Kornhuber, J.2; Friedland, K.2
1 Molecular
and Clinical Pharmacy, Department of Chemistry and Pharmacy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen,
Germany
2 Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
Low adherence and Drug-Related Problems (DRP) are important issues, which limit the clinical outcome in psychiatric
patients. To address these problems we designed a clinical trial, which was conducted in the psychiatric university
hospital in Erlangen. Patients aged >18 years, taking medications and who gave written consent were allocated to
control or intervention group depending on the date of their admission. Control patients (09/2012-03/2013) received
usual care whereas patients in the intervention group (05/2013-12/2013) benefited from a structured extended medication management program.
The medication management program consisted of two different components. Firstly, individualized patient counselling
by two pharmacists was conducted regarding the pathophysiology of the psychiatric disease, mechanism of action of the
psychoactive drugs, and information about the clinical relevance of side effects in two patient consultation during hospital
stay. Secondly, DRP were identified in both groups in the course of structured clinical reviews of patients’ charts at
admission, weekly during the hospital stay, at discharge as well as three months after discharge including a comprehensive patient interview, the assessment of laboratory parameters and adverse drug events by the two pharmacists. The
detected DRP were classified according to a validated classification system with domains to classify the problem, the
intervention and the outcome (1). Causing drugs were documented and the relevance of every DRP was estimated as
minor, moderate or high in concordance with criteria used in other pharmaceutical care studies in psychiatry (2). Besides
DRP, adherence was investigated during the patients hospital stay and for three months after discharge. Adherence was
measured by the self-report questionnaires “Medication Adherence Report Scale” (MARS) and “Drug Attitude Inventory”
(DAI) at admission, discharge and 3 months after discharge. The primary outcome measures were the total number of
DRP per patient and the differences of change in MARS and DAI. Results were adjusted for age, gender, comorbidities
and baseline values for MARS and DAI and additionally for length of stay and number of drugs at admission for DRP.
Depending on their time of admission 269 patients were allocated to receive either usual psychiatric care (control) or the
structured medication management program (intervention). 419 and 396 DRP were identified within the hospital stay
corresponding with a median of 3 (IQR = 1-5) and 2 (IQR = 1-4) DRP per patient in control and intervention group.
Differences were not statistically significant (p = 0.487). After the entire study period 303 DRP (median = 2 unsolved
DRP per patient, IQR = 1-3) remained unsolved in control patients in contrast to 50 (median = 0, IQR = 0) in intervention
group. The adjusted number of unsolved DRP in intervention group was 1.82 (95% CI: 1.52 – 2.14) less compared to
usual care. Regarding adherence, the mean MARS score of control patients increased during hospitalization from 22.23
(2.87) by 4.84% to 23.44 (2.34) at discharge. Three months after discharge, the MARS score decreased by 3.9% near to
his original level, 22.47 (2.99). In contrast, the mean MARS score improved in the intervention group from 22.02 (3.42) at
admission by 11.60% to 24.34 (1.61) at discharge. This value declined by 2.36% to 23.75 (2.08) at the follow-up. Taking
into account the improvement of control group, the adjusted change in the MARS score for intervention patients was
1.33 points (6.65%, 95% confidence interval 0.73 – 1.93, P < 0.001 ), significantly higher than in control patients. 53.4%
of intervention group patients in comparison to 31.1% of patients in the control group achieved the possible maximum
value of 25 points in the MARS indicating a perfect adherent behavior three months after discharge.
In conclusion, our study provides first evidence that a structured medication management for psychiatric patients may be
effective to reduce DRP and to improve patient adherence.
References:
1. Ganso, M. et al.: Krankenhauspharmazie 2009, 30(7): 349-362.
2. Campell, A.R. et al.: Am J Pharm Educ. 2011, 10;75(1): 8.
87
Evidence-based medication management in cancer patients
Wilmer, A.1; Tasar, A.2; Fleckenstein, K.3; Hack, C.3; Ruberg, K.2; Ko, Y.D.3; Jaehde, U.1
1 Institute
of Pharmacy, Clinical Pharmacy, University of Bonn, Germany
Pharmacy Marxen, Wesseling, Germany
3 Department of Internal Medicine, Johanniter Hospital, Bonn, Germany
2 Kronen
Many studies have shown that there is a strong need for specific interventions assuring medication safety, especially in
patients receiving complex drug treatments such as cancer patients. In order to establish risk-minimizing interventions, it
is crucial to generate evidence for their effectiveness in appropriately designed studies.
We implemented a multiprofessional best-practice model at the oncology outpatient ward of the Johanniter Hospital in
Bonn providing a structured and standardized medication management for cancer patients. It was the aim of this study to
provide first data on the effectiveness of this intervention to enhance patient safety.
The model was developed in a multiprofessional quality circle to define ‘best practice’. Care modules were developed for
medication review and interaction check (basic module), malnutrition, and for the management of four common adverse
events: nausea/emesis, mucositis, fatigue, and pain (supplementary modules). All modules include evidence-based
recommendations for supportive care, written patient information, and an algorithm illustrating the care process. They
can be applied individually for each patient according to the anticipated toxicity. After implementation, the feasibility of
the model was evaluated in a pilot study. Among others, the effectiveness was evaluated using the patient-reported
outcome (PRO) version of the CTCAE criteria [1].
The best-practice model was evaluated in a randomized two-arm interventional study. 51 outpatients with solid tumors
were randomized either to the control group or the intervention group with multiprofessional and modular care, and
monitored for a maximum of 5 cycle of chemotherapy. Primary endpoint of the study was the time to first occurrence of
severe symptoms (PRO-CTCAE grade 3 to 4). Secondary endpoints were health-related quality of life and patient
satisfaction with information received.
The results suggest that the best-practice model may delay the time to first occurrence of grade 3 to 4 symptoms.
However, the effect was not statistically significant due to the small sample size. It is remarkable that the delaying effect
was especially observed for symptoms for which the supplementary care modules were developed (mucositis, nausea
and vomiting, and fatigue). There was no difference between the two groups regarding the secondary endpoints healthrelated quality of life and patient satisfaction.
In conclusion, our study provides first evidence that a structured and standardized medication management for cancer
patients may be effective to enhance patient safety. However, a larger number of patients is needed in order to prove its
effectiveness.
References:
1. US Department of Health and Human Services. National Cancer Institute: Development of the Patient-Reported Outcomes Version of the
CTCAE. Available from: https://wiki.nci.nih.gov/x/cKul. Last access: 29 August 2014
88
PHARMAGRIPS: Structured pharmaceutical counseling in the self-medication of the common cold. A
randomised controlled study (RCT)
Laven, A.1; Schäfer, J.2; Läer, S.3
1,2,3 Institut für Klinische Pharmazie und Pharmakotherapie,
Heinrich-Heine-Universität Düsseldorf
Background: Many minor ailments are treated in Germany by self-medication. Most drugs dispensed by pharmacy staff
are those for the common cold, general pain and gastrointestinal disorders. Whilst pharmacists express their need for
further training in counseling on side effects, interactions and contraindications, they tend to receive feedback from
patients to the effect that the drugs used have not worked.
Methods: From July to October 2013 we carried out a prospective, single-blind, quasi-randomised controlled study on
the effect of training on structured pharmaceutical advice in self-medication of the common cold (PHARMAGRIPS
Study). Using a controlled, interventional study design we investigated whether it is possible to improve the pharmaceutical counseling in self-medication within a short time, by using an appropriate teaching method. The counseling should
be made in a systematic way and refer to evidence-based content in order to avoid incorrect advice. We enrolled 86
pharmacists and assigned them randomly into the study protocol. Of those, 56 completed the study as planned and were
analysed.
In this study, we reviewed the structure of the average pharmaceutical consultation and added evidence-based content
from the Cochrane Reviews on common cold. We then integrated this structured consultation in a methodical modern
training program consisting of e-learning and live classes which we evaluated scientifically. For this purpose, we conducted telephone interviews with the participating pharmacists by using standardized case report forms. The case report
forms contained the questions that the participants were supposed to ask. For every question asked, the participant
received a certain amount of points, 18 in total.
The training was stated to be successful at the primary endpoint when an improvement of at least 3.5 out of 18 points
was achieved on average. The secondary endpoints were related to various aspects of the interview process (medical
history, limits of self-medication, evidence-based drug selection and integration of customer input in the consultation
interaction).
Results: The training group improved in the primary endpoint by an average of 5,93 points (p < 0,001) and compared to
the control group significantly in all secondary endpoints, with one participant managing to achieve the full score.
The participants recognized the importance and practical relevance of the exercise in a short time and were able to
implement even integrate complex content in their consultations and to give the customer appropriate advice.
References:
1. Laven, A.; Läer, S.: Med Monatsschr Pharm. 2013, 36: 102-110.
2. Laven, A.; Schäfer, J.; Läer, S.: PHARMAGRIPS: Med Monatsschr Pharm. 2014, 37: 209-220.
89
OPTIMIZING ORAL DRUG PERFORMANCE
Of Springs and Parachutes – Improving Oral Bioavailability
Brewster, M.E.
Janssen Research and Development, Johnson & Johnson, Turnhoutseweg 30, Beerse 2340, BELGIUM
Contemporary drug pipelines are increasingly populated with difficult-to-formulate drug substances. The genesis of this
heightened complexity is rooted in three confluent trends including (1) the reliance of the drug discovery process on high
throughput screening, (2) growing issues with drug form and (3) the nature of current drug targets which are often
associated with structure-activity relationships (SAR) which deviate or disconnect from the chemical space associated
with good oral bioavailability and acceptable biopharmaceutical fitness. Taken in aggregate, this evolution in dosage
form development challenges has forced formulators to innovate with a variety of novel approaches promulgated in the
last few years. Enabling formulation strategies including the use of nanotechnology, lipid-based dosage forms giving rise
to self-micro or nano-emulsifying drug delivery systems (S (M, N) EDDS) and amorphous solid dispersions in which a
drug is rendered non-crystalline in a glassy matrix [1]. In many of these dosage forms, a postulated element critical to
their biopharmaceutical success is the ability of the formulation to generate a supersaturated solution in the gastric or
intestinal compartment [2]. This so-call “spring” effect (that is pushing concentrations of the drug, in excess of their
thermodynamic solubility, into the media) is then modified with a formulation component which hinders precipitation or
crystallization of the supersaturated, metastable solution (i.e., a parachute). In the case of an amorphous solid dispersion, the glassy dispersant may also serve the dual role of acting as a parachute. In both designing and producing
amorphous solid dispersions, appropriate assays are requires that can assess the effectiveness of an excipient in
generating a stable dispersion, of generating a supersaturated solution in an appropriate media and the stability profile of
the formed metastable system [3]. Such in vitro approaches include methodologies designed around solvent-shift
approaches as well as non-crystalline solid approaches where supersaturation is induced, respectively, by adding a drug
in a water-miscible solvent to an aqueous matrix or by adding the amorphous phase directly to the aqueous system.
These approaches, while useful, can underestimate supersaturation stability in vivo [4]. This may be due to ignoring
drug absorption which can impact the supersaturation ratio. Dissolution apparatus with an absorption compartment can
be useful from that standpoint [5]. In vivo assessments of supersaturation rely on drug formulation administration followed by gastric and intestinal sampling through the use of specialized catheters with simultaneous assessments of
bioavailability and pharmacokinetic parameters. Comparative assessments of in vivo and in vitro tools can be helpful in
optimizing IVIVC and dosage form design. A number of amorphous solid dispersions have reached the market. These
include dosage forms in which the drug is rendered amorphous and mixed with various excipients/carriers through the
use of a number of processing techniques such as melt extrusion, melt blending, bead coating, solvent processing,
spray-drying and antisolvent precipitation (i.e., the microprecipitated bulk powder approach). In all cases, post-process
manipulation is needed to generate the final dosage form whether that is a tablet or capsule. Based on the traditional
uncertainty of translate-ability of animal models, dosage form testing in man is often completed to ensure that the best
concept is developed and forwarded to the market [2]. In these studies, indirect and increasingly, direct methods are
being applied to better understand the applied supersaturating drug delivery system and its mechanism for augmenting
oral bioavailability [3]. As was suggested, presenting the drug in a physically stable amorphous format meets only part
of the challenges associated in the development of these systems. An appropriate processing approach also needs to
be identified, confirmed and implemented and biorelevant approaches which translate to man are a sine qua non for
successful drug development.
References:
1. Branchu, S. et al.: Eur. J. Pharm. Sci. 2007, 22: 128-139.
2. Brouwers, J. et al.: J. Pharm. Sci. 2009, 98: 2549-2572.
3. Bevernage, J. et al.: Int. J. Pharm. 2013, 453: 25-35.
4. Psachoulias, D. et al.: Pharm. Res. 2011, 28: 3145-3158.
5. Bevernage, J. et al.: Eur. J. Pharm. Biopharm. 2012, 82: 424-428.
90
Assessing the gastrointestinal „Spring“ effect – the media
Leigh, M.
91
Will the parachute crash? – transfer models for assessing performance of optimized formulations
Kostewicz, E.S.; Ruff, A.
Institute for Pharmaceutical Technology, Goethe University, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany
Given the greater prevalence of poorly-soluble compounds in contemporary drug discovery pipelines, formulation approaches that enhance supersaturation or minimize gastrointestinal (GI) precipitation within the GI environment are
becoming more widely adopted as a strategy for enhancing bioavailability. As the options for delivering poorly soluble
drugs have become more innovative, predictive and reliable in vitro tools for appropriate evaluation of formulation behaviour are needed.
Due to the complexity of the supersaturation and precipitation process occurring along the GI lumen, there are a multitude of factors that need to be considered when evaluating these parameters in vitro. For supersaturation and precipitation, luminal concentrations may be influenced not only by gastric emptying, permeability, ionization characteristics of the
API, solubilisation by bile acid micelles, the dissolution characteristics of the formulation used, but also by the sharp pH
change observed between stomach and intestine, prevailing composition and volume of the luminal fluids, dilution of the
formulation with GI luminal fluids, digestion of solubilizing excipients and the nature of excipients used in the formulation,
to name just a few (1).
The critical steps that need to be considered in an in vitro model therefore include the assessment of the formulations’
rapidly dissolving supersaturation “spring” with a precipitation inhibition or retarding “parachute” that can occur in vivo
along the GI tract. The transfer model is an in vitro method that can be used for assessing the performance of such
optimized formulations. This model utilizes an USP II dissolution apparatus containing two compartments simulating the
stomach and intestine, respectively. To simulate the transfer of drug out of the stomach and into the intestine, the drug in
the simulated gastric fluid compartment is pumped into a simulated intestinal fluid compartment in which supersaturation
and/or precipitation is evaluated by concentration versus time measurements. In this model, the influence of transfer
rate, bile salt concentration, pH and compartmental volumes on the supersaturation and precipitation behaviour of the
formulation can be evaluated.
As part of this presentation, I will provide an overview of the current status of the transfer model, present a few examples
utilizing this method for evaluating the supersaturation and precipitation behaviour of optimized formulations and discuss
the in vivo relevance of this tool.
Reference:
1. Kostewicz, E.S. et al.: Eur. J. Pharm. Sci. 2014, 57: 342-366.
92
Connecting oral formulation performance to therapeutic effect
Cristofoletti, R.
93
MULTITARGET DRUGS
An introduction to polypharmacology in drug discovery
Peters, J.-U.
Roche Pharmaceutical Research and Early Development, Discovery Chemistry, Innovation Center Basel, Building 92 / 3.64C, CH-4070 Basel,
Switzerland
Polypharmacology has been receiving an ever-increasing interest over the last 10 years, and has been proposed as the
“next paradigm in drug discovery”.1 This is mainly due to the advent of modern polypharmacological drugs,2 but also to
the recognition of unintended polypharmacology as a source of adverse drug effects.3 This talk gives an introduction to
polypharmacology, its opportunities and risks, and its role in modern and historic drug discovery. Topics include: (1) the
prevalence of polypharmacology in drug-like compounds; (2) the significance of polypharmacology for safety; (3) how to
recognize polypharmacological compounds early; (4) why polypharmacological drugs may be more efficacious; (5)
opportunities of polypharmacology: historic and modern examples; (6) strategies for the discovery of polypharmacological drugs.
References:
1. Hopkins, A.L.: Nat. Chem. Biol. 2008, 4(11): 682–690.
2. Peters, J.-U.: J. Med. Chem. 2013, 56(22): 8955–8971.
3. Peters, J.-U. et al.: Drug Discovery Today 2012, 17(7–8): 325–335.
94
Multifactorial activity of the naphthoquinone shikonin against cancer cells
Efferth, T.
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz;
E-mail: [email protected]
Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of
currently used therapeutics, new candidate compounds are required for improvement of therapy success. In a screen of
40 phytochemicals derived from medicinal plants used in Japanese Kampo medicine, shikonin was the most cytotoxic
compound [1]. This is a naphthoquinone derived from Lithospermum erythrorhizon, which is used in traditional Asian
medicines for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of
shikonin.
Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle,
mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent
fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution
using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this
accumulation was associated with a shikonin-dependent deregulation of cellular Ca2+ and ROS levels. This deregulation
led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis [2].
Next, we included micro-RNA microarrays and stable-isotope dimethyl labeling for quantitative proteomics. The integration of bioinformatics and the three "-omics" assays showed that the PI3K-Akt-mTOR pathway was affected by shikonin.
Deregulations of this pathway are frequently associated with cancerogenesis, especially in a wide range of hematological malignancies. The effect on the PI3K-Akt-mTOR axis was validated by demonstrating a decreased phosphorylation
of Akt and a direct inhibition of the IGF1R kinase activity after shikonin treatment. Our results indicate that inhibiting the
IGF1R-Akt-mTOR signaling cascade is a new cellular mechanism of shikonin strengthening its potential for the treatment
of hematological malignancies [3].
References:
1. Efferth, T. et al.: Cancer Genomics Proteomics 2007, 4: 81-91.
2. Wiench, B. et al.: eCAM 2012: 726025.
3. Wiench, B. et al.: eCAM 2013: 818709.
95
Polypharmacology: In silico recognition vs. rational design
Proschak, E.
Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue Str. 9, D-60438,Frankfurt a.M., Germany
The „one drug – one target – one disease“ paradigm in drug discovery has been reconsidered during the last decade.
This paradigm change was mainly caused by high attrition rates in drug approvals due to toxicity and lack of efficacy. On
top of that, the results of post-genomic and network biology showed that putative drug targets rarely act within isolated
systems but rather as a part of a highly connected network.[1] Furthermore, the efficacy of several approved drugs has
been traced back to the interaction with multiple targets.[2] Computational techniques play an important role in prediction
and recognition of novel targets for approved drugs. We will discuss two machine learning approaches – self organizing
maps and inverse distance weighting – for polypharmacological profiling of bioactive compounds, exemplified by two
prospective studies [3,4].
While the recognition of occasional polypharmacological behavior is an established task, the rational design of multitarget ligands remains challenging. Dual or multi-target ligands have several advantages compared with selective compounds, including improved efficacy and more simple pharmacokinetic and pharmacodynamic properties in comparison
to the combination of several drugs [5]. In this context we present three methodic approaches to design dual inhibitors of
5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). In our first study we connected previously pubished 5-LO
and sEH pharmacophores, an imidazo-[1,2a]-pyridine core with an aryl urea moiety via flexible propyl linker [6]. The
second study contains the discovery of a benzimidazole-based dual 5-LO/sEH inhibitor by means of in silico screening
[7]. The strategy of the virtual screening protocol was an exhaustive pairwise evaluation of pharmacophore models for
both targets to obtain a dual-target pharmacophore model. Our last study deals with the development of a fragment
based strategy for dual-target drug discovery. Here, we applied a modified self-organizing map algorithm for in silico
recognition of molecular fragments binding both targets. The predicted properties were confirmed by complementary
screening techniques: STD-NMR and enzyme assay. The enlargement of the fragment hit led to submicromolar dual
target inhibitor of sEH and 5-LO.[8]
References:
1. Jeong, H. et al.: Nature 2001, 411: 41-42.
2. Zimmermann, G.R.; Lehár, J.; Keith, C.T. : Drug. Discov. Today 2007, 12 : 34-42.
3. Paulke, A. et al.: J. Ethnopharmacol. 2013, 148: 492-497.
4. Steri, R.: Biochem Pharmacol. 2012, 83: 1674-1681.
5. Morphy, R.; Rankovic, Z.: J. Med. Chem. 2005, 48: 6523-6543.
6. Meirer, K. et al.: J. Med. Chem. 2013, 56: 1777-1781.
7. Moser, D. et al.: ACS Med. Chem. Lett. 2012, 3: 155-158.
8. Achenbach, J. et al.: ACS Med Chem Lett 2013, : 1169-1172.
96
Study of the anxiolytic actions of Valeriana officinalis L., Melissa officinalis L., Passiflora incarnata L.
and their combination STW 32 in experimental models of anxiety
Okpanyi, S.N.1; Kelber, O.1, Weischer, M.-L.2
1 Steigerwald
2 Institute
Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany
for Pharmacology and Toxicology, University of Münster, Germany
Several well established psychotropic herbal medicinal preparations, such as of Melissae folium, Passiflorae herba and
Valerianae radix, are recognised and recommended officially both in the German Commission E and in the ESCOP
Monographs for the treatment of tenseness, restlessness and irritability, with difficulty in falling asleep. Being that these
phytomedicines do not negatively influence vigilance and reaction time, the primary mode of their calmative action is
more likely to be anxiolytic and not sedation like benzodiazepines and barbituric acid derivatives.
The anxiolytic actions of the three hydroethanolic extracts and their combination were investigated in elevated plus maze
(EPM) and social interaction of the mouse. Influence on exploratory behaviour (vigilance, rearing and locomotory activity)
was tested. Diazepam 1.0 mg/kg b.w. was used as a standard anxiolytic agent.
Significant results of the EPM test were as follows: Diazepam increased no. of entry 2  0.4 (control) to 9  3.7 (p 
0.01), duration of stay 13  3.1 to 49  14.3 (p 0.05). Passiflora (P) 350 mg/kg b.w. increased no. of entry 1.8  0.8
(control) to 5.0  0.6 (p  0.01), duration of stay 25  7.6 (control) to 91  19.3 (p  0.01). Valeriana (V) 1040 mg/kg
b.w. increased entry 2  0.6 (control) to 4  1.2 (p  0.05), duration of stay 20  7.6 (control) to 57.7 (p  0.01). The
effect of the combination STW 32 (P 40 %, V 20 %, M 40 % of fluid extract) was very pronounced at a low dose: 60
mg/kg b.w. increased entry from 1.5  0.4 to 3.3  0.5 (p  0.05), duration from 11  4.8 to 29  6.0 (p  0.05). The
higher doses 120 mg and 240 mg/kg b.w. also significantly increased all effects.
The test plant extracts and their combination did significantly increase social interactions to as well as vigilance and
locomotory activity. A clear synergistic action was manifest in the combination.
These results bear evidence that the calmative and sleep inducing effects of the herbal extracts and their combination
STW 32 are due to an anxiolytic effect.
97
Motility modulation beyond MCP: Mechanisms of action of a clinically proven herbal medicinal product, STW 5, in functional GI diseases
Kelber, O.1; Hoser, S.2; Abdel-Aziz, H.1; Okpanyi, S.N.1; Nieber, K.2
1 Scientific
Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany
Institute, Leipzig University, Leipzig, Germany
2 Pharmaceutical
INTRODUCTION: Motility-modulating drugs have been an important therapeutic approach in functional GI diseases. This
approach has been questioned by the withdrawal of the prokinetic drug cisapride from the market due to cardiac side
effects and now again by the referral for metoclopramide (MCP) due to neurological side effects leading to the omission
of functional dyspepsia and gastrooesophageal reflux disease from its therapeutic indications.
Therefore it is of increasing importance to identify other treatment options with proven clinical efficacy but a more favourable safety profile. As has been recently shown by a clinical review publication (1), an herbal medicinal product, STW 5,
is a safe and therapeutically effective treatment option in these indications, as has been shown by randomized controlled
clinical trials in functional dyspepsia (FD) as well as in irritable bowel syndrome (IBS).
AIMS & METHODS: For identifying the mechanisms of action underlying its clinical effect, a systematic database search
regarding its effect on GI motility was conducted in accordance to the PRISMA statement and checked for completeness
by means of hand searching and cross referencing.
RESULTS: A large number of publications on STW 5 [2] and on the components of the product were identified. Already
the first mechanistic studies on the combination [2, 3] suggested a dual mechanism of action on motility, with a spasmolytic effect in acetylcholine induced contractions and a tonicising effect in the relaxed state. This has been confirmed
[4, 5] in human isolated intestinal segments [6] and in inflamed intestinal tissue in vitro and in vivo [7-10]. In the stomach,
a region specific action was described in vitro, based on an inhibition of Ca influx via SOC channels in the gastric fundus
and on a stimulation of Ca influx via L type Ca channels in the antrum [11]. This region specific action has been confirmed in a human study in vivo [12]. In the lower esophageal sphincter, a tonicising action mediated by L type Ca
channels has been shown in vitro [6]. The components of the herbal components have been shown to act synergistically
by pharmacological studies [13].
CONCLUSION: The in vitro-, in vivo- and human studies showed spasmolytic as well as tonicising-prokinetic effects
possibly relevant for the well documented clinical effect of STW 5 in functional GI diseases, so supporting the use of this
medicine in these diseases, which previously have been treated with MCP or cisapride.
References:
1. Ottillinger et al.: WMW 2013, 163:65.
2. Brierley, Kelber: Curr Opin Pharmacol, 2011, 11: 604.
3. Okpanyi et al.: Acta Hort. 1993, 332: 227.
4. Ammon et al.: Phytomed 2006, 13: SV: 67.
5. Heinle et al.: Phytomed 2006, 13: SV: 75.
6. Kelm et al.: ZPT 2013, 34(S1): S31.
7. Schemann et al.: ZGastroenterol 2008, 46: 1039.
8. Michael et al.: Phytomed 2009, 16: 161.
9. Sibaev et al.: ZPT 2013, 34(S1): S31.
10. Wadie et al.: Int J Colorectal Dis 2012, 27: 1445.
11. Hohenester et al.: Neurog Motil 2004, 16: 765.
12. Pilichiewicz et al.: Am J. Gastroenterol 2007, 102: 1.
13. Hoser, S. et al.: Planta Med 2013, 79: 1258.
98
NON-CANONICAL GPCR-SIGNALING
Dynamic ligand binding
Bock, A.
99
Voltage-dependent GPCR-activation
Rinne, A.
100
Mechanosensitivity of histamine H1 receptor
Mederos y Schnitzler, M.
101
Signaling in malaria parasites: A non- canonical G-protein from plasmodium falciparum
Kaiser, A.*; Langer, B.; Kersting, D.; Krüger, M.
Institute for Pharmacogenetics, University of Duisburg-Essen, Hufelandstrasse 55, 45177 Essen, Germany
During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts like
the blood stream, the human liver and the midgut of the mosquito. Key cellular mechanisms involving cAMP or cGMP
dependent signal transduction chains [1] are assumed to act at these interfaces. Previous findings showed that the
parasite uses the G-protein from the human host for invasion [2]. We here describe the first cloning and expression of a
guanine-nucleotide-binding protein (G-protein) from Plasmodium.
The protein reveals an open reading frame of 2733 bp encoding a protein of 911 amino acids and has a theoretical pI of
8.68 and a molecular weight of 108.57 kDa. Transcript formation and expression are significantly increased in the late
developmental stages during schizont and gametocyte formation suggesting its role in stage conversion and transmission. Most notably, the G-protein has GTP binding capacity and Gtpase activity due to an EngA domain which is also
present in small Ras-like GTpases in a variety of Bacillus species and Mycobacteria. By contrast, P. falciparum G-protein
is divergent from any human alpha-subunit [3]. The G-protein was expressed in E. coli as a histidine-tagged fusion
protein with a short half life of 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and separation by Blue Native page gel electrophoresis. Binding of a fluorescent GTP analogue BODIPY®
FL guanosine 5’O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan [4] stimulated GTP binding
in the presence of Mg2+. The determined GTpase activity of the human paralogue was 50% higher than the activity of the
parasitic enzyme.
In light of these significant results a non-canonical cGMP-regulated, rudimentary signaling pathway seems to be present
in Plasmodium with a functional, non-heterotrimeric G-protein. A current research for the more prevalent receptor will
delineate this pathway with respect to transmission and relevance to antimalarial chemotherapy.
References:
1. Baker, D.A.: Cell Microbiol. 2011, 13:331-339.
2. Murphy, S.C et al.:PLoS Med 2006, 3: e528.
3. Kimple, A.J. et al.: Pharmacol Rev.2011, 63:728-749.
4. Higashijima, T.; Burnier, J.; Ross, E.M.: J Biol Chem, 24: 14176-14186.
102
BIOPHARMACEUTICALS/BIOTECHNOLOGY
Major trends and challenges in biotherapeutic product development: "polysorbate degradation" and
"drug-device combination product development"
Adler, M.
103
Finding the right candidate – integrated lead ID of next-generation molecules
Ruberg, E.-M.
104
Non‐immunogenic messenger RNA therapeutics
Rudolph, C.
ethris GmbH, Lochhamer Str. 11, 82152 Martinsried
Replication-deficient viruses have been used successfully in the field of gene therapy because of their high transfection
efficiency. However, the risk of insertional mutagenesis and induction of undesired immune responses remain critical
obstacles for their safe medical application. On the other hand, nonviral vectors have been intensively investigated for
plasmid DNA (pDNA) delivery as a safer alternative, although their gene transfer efficiency is still many folds lower than
for viral vectors, which has been predominately attributed to the insufficient transport of pDNA into the nucleus. Instead
of pDNA, messenger RNA (mRNA) has recently emerged as an attractive and promising alternative in the field of
nonviral gene delivery1. This strategy includes several advantages compared to the use of pDNA: i) the nuclear membrane, which is a major obstacle for pDNA, can be avoided because mRNA exerts its function in the cytoplasm; ii) the
risk of insertional mutagenesis can be excluded; iii) the determination and use of an efficient promoter is omitted; iv)
repeated application is possible; v) mRNA is also effective in non-dividing cells, and vi) vector-induced immunogenicity
may be avoidable. In particular, immunogenicity concerns have been successfully solved by inclusion of chemically
modified nucleotides in the mRNA molecule during in vitro transcription. In our approach, replacement of 25% of uridine
and cytidine by 2-thiouridin and 5-methylcytidin, respectively, largely reduced mRNA binding to sensors of the innate
immune system, including Toll-like receptors and RIG-I, which largely reduced mRNA immunogenicity in vivo after
intravenous and intrapulmonary application2. According to these characteristics, we termed these chemically modified
mRNA stabilized non-immunogenic messenger (SNIM®) RNA which are now commercialized by the company ethris
GmbH, for development of “Transcript therapies” for the treatment of various inherited diseases and application in
regenerative medicine.
The potential range of programs to which the technology can be applied is very broad spanning metabolic or hereditary
monogenetic disorders to regenerative medicine. We have successfully developed technologies for the pulmonary
delivery of SNIM® RNA, thus demonstrating SNIM® RNA products can be delivered conveniently and efficiently by a
number of routes that will support patient compliance and quality of life.
References:
1. Yamamoto, A. et al.: Eur J Pharm Biopharm. 2009, 71(3): 484-489.
2. Kormann, M.S. et al.: Nat Biotechnol. 2011, 29(2): 154-157.
105
Divergent Pathways for the Biosynthesis of Merochlorins, Cyclic Meroterpenoid Antibiotics from a
Marine Streptomycete
Kaysser, L.1,2; Bernhardt, P.2; Nam, S.-J.2; Teufel, R.2; Diethelm, S.2; Loesgen, S.2; Fenical, W.2; Moore, B.S.2,3
1 German
Centre for Infection Research (DZIF) at the Department of Pharmaceutical Biology, University of Tuebingen, Auf der Morgenstelle 8,
72076 Tuebingen, Germany. Email: [email protected]
2 Scripps Institution of Oceanography and
3 Skaggs School of Pharmacy, University of California San Diego, 9500 Gilman Drive, La Jolla 92037, CA, USA
Meroterpenoids are mixed polyketide-terpenoid natural products with a broad range of biological activities. Four new
meroterpenoid antibiotics, merochlorins A–D, have been isolated from the marine bacterium Streptomyces sp. strain
CNH-189, which possess novel chemical skeletons unrelated to known bacterial agents.[1,2] A distinctively cyclized
sesquilavandulyl side chain in the merochlorins A, B and C indicated a sophisticated biosynthetic machinery for the
formation, transfer and the conversion of this moiety. Draft genome sequencing, mutagenesis and heterologous expression provided the merochlorin biosynthetic gene cluster, encoding two rare bacterial vanadium-dependent haloperoxidases (VHPO).[2] Genetic engineering and in vitro biochemistry gave detailed insights into assembly of the merochlorins
including the divergence of the biosynthetic pathways and the complex cyclization mechanisms.[2-5]
References:
1. Sakoulas, G. et al..: PLoS ONE 2012, 7: e29439.
2. Kaysser L. et al.: J. Am. Chem. Soc. 2012, 134(29): 11988–11991.
3. Teufel, R. et al.: Angew. Chem. Int. Ed. 2014 (submitted).
4. Diethelm, S. et al.: Angew. Chem. Int. Ed. 2014 (submitted).
5. Kaysser, L. et al.: (in preparation).
106
Combination strategies for targeting the oncogenic Pim1 kinase
Grünweller, A.1; Lange-Grünweller, K.1; Weißer, A.1; Weirauch, U.2; Aigner, A.2; Hartmann, R.K.1
1 Institut
für Pharmazeutische Chemie, Pharmazie, Philipps-Universität Marburg, 35032 Marburg, Germany
für Pharmakologie und Toxikologie, Klinische Pharmakologie, Universität Leipzig, 04107 Leipzig, Germany
2 Rudolf-Boehm-Institut
Pim1, a constitutively active serine/threonine kinase, is overexpressed in various aggressive solid tumors and lymphomas. Pim1 is an effective inhibitor of apoptosis and an activator of cell proliferation, and its overexpression correlates
with a bad prognosis for patients. Knockout of the Pim kinase family (Pim1, 2 and 3) resulted in fertile mice with only mild
phenotypic changes, thus Pim kinases seem to have no essential function in healthy cells and adult tissues. Interestingly, the 3´-untranslated region (3´-UTR) of the Pim1 mRNA has several conserved putative binding sites for microRNAs
(miRNAs). We have recently shown that Pim1 is a target of miRNA regulation by miR-33a and miR-15b [1-3]. Furthermore, Pim1 is an epigenetic regulator by phosphorylating histone H3 at serine 10 to activate transcription in a c-Mycdependent manner and by phosphorylation of heterochromatin protein HP1γ.
We have applied several RNA-based strategies to explore Pim1 as a new tumor target in mouse xenografts of colon
carcinoma and glioblastoma. Delivery of miR-33a or Pim1-specific siRNAs into tumors was achieved by forming nanoplexes of RNA with branched low molecular weight polyethylenimine (PEI). This approach was also used to explore a
new antisense strategy in vivo which is called U1-Interference (U1i) [4]. Our RNA-based strategies against Pim1 reduced
tumor growth substantially without changing liver enzyme activities or inducing unwanted immune responses.
Efficient and successful antitumor strategies require combinatorial approaches to reduce the risk of chemoresistance
and to lower unwanted side effects. The development of well-tolerated new combination strategies to target oncogenes
hold great promise for the clinic. We are currently exploring combinations of RNA-Interference, statins, natural compounds and 5-FU to lower the respective doses of these compounds for Pim1 inhibition. Statins, which block the mevalonate pathway by inhibition of HMG-CoA-reductase, have pleiotropic antitumor effects at low micromolar concentrations.
However, strategies combining statins with siRNA or other Pim1-inhibitors improve statin-dependent effects on Pim1,
thus lowering the statin concentrations for Pim1 inhibition towards the sub-micromolar range. We will now further evaluate statin effects on Pim1 in mouse xenografts of colon carcinoma and glioblastoma.
References:
1. Thomas, M. et al.: Oncogene 2012, 31(7): 918-928.
2. Ibrahim, A.F. et al.: Cancer Research 2011, 71(15): 5214-5224.
3. Weirauch, U.: Neoplasia 2013, 15(7): 783-794.
4. Weirauch, U. et al.: Nucleic Acids Therapeutics 2013, 23(4): 264-272.
107
SHORT LECTURES
108
Design, synthesis and biological evaluation of a stabilized RvE2 analog
SL.01
Fukuda, H.; Takakura, Y.; Ishimura, K.; Hirao, T.; Muromoto, R.; Matsuda, T.; Arisawa, M.; Shuto, S.
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Resolvins are lipidic chemical mediators that are metabolites of -3 fatty acids EPA or DHA. Because of the highly
potent anti-inflammatory activities, they can be leads to develop novel anti-inflammatory drugs. However, resolvins have
some problems for their use as clinical drugs that they are unstable to oxygen and to light due to the polyunsaturated
bonds, and also that they are gained only a trace from mammals.
We addressed a challenge to develop stabilized derivatives of resolvins. Therefore, we expected that resolvins can be
stabilized without losing the biological activity by replacing the Z-olefin of resolvin structure with a chiral cis-cyclopropane
to reduce number of the unsaturated bonds.
Firstly, we achieved a total synthesis of resolvin E2 (RvE2) in short steps, and then succeeded to synthesize a cyclopropane analog of RvE2 (CP-RvE2), in which the C11-C12 Z-olefin moiety of RvE2 was replaced with a chiral ciscyclopropane structrure.
The stability and anti-inflammatory activity of the analog will be reported.
109
Biosynthetic studies on fungal meroterpenoids and their fascinating chemistry
SL.02
Matsuda, Y.; Akakawa, T. ; Mori, T. ; Wakimoto, T. ; Abe, I.
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan
Fungal natural products, represented by penicillin, lovastatin, and ciclosporin, have been a rich source of biologically
active substances, and are still promising leads for future drug discovery and development. Fungal metabolites classified
as “meroterpenoids” exhibits a wide range of biological activities; mycophenolic acid is used as an immunosuppressant
agent, and pyripyropenes and ascofuranone have been intensively studied to develop as cholesterol-lowering and antitrypanosomal drugs, respectively.
Besides their biological activities, diverse chemical structures are also important characteristics of fungal meroterpenoids, and those derived from 3,5-dimethylorsellinic acid (DMOA) include an especially large variety of molecules often
with complex carbon skeletons,1 as exemplified by the spiro-lactone system of austinol and the unique bridged-ring of
anditomin. Understanding the molecular basis for their biosynthetic systems would not only facilitate the discovery of
enzymes catalyzing an intriguing reaction but also provide genetic building blocks that can be used to obtain novel
attracting compounds by metabolic engineering or synthetic biology approaches.
We established the biosynthetic pathways of austinol and anditomin by reconstituting their pathways using heterologous
expression systems in Aspergillus oryzae and by in vitro assays with selected enzymes.2 In the course of our study, we
identified two unique enzymes that catalyze intriguing reactions in both the austinol and anditomin pathways. In the
austinol biosynthesis, AusE, which is a Fe2+ and α-ketoglutarate-dependent dioxygenase, was found to convert preaustinoid A1 into preaustinoid A3 by oxidative rearrangement to generate spiro-lactone structure of austinol. The anditomin
also involves a dioxygenase AndA with a distinct activity. AndA utilizes preandiloid B as its substrate and yields the
bicyclo[2.2.2]octane system of andiconin with an unprecedented structural reconstruction.
In summary, our study provides insight into how structurally diverse metabolites are generated by enzymes with a similar
activity. Investigation of the biosyntheses of other related natural products as well as the construction of unnatural
metabolisms by mixing the obtained genetic components will be performed in our future studies.
References:
1. Geris, R.; Simpson, T.: Nat. Prod. Rep. 2009, 26(8): 1063-1094.
2. Matsuda, Y. et al.: J. Am. Chem. Soc. 2013, 135(30): 10962-10965.
110
SL.03
Destruxin E, a Potent Negative Regulator of Osteoclast Morphology: Solid-Phase
Library Synthesis and Biological Evaluation
Yoshida, M.1; Ishida, Y.1; Sato, H.1,2; Murase, H.3; Nakagawa, H.3; Doi, T.1
1 Graduate
School of Pharmaceutical Sciences, Tohoku Uniniversity, 6-3 Aza-Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
Tanabe Pharm Corporation, 2-2-50 Kawagishi, Toda-shi, Saitama 335-8505, Japan
3 Department of Applied Biological Chemistry, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan
2 Mitsubishi
Destruxin E (1), isolated from Metarhizium anisopliae, is a 19-membered cyclodepsipeptide consisting of five amino
acids (-alanine, L-NMe-alanine, L-NMe-valine, L-isoleucine and L-proline) and -hydroxy acid derivative [1]. The structural features of destruxin E (1) are a 19-membered macrolactone and a terminal epoxide in the side chain, and the
stereochemistry of the epoxide has not been elucidated until our total synthesis [2]. To date, several kinds of destruxin
derivatives have been isolated, destruxin E (1) exhibits the most potent V-ATPase inhibitory activity (IC50 0.4 M) among
the destruxin family [3]. Because of the unique structural features and biological activity, we became interested in the
total synthesis of 1 and its derivatives. We have recently achieved the solid-phase total synthesis and structure determination of 1, and we also found that destruxin E (1) exhibits 10-fold stronger V-ATPase inhivitory activity than the stereoisomer of the epoxide epi-1 [2].
DIC
MeAla
N
Fmoc
O
PyBroP
b-Ala
O
O
1) SPPS
2) Cleavage
O
NFmoc
HO
O
O
PyBroP
NH HO
Ile Fmoc
TBSO
O
N
PyBroP
O
*
N
O
O
O
HA-Pro-OH 2
H
N
N
O
OH
O
MNBA
O
*
O
N
DMAPO
N
N
H
O
OH
H
N
N
OH
MeVal
O
Polymer-support
OH H2N
Macrolactonization
3
O
O
O
O
O
O
O
O
*
O
3) K2CO3
N
N
H
4
H
N
N
1) H3O+
2) TsCl
N
O
O
N
H
O
O
O
O
O
O
*
N
1g
Destruxin E (1) (1gS)
epi-Destruxin E epi-(1) (1gR)
Destruxin E (1) exhibits 10-fold V-ATPase inhibitory activity than epi-destruxin E epi-(1).
Scheme 1. Solid-Phase Total Synthesis of Destruxin E (1)
Recently, it has also been reported that destruxin E (1) intriguingly induces morphological changes in osteoclasts-like
multi nuclear cells (OCLs) at low concentration without affecting the V-ATPase activity of the OCLs [4]. Therefore,
destruxin E (1) would be a good candidate for a new type of antiresortive agent. In order to elucidate the mode of action
in OCLs, we next attempted the library synthesis of destruxin E (1) and elucidation of the structure-activity relationships.
We designed 18 derivatives based on the structure of 1, and the library synthesis was performed by solid-phase synthesis using split & pool method. 19-membered macrolactone was formed by MNBA-mediated macrolactonization [5], and
formation of the epoxide in parallel furnished the desired destruxin derivatives. Details of the synthesis and biological
evaluation of destruxin derivatives will be presented.
Acknowledgement:
This work was supported by a Grant-in-Aid for Young Scinentists (B) (M.Y.) from Japan Society for the Promotion of Science and was also partially
supported by Protein Research Foundation (M.Y.).
References:
1. Päis, M. et. al.: Phytochemistry 1981, 20(4): 715–723.
2. Yoshida, M. et. al.: Org. Lett. 2010, 12(17): 3792–3795.
3. Vázquez, M.J. et. al.: Chem. Biodiversity, 2005, 2(1): 123–130.
4. Nakagawa, H. et. al.: Bone 2003, 33(3): 443–455.
5. Shiina, I. et. al.: Chem. –Eur. J. 2005, 11(22): 6601–6608.
111
SL.04
Design and synthesis of NF-κB inhibitors carring epoxyquinol moiety
Saitoh, T.1,2; Ohta, E.2; Umezawa, K.3; Nishiyama, S.2
1 International
Institute for Integrative Sleep Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba 305-8577, JAPAN
of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, JAPAN
3 Department of Molecular Target Medicine Screening, School of Medicine, Aichi Medical University, Nagakute, Aichi 408-1195, JAPAN
2 Department
The NF-κB signaling pathway plays a central role not only in inflammation but also in the development of cancer. Therefore, NF-κB inhibitors are expected to be novel candidates as chemotherapeutic agents for inflammatory and cancer
diseases, as well as bioprobes for the characterization of intracellular biological responses and cell function. 1 Over the
past decade, a number of structurally diverse small molecules that block the NF-κB signaling pathway, have been
identified. Especially, the epoxyquinol class NF-κB inhibitors, such as DHMEQ (1),2 cycloepoxydon (2),3 and panepoxydon (3),4 were found to exhibit remarkable inhibitory activity against NF-κB activation (Fig. 1). Despite their structural
similarity (Fig. 1), all of them showed completely different modes of action in the inhibition of NF-κB activation. Whereas
DHMEQ (1) exhibits inhibitory activity by covalently binding to the NF-κB components, panepoxydon (2) and cycloepoxydon (3) show inhibition by interfering with the degradation of IκB-α and activation of IκB kinase (IKK).3, 4 In this
study, we synthesized several epoxyquinol and non-epoxyquinol derivatives based on the structure of DHMEQ (1) to
examine its structure–activity relationship.
O
O
OH
Receptor
O
OH
OH
TAK1
TAB1TAB2
O
NH
O
O
IKKg
OH
O
HO
Phosphorylation
Panepoxydon
Cycloepoxydon
Phosphorylation
IKKa IKKb
IkBa
O
p50 p65
Degradation
HO
p50 p65
DHMEQ (1)
Panepoxydon (2)
Inhibition of DNA binding of NF-kB
Cycloepoxydon (3)
DNA binding
p50 p65
Inhibition of phosphorylation of IkB
DHMEQ
Figure 1
We synthesized epoxyquinol and salicylic amide analogs carring the partial structure of DHMEQ (1) and evaluated these
biological activities (Fig. 2).5, 6 Although some epoxyquinol analogs, parasitenone (4) and amino epoxyquinols (5, 6)
showed the potent NF-κB inhibitory activity, salicylic amide analogs (7) did not inhibit the activitation of NF-κB. The
results of the biological investigation of epoxyquinol analogs (4-6) indicated that these modes of action were slitely
different from each other and it depended on the structure of side chain unit and syn or anti relative configuration. The
presentation will provide an overview of synthesis and biological activity of these analogs.
OH
OH
OH
O
O
Parasitenone (4)
O
H
N
Epoxyquinol unit
OH
R
O
R = Ac: NAcEQ (5)
R = Alloc: NAllocEQ (6)
O
H
N
H
N
O
Salicylic acid unit
O
DHMEQ (1)
Figure 2
References:
1. Kataoka, T.: J. Antibiot. 2009, 62: 655.
2. Ariga, A. et al.: J. Biol. Chem. 2002, 277: 24625.
3. Gehrt, A. et al.: J. Antibiot. 1998, 51: 455.
4. Erkel, G.; Anke, T.; Sterner, O.: Biochem. Biophys. Res. Commun. 1996, 226, 214.
5. Saitoh, T. et al.: Bioorg. Med. Chem. Lett. 2009, 19: 5383.
6. Saitoh, T. et al.: Bioorg. Med. Chem. Lett. 2010, 20: 5638.
112
OH
R
O
OH
Salicyl amides (7)
Structure-activity relationship studies on small molecule Bid-inhibitors
SL.05
Barho, M.T.1; Oppermann, S.2; Schrader, F.C.1; Degenhardt, I.1; Elsässer, K.2; Wegscheid-Gerlach, C.1; Culmsee, C.2;
Schlitzer, M.1
1
2
Institut für pharmazeutische Chemie, Philipps-Universität Marburg,, Marbacher Weg 6, 35032 Marburg, Germany
Institut für Pharmakologie und klinische Pharmazie, Philipps-Universität Marburg,, Karl-von-Frisch Str. 1, 35033 Marburg, Germany
Apoptosis is the underlying pathomechanism of several diseases affecting the central nervous system like Alzheimer's or
Parkinson's disease and also plays an important role after traumatic injuries of the brain or injuries after an ischemic
stroke [1,2]. In this context, induction of apoptosis, triggered by several mechanisms, will lead to an irreversible loss of
neuronal tissue. Especially, mitochondrial demise is considered as the “point of no return” in programmed cell-death in
neurons [3]. The BH3-only protein Bid has been established as an important regulator of mitochondrial integrity, thereby
presenting Bid as a promising target for the development of neuroprotective substances [4,5]. In order to gain a deeper
insight into SAR and to identify novel neuroprotective compounds, a known Bid-inhibitor, which resulted from a fragment
based NMR approach [6,7], has been critically evaluated and was used as template. Supported by molecular docking
into the NMR structure of murine Bid [8], moieties have been exchanged, respectively modified by addition or removal of
functional groups. Also combinations of new substructures have been prepared, to screen for additive effects. All compounds have been tested for their ability to protect cultured neurons from glutamate induced apoptosis using the MTT
Assay. The best compounds displayed significant neuroprotection in concentrations as low as 1 µM [9].
Scaffold of investigated compounds
References:
1. Mattson, M.P. et al.: Apoptosis. 2001, 6: 69-81.
2. Culmsee, C.; Landshamer, S.: Curr. Alzheimer Res. 2006, 3: 269-283.
3. Landshamer, S. et al.: Cell Death Differ. 2008, 15: 1553-1563.
4. Grohm, J.; Plesnila, N.; Culmsee, C.: Brain Behav. Immun. 2010, 24: 831-838.
5. Culmsee, C.; Plesnila, N.: Biochem. Soc. Trans. 2006, 34: 1334-1340.
6. Becattini, B. et al.: Chem. Biol. 2004, 11(8): 1107-1117.
7. Becattini, B. et al.: Proc. Natl. Acad. Sci. U.S.A 2006, 103(33): 12602-12606.
8. McDonnell, J.M. et al.: Cell 1999, 96: 625-634.
9. Barho, M.T. et al.: ChemMedChem 2014, (accepted).
113
SL.06
Synthesis and Structure Affinity Relationships of Dual Chemokine Receptor 2 and
Chemokine Receptor 5 Antagonists and Development of a Selective, Fluorinated CCR2 Ligand for PET
Studies
Junker, A.1; Schepmann, D.1; Yamaguchi, J.2; Itami, K.2,3; Faust, A.4; Wagner, S.5; Wünsch, B.1
Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstr. 48, D-48149 Münster, Germany
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: [email protected]
2 Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan
3 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Chikusa-ku, Nagoya 464-8602 Japan
4 European Institute for Molecular Imaging (EIMI), Mendelstr. 11, D-48149 Münster, Germany
5 Klinik für Nuklearmedizin, Albert-Schweitzer-Campus 1, Gebäude A1, D-48149 Münster, Germany
1
The chemokine receptor subtypes 2 (CCR2) and 5 (CCR5) are two G-protein coupled CC chemokine receptors, that play
a crucial role in the trafficking of monocytes, macrophages and in the functions of other cell types relevant for the development and progression of several diseases, such as rheumatoid arthritis (RA) [1], atherosclerosis [2], asthma [3], and
HIV-1 (AIDS) [4]. Strong preclinical evidence indicates greater efficacy for dual targeting of CCR2 and CCR5, than
targeting CCR2 or CCR5 alone [5].
Herein, we present the design, synthesis and evaluation of biological activities of highly potent, benzo[7]annulene-based,
dual CCR2 and CCR5 antagonists and the development of a highly selective, [ 18F]-labeled CCR2 receptor radioligand as
a new diagnostic tool for positron emission tomography (PET) [6].
0.2
Time [min]
32-42
110-120
Figure 1: Whole body PET images of
biodistribution studies of [18F]7b (10
MBq i.v.) after intravenous injection into
C57BL/6 –WT mouse.
Scheme 1: Synthesis of various benzo[7]annulen-based, dual CCR2-CCR5 antagonists 6a-u.
(a) Suzuki-Miyaura cross-coupling reaction (b) Amide coupling reaction.
In order to introduce broad diversification in the last step of the synthesis, two different strategies (A and B) were developed. Strategy A allowed the variation of the amino part R1 of the benzo[7]annulen-8-carboxamides 6a-u, by amide
coupling reactions of acid 4 with different amines. Whereas, strategy B provided a broad exploration of the aryl part Ar
by Suzuki-Miyaura cross-coupling reaction of the aryl bromide 5 with various arylboronic acids.
The CCR2 and CCR5 affinities of the novel compounds were determined in competitive radioligand receptor binding
assays ([3H]TAK-779 (CCR5), [125I]-CCL2 (CCR2)). Additionally, CCR2 receptor potencies were recorded using the
intracellular Ca2+ mobilization assay (hCCR2) and the β-arrestin recruitment assay (mCCR2). A highly potent and selective CCR2 antagonist was further converted into a fluorinated PET ligand [18F]7b.
References:
1. (a) García-Vicuña, R. et. al.: Arthritis Rheum. 2004, 50 (12): 3866-3877. (b) Dawson, T. et. al.: Atherosclerosis 1999, 143 (1): 205-211.
2. Szalai, C. et al.: Atherosclerosis 2001, 158 (1): 233-239.
3. Maus, U.A. et al.: J. Immunol. 2003, 170 (6): 3273-3278.
4. (a) Deng, H. et al.: Nature 1996, 381 (6584): 661-666. (b) Liu, R. et al.: Cell 1996, 86 (3): 367-77.
Financial support of the IRTG and this project
by the DFG is gratefully acknowledged. We are
5. (a) Yang, Y.-F. et al.: Eur. J. Immunol 2002, 32(8): 2124-2132.
also grateful for the financial support by the
(b) Tokuyama, H. et al.: Intern Immunol 2005, 17(8): 1023-1034.
Collaborative Research Center 656 MoBil.
(c) Schröppel, B. et al.: J. Am.Soc. Nephrol. 2004, 15 (7): 1853-1861.
(d) Junker, A. et al.: Topics in Medicinal Chemistry (Springer) 2014; 1-55.
6. (a) Junker, A.; et al.: OBC 2014, 12 (1): 177-186. (b) Junker, A. et al.: J. Org. Chem. 2013, 78 (11): 5579-5586.
114
Testing of potential inhibitors of human heparanase in a fluorescence activity assay
SL.07
Schoenfeld, A.; Vierfuß, S.; Lühn, S.; Alban, S.
Pharmaceutical Institute, Christian-Albrechts-University, Gutenbergstr. 76, 24118 Kiel, Germany
Heparanase, an endo-β-glucuronidase cleaving heparan sulfate (HS) chains at cell surfaces and in the extracellular
matrix (ECM), is involved in angiogenesis, tumor progression and metastasis as well as in inflammation and kidney
dysfunction. Therefore, heparanase is considered a promising therapeutic target and diagnostic marker. Recently, we
have developed a simple, rapid, fully automatable fluorimetric activity assay using the synthetic sulfated pentasaccharide
fondaparinux as substrate and bacterial heparinase II (HEP-II) instead of human heparanase (hHEP). The aim of this
study was to evaluate this assay for inhibitor testing as well as to check whether the assay principle is applicable to
measure the activity and inhibition, respectively, of the actual target enzyme hHEP.
Besides the known hHEP inhibitor suramin and the antiinflammatory and antimetastatic PS3, two series of β-1,3-glucan
sulfates differing in their chain length and degree of sulfation, further semisynthetic sulfated glycans, and two sulfated
polysaccharides isolated from algae were included to examine structure-activity relationships.
The inhibitory activity of sulfated glycans showed to be greatly dependent on both their degree of sulfation and their
basic glycan structure, but independent of their molecular size. The β-1,3-glucan sulfates were superior to suramin as
well as to the other glycans with similar degree of sulfations. The most active inhibitor was found to be the β-1,3-glucan
sulfate PS3 (IC50 = 0.017 µM). By using hHEP instead of HEP-II comparable results were obtained. With an IC50 being
about 160 times lower than that of suramin, PS3 exhibited again the strongest inhibitory effects. Inhibition of hHEP may
therefore contribute to the potent antiinflammatory and antimetastatic activities of PS3 in vivo. In conclusion, the fluorimetric hHEP activity assay proved to be a simple, fully automatable tool for testing potential inhibitors. In case of HS
mimetic inhibitors, the assay variant with HEP-II may provide a fast and inexpensive option for initial screening purposes.
115
Monitoring Conformational Changes in PPARβ/δ by Cross-linking and
Mass Spectrometry
SL.08
Schwarz, R.; Tänzler, D.; Kölbel, K.; Ihling, C.; Sinz, A.
Institute of Pharmacy, Department of Pharmaceutical Chemistry & Bioanalytics, Martin-Luther University Halle-Wittenberg, Germany
Chemical cross-linking, combined with an enzymatic digestion and mass spectrometric analysis of the reaction products,
has evolved into an alternative strategy to identify protein-protein and protein-ligand interactions [1]. Peroxisome proliferator-activated receptors (PPARs) belong to the subfamily of nuclear receptors that are involved in metabolic processes.
One subtype, PPARβ/δ, is thought to be involved in the development of several chronic diseases and presents an
important drug target [2]. Here, we present the monitoring of conformational changes in PPARβ/δ upon ligand binding by
cross-linking studies combined with mass spectrometry.
Expression and purification of the ligand binding domain of PPARβ/δ (amino acids 166-441) was optimized for obtaining
high protein yields. Cross-linking reactions were performed using the amine-reactive homobifunctional cross-linker
bis(sulfosuccinimidyl)glutarate (BS²G). After in-gel digestion, peptides were analyzed by high-resolution mass spectrometry (Orbitrap Fusion Tribrid, Thermo Fisher Scientific). All cross-links were evaluated with the in-house software StavroX [3]. The distance constraints imposed by the cross-links served to monitor conformational changes in PPARβ/δ upon
binding of the agonists GW1516 (Fig.1) and GW0742 (Fig.2).
In the absence of ligands a higher number of cross-links were identified, indicating a high flexibility of the ligand binding
domain in the unbound state. After ligand binding, cross-links between lysines K322 and K422/K423 to the N-terminus
suggested a large conformational change in PPARβ/δ. Apparently, amino acids 185-210 are easily accessible to the
cross-linker as in the absence of ligands as well after ligand binding most of the cross-links were found in that region.
In addition, photo-affinity labeling studies with the unnatural photo-amino acid para-benzoyl-L-phenylalanine (Bpa) are
currently performed. Bpa was incorporated at specific positions into PPARβ/δ in the high flexible Ω-loop or the activation
function helix 2 to investigate conformational changes upon ligand binding. Large hydrophobic amino acids, such as
phenylalanine or tryptophan, are preferrably exchanged for Bpa. To inspect the conformation behavior of the activation
function helix 2 the C-terminal tyrosine (Y441) was exchanged for Bpa.
Fig. 1: PPARβ/δ agonist GW1516
Fig. 2: PPARβ/δ agonist GW0742
References:
1. Sinz, A.: Mass Spectrom. Rev. 2006, 25(4): 663-682.
2. Berger, J. et al.: Annu. Rev. Med. 2002, 53: 409–435.
3. Götze, M. et al.: J. Am. Soc. Mass Spectrom. 2012, 23(1): 76-87.
116
A dynamic pH junction method for monitoring the catalytic activity of cerebroside
sulfotransferase
SL.09
Li, W.1; Zech, I.2; Gieselmann, V.2; Müller, C.E.1
1 PharmaCenter
2 Institut
Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
für Biochemie und Molekularbiologie, University of Bonn, Nussallee 11, 53115 Bonn, Germany
Cerebroside sulfotransferase (CST) is a promising new therapeutic target for metachromatic leukodystrophy (MLD), a
rare and severe genetic disease. CST catalyzes the transfer of a sulfate group from the coenzyme 3′phosphoadenosine-5′-phosphosulfate (PAPS) to cerebroside as a substrate yielding cerebroside sulphate and adenosine-3′,5′-diphosphate (PAP).1 So far only a few weakly potent competitive inhibitors of the cosubstrate PAPS have been
published, whereas no CST inhibitors competitive for the substrate cerebroside have been described. 2 In the present
study we developed a capillary electrophoresis- (CE-) based assay for monitoring the catalytic activity of CST. By using
dynamic pH junction stacking, a low nanomolar limit of detection (LOD = 66.6 nM) was achieved for the reaction product
PAP. Our CE method was sensitive, robust and suitable for CST inhibitor screening, Ki value determination, and enzyme
kinetic studies. Several reference compounds were tested including cerebrosides, psychosine and Congo Red2,3 in order
to validate the method. The newly developed CE method will be used to develop novel CST inhibitors, which are urgently
needed for the treatment of MLD, a devastating disease that in the absence of therapy leads to early death of the young
patients.
References:
1. Eckhardt, M.: Mol. Neurobiol. 2008, 37: 93–103.
2. Zaruba, M.; Hilt, D.; Tennekoon, G.: Biochem. Biophys. Res. Commun. 1985, 129: 522–529.
3. Honke, K. et al.: J. Biol. Chem. 1997, 272: 4864–4868.
117
Confocal Raman microscopic (CRM) methodology for the analysis of the penetration
of pharmaceutical actives into the skin
SL.10
Lunter, D.J.
Pharmaceutical Technology, Eberhard Karls University of Tuebingen, Auf der Morgenstelle 8, Tuebingen, 72076, Germany
CRM is increasingly used for the detection of xenobiotics within the skin. Conventionally, the skin is scanned from the
stratum corneum downwards (depth scan/virtual cross section). In this context an alternative methodology is presented.
It employs CRM on ex vivo cross sections of porcine skin. To investigate the usefulness of the methodology procaine
was chosen as a model drug. It exhibits poor skin penetration as well as good detectability by CRM. These two features
make it an ideal model substance to study penetration enhancement by CRM. Semisolid preparations that contained
procaine HCl and the penetration enhancers propylene glycol or polyoxyethylene-23-lauryl ether (POE-lauryl ether) were
used as model formulations. POE-lauryl ether was chosen as Shin et al. showed that it enhanced the penetration of local
anesthetics [1]. They drew this conclusion on the basis of permeation experiments and the calculation of the enhancement ratio as the ratio of the flux with enhancer and the flux without enhancer. Propylene glycol was chosen as it is
known to enhance the penetration/permeation of a variety of drugs and is frequently used in dermal preparations.
Excised postauricular porcine skin was incubated in Franz diffusion cells with semisolid formulations that contained
either enhancer. An enhancer free formulation was used as control. After the incubation, the skin was cleaned, frozen
and saggital cuts were made with a cryo-microtome (HM 560 Cryo-Star; Thermo Fisher, D-Langenselbold). The cuts
were examined with a confocal Raman microscope (alpha 500, WiTec, D-Ulm) equipped with a 532 nm laser and a 10x
(NA: 0.25) objective. Areas of 20x70 µm were mapped with a spatial resolution of 1.3 µm and a spectral resolution of
1 cm-1. Colour coded images that visualize the distribution of procaine within the skin were calculated. For a comparison
of the relative procaine amount that was delivered to the skin by the penetration enhancers, the normalized peak areas
of procaine in the scans were calculated and compared to the value that was obtained without enhancer. For comparison, a permeation experiment was performed and the enhancement ratios were calculated.
The CRM investigation revealed that propylene glycol did not enhance the penetration of procaine whereas POElauryl ether had a distinct enhancing effect on the amount of procaine in the epidermis. Furthermore, it could be shown
that procaine was predominantly located in the lipid rich domains in the skin. A behaviour that can be explained by the
high partition coefficient of procaine (kp=100). In contrast, the permeation experiment and the enhancement ratios
derived therefrom did not reveal any enhancement by propylene glycol or POE-23-lauryl ether.
It can therefore be concluded that CRM can give additional information in the investigation of dermal penetration. Information that is not accessible by the conventional method of calculating the enhancement ratio on the basis of permeation data. Furthermore, the proposed CRM-methodology can visualize the distribution of the drug within the skin and
enhance the depth from which Raman spectra can be collected.
Acknowledgements: Institute of Experimental Medicine, University of Tuebingen, Schenk, M.
Reference:
1. Shin, C.-S. et al.: Int. J. Pharm. 2004, 287: 73-78.
118
Determination of the Dissolution Behaviour of Celecoxib-Eudragit E 100-Nanoparticles
using Cross-Flow Filtration
SL.11
Schichtel, J.1,2, Prinz, E.-M.1, Tuereli, A.E.1, Langguth, P.2
1 MJR
PharmJet GmbH, Saarland University Medical Center, 66424 Homburg, Germany
Technology and Biopharmaceutics, Johannes Gutenberg-University, 55099 Mainz, Germany
2 Pharmaceutical
This study deals with the development of a dissolution test method for nanoparticulate dosage forms. Thereto, nanoparticles (NPs) consisting of celecoxib as model drug and Eudragit E 100© were prepared using Microjet Reactor©. This
technology (assembly depicted in Figure 1) uses two opposed high velocity linear jets. One jet conveys the solvent with
API and polymer, the other jet the non-solvent. A third jet carrying inert gas facilitates fast depletion of the organic
solvent. The produced particles have a Z-Average ranging from 100 to 250 nm, polydispersity index below 0.2 and
entrapment efficiency of 70 to 80 % (scanning electron microscopy image of NPs shown in Figure 2). Dissolution tests
were conducted using cross-flow filtration, a new approach in pharmaceutical dissolution testing. Current compendial
methods implement the usage of dead-end filters to separate undissolved particles of the dosage form from dissolved
drug molecules. The application of dead-end filters bears the risk of filter clogging and is extensive since new filters have
to be used for every dissolution test. Prior to dissolution testing, the applicability of cross-flow modules with respect to
their ability to retain NPs was successfully tested. Thereto, comparative dynamic light scattering and UV-spectroscopic
measurements of nanosuspension and filtrate were performed at neutral pH where dissolution of Eudragit E 100© does
not occur. These experiments revealed that the used filter modules are impermeable for the tested nanoparticles. For
analysis of celecoxib an UV-Vis-spectroscopic method was developed which facilitates the quantification of celecoxib
besides Eudragit E 100©. As dissolution medium hydrochloric acid of pH 1.2 was used since the stomach is the chosen
site of release in vivo. Furthermore, Eudragit E© is only soluble at pH 5 or less [2]. Additionally, cetrimide, a quaternary
ammonium salt, was added in a concentration of 0.3 % (m/v) to the medium. In this way, sink conditions for celecoxib
dissolution are given [3]. To compare the effect of different filters dissolution tests were performed using three different
filters: two cross-flow filtration modules with two different filter pore sizes as well as syringe filter holders to represent
dead-end filtration. The suitability of the syringe filter holders to retain undissolved NPs was successfully tested as
described above. The obtained results reveal that dead-end filtration well correlates with cross-flow filtration at larger
pore size. In summary, it can be stated that cross-flow filtration can be a well appropriate alternative to classical deadend filtration in pharmaceutical dissolution testing.
Figure 1: Schematic of MJR (mode of operation)
Figure 2: SEM image of celecoxib-Eudragit E 100 NPs
References:
1. Baumstümmler, B.; Penth, B.; Penth, F.; Türeli, A.E.: US Patent (US20130012551 A1) 2013.
2. Rowe, R.C.; Sheskey, P.J.; Owen S.C.: Handbook of Pharmaceutical Excipients (American Pharmaceutical Association and Pharmaceutical
Press) 2006.
3. Albers et al.: Eur. J. Pharm. Biopharm. 2009, 71(2): 387–394.
119
Sensitivity of concentration-effect versus dose-effect analysis to detect small
magnitudes of QTc prolongation in preclinical cardiovascular safety setting
SL.12
Gotta, V.1; van Ammel, K.2; Cools, F.2; Gallacher, G.J.2; Visser, S.A.G.3; Morissette, P.4; Sannajust, F.4; Danhof, M.1; van
der Graaf, P.H.1
1 Systems
Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden, The Netherlands
Safety Pharmacology, Janssen Research & Development, Janssen Pharmaceutica NV, Belgium
3 Quantitative Pharmacology and Pharmacometrics / Merck Research Laboratories, Merck & Co., Inc., Upper Gwynedd, PA, USA
4 SALAR-Safety and Exploratory Pharmacology / Merck Research Laboratories, Merck & Co., Inc., West Point, PA, USA
2 Global
The heart-rate corrected QT interval (QTc) on the electrocardiogram is a simple biomarker for potential proarrhythmic
risk. A QTc prolongation (∆QTc) of 2-8 ms in the dog correlates with a significant 10 ms prolongation in humans[1,2].
This simulation study aimed to investigate the sensitivity of concentration-effect (pharmacokinetic-pharmacodynamic,
PKPD) analysis to detect small magnitudes of QTc-prolongation in a typical preclinical cardiovascular (CV) safety study
in the conscious telemetered dog (crossover study in 4-8 animals receiving by oral route a vehicle and three dose levels,
followed each by a wash-out phase). Results were compared with conventional dose-effect analysis (analysis of covariance, ANCOVA)[3,4].
A PKPD model predicting individual QTc was first developed from vehicle arms of 28 typical CV safety studies and one
positive control study (D,L-sotalol administered orally). The model quantified between-animal, inter-occasion and withinanimal variability and described QTc as a function of circadian variation and drug concentration (direct effect). This “true”
model was used to repeatedly (n=500) simulate studies with typical drug-induced ∆QTc of 1 to 12 ms at high-dose peak
concentrations. Simulated studies were re-analyzed by both PKPD and ANCOVA. Sensitivity (=true positive rate/power)
was calculated as the percentage of studies in which a significant (α=0.05) drug effect was found (PKPD: likelihood-ratio
test for linear concentration-effect slope; ANCOVA: comparison of dose versus vehicle and linear trend test for dose).
One simulation scenario did not include a concentration-effect relationship and served to investigate false-positive rates.
PKPD analysis/ANCOVA had a sensitivity of 80% (horizontal dashed line) to detect effects of 7/12 ms (n=4 animals),
5/10 ms (n=6) and 4.5/8 ms (n=8), respectively (Figure below). False-positive rates (estimates at true typical ∆QTc=0
ms) were higher using ANCOVA (39%) compared to PKPD analysis (1%).
Results suggest superior sensitivity and specificity of PKPD approaches to quantify small QTc effects in preclinical safety
testing. Their use may increase the confidence in observed effects and potentially allow reducing the number of animals
while maintaining required study sensitivity.
Acknowledgements: This project was supported by the Dutch Top Institute Pharma (TIPharma) PK-PD platform 2.0.
References:
1. Chain, A. et al.: Br. J. Clin. Pharmacol. 2013, 76(5): 708-724.
2. Parkinson, J. et al.: J. Pharmacol. Toxicol. Methods. 2013, 68(3): 357-366.
3. Aylott, M. et al.: Pharm Stat. 2011, 10(3): 236-249.
4. Sivarajah, A. et al.: J. Pharmacol. Toxicol. Methods. 2010, 62(1): 12–19.
120
POSTERS
121
ANTIINFLAMMATORY DRUGS (AD01-AD19)
AD.01
Ceramide-1-Phosphate: a new player in modulating DC in allergic
inflammation?
Baudiß, K.; Ayata, K.; Vieira, R.P.; Idzko, M.
models. Immunostaining revealed additional down regulation of filaggrin
expression in FLG- models.
By combining two characteristics of atopic skin, we were able to demonstrate
that FLG- models have a higher susceptibility to inflammatory stimuli. Further
investigations will show if the skin models are suitable test systems to assess
anti-inflammatory effects in vitro and how Th2-derived cytokines further
influence the skin homeostasis.
Department of Pneumology, University Hospital Freiburg, Breisacherstr. 66, 79106
Freiburg, Germany
Acknowledgement: Financial support by the Collaborative Research Center 1112 for the
project C02 is gratefully acknowledged.
Introduction: Sphingolipids are playing an essential role in normal cell und
tissue homeostasis as well as in the development and progress of various
diseases and disorders. The central molecule in the sphingolipid metabolism is
ceramide, which can be converted into Shingosine-1-Phosphate (S1P) and
Ceramide-1-Phosphate (C1P). While the role of S1P in the pathogenesis of
airway inflammation has been extensively studied, little is known about C1P
[1].
Aim: The aim of the current study was to elucidate wheater C1P can inhibit
cardinal features of acute experimental asthma and might be a new modulator
for DC in allergic inflammation.
Methods and Results:.
Balb/c mice were sensitized with ovalbumin-alum (OVA-alum) and challenged
with OVA-aerosols for 3 days. After OVA challenge mice were sacrificed and
bronchoalveolar fluid (BALF) was collected. The distribution of different cells
were analysed by flow cytometer. The cytokine content in BAL fluid was
measured by Elisa. C1P treated mice showed less inflammatory cells in BALF
and lung tissue sections accompanied by decreased Th2 cytokine production
in the mediastinal lymphnodes compared to vehicle treated mice. Furthermore
C1P pulsed dendritic cells showed a limited priming capacity of Th2 immunity
in a DC driven model of allergic airway inflammation in vivo. Mechanistically
C1P inhibited the OVA-induced NF-kB activation in vitro and in vivo. Parametric test were applied for statistical analysis
Conclusion: In summary, C1P reduces the development of allergen-induced
asthma in a mouse model by attenuating NF-kB activation and influencing
dendritic cells. Our results suggest that C1P might be a therapeutic target for
treatment of asthma.
References:
1. Palmer, C.N. et al.: Nat Genet. 2006, 38(4): 441-446.
2. Kuchler, S. et al.: Altern Lab Anim. 2011, 39(5): 471-480.
3. Vavrova, K. et al.: J Invest Dermatol. 2014, 134(3): 746-753.
4. Oyoshi, M.K. et al.: Adv Immunol. 2009, 102: 135-226.
References:
1. Gangoiti, P. et al.: Translational Oncogenomics 2008, 3: 81-98.
AD.02
Effects of TH2 Cytokines on Filaggrin Deficient Skin Constructs
Hönzke, S.; Schäfer-Korting, M.; Küchler, S.
Institute for Pharmaceutical Sciences, Pharmacology & Toxicology, Königin-Luise-Straße
2+4, 14195 Berlin, Germany
Atopic dermatitis (AD) is a chronic inflammatory skin disease which is
characterized by an impaired skin barrier function. Loss-of-function mutations
in the filaggrin gene (FLG) are a major predisposing risk factor for the
manifestation of AD [1]. However, the exact pathogenesis is still ambiguous. In
order to unravel the impact of filaggrin deficiency on the skin homeostasis, we
established a FLG knock down skin model which exhibits a disturbed
epidermal maturation and differentiation, altered skin lipid composition and
organization as well as altered dermal drug absorption [2-3]. Aside from
specific histological features, AD is characterized by an overshooting immune
response. Particularly high levels of T helper cells (TH2) derived inflammatory
cytokines contributes to the pathogenesis of AD [4]. Nevertheless, the effects
of TH2 cytokines such as IL-4 and IL-13 on the skin barrier function and
especially interdependencies with the FLG deficiency are not yet fully
understood.
Therefore, we systemically stimulated the FLG deficient skin model with TNF
alpha, IL-4 and IL-13 for four days in order to induce inflammatory conditions in
vitro. We detected significantly increased levels of the pro-inflammatory
cytokines IL-6 and IL-8, whereas levels were even higher in the FLG deficient
skin models compared to the normal models (503.4 ± 55.09 ng/ml vs 335.3 ±
11.86 ng/ml). Interestingly, even untreated FLG- models released slightly
higher amounts of IL-8 and IL-6. Histological evaluations revealed major
structural changes such as the induction of spongiosis, parakeratosis and an
increase of epidermal thickness which was again most pronounced in the FLG-
122
AD.03
Treatment with Peroxisome Proliferator-Activated Receptor
Agonist Docosahexaenoic Acid Normalizes Filaggrin Expression
in a Filaggrin Knock Down Skin Model
Wallmeyer, L.1; Lehnen, D.1; Sochorová, M.2; Školová, B.2; Schäfer-Korting,
M.1; Vávrová, K.2; Küchler, S.1
1 Freie
Universität Berlin, Institute of Pharmacy, Pharmacology and Toxicology, KöniginLuise-Straße 2+4, 14195 Berlin, Germany
2 Charles University Prague, Faculty of Pharmacy, Heyrovského 1203, 50005 Hradéc
Králové, Czech Republic
Loss-of-function mutations in the gene encoding for filaggrin (FLG) are the
major predisposing factor for atopic dermatitis (AD). As for today, therapeutic
options for FLG associated skin diseases only alleviate the symptoms such as
dry and itchy skin or reduce the inflammation. No therapy exists preventing the
development of these symptoms or restoring the disturbed skin barrier
function. Peroxisome proliferator-activated receptor (PPAR) agonists are not
only important therapeutics for the treatment of lipid disorders and diabetes but
also exhibit beneficial effects in patients suffering from inflammatory skin
diseases like AD. PPAR agonists are known to increase the expression of FLG
in skin and positively influence the skin barrier homeostasis, skin barrier
recovery and stratum corneum (SC) integrity [1]. The underlying mechanism,
however, is still ambiguous.
In order to study the effects caused by a lack of FLG in vitro, we established a
FLG knock down skin model [2, 3]. Here, we evaluated the effects of the PPAR
agonist docosahexaenoic acid (DHA) in a FLG deficient (FLG-) skin model in
terms of FLG expression, skin lipid organization and composition and skin
permeability. We detected an about 15.72-fold upregulation of FLG in DHA
treated normal skin models (FLG+). FLG expression increased significantly
about 2.69-fold in FLG- models upon DHA treatment even exceeding the FLG
expression of DHA untreated FLG+ samples. These results were confirmed on
the protein level and histological examination revealed a thickening of the SC
upon DHA treatment (FLG-/DHA- 8 ± 1.2 µm vs. FLG-/DHA+ 12.8 ± 1.5 µm).
In terms of skin lipid composition, a treatment with DHA normalized the
pathologically increased free fatty acid (FFA) levels: FFA amounts were
reduced from 22.0 ± 2.7 μg/mg to 15.3 ± 1.0 μg/mg in FLG- models following
DHA treatment (FLG+ 13.3 ± 1.5 μg/mg). Furthermore, the skin lipid organization was significantly improved in FLG- constructs as determined by ATRFTIR. Interestingly, skin absorption studies did not show an improvement of
the skin barrier after DHA treatment. The beneficial effects on the skin barrier
homeostasis are undoubted but further studies are necessary to completely
understand the effects of PPAR agonists on the skin barrier function.
Acknowledgements: Financial Support by the Foundation SET (Stiftung zur Förderung der
Erforschung von Ersatz- und Ergänzungs-methoden zur Einschränkung von Tierversuchen) is gratefully acknowledged. This work was supported by the Czech Science
Foundation (project No. 207/11/0365) and Charles University (SVV 265 001).
References:
1. Michalik, L.; Wahli, W.: Biochimica et biophysica acta. 2007, 1771: 991-998.
2. Küchler, S. et al.: Altern Lab Anim. 2011, 39: 471-480.
3. Vávrová, K. et al.: J Invest Dermatol. 2014, 134: 746-753.
AD.04
Induction of glucocorticoid-induced leucine zipper (GILZ)
contributes to anti-inflammatory effects of the natural product
curcumin in macrophages
Hoppstädter, J.; Hachenthal, N.; Sauer, K.; Kiemer, A.K.*; Diesel, B.
Pharmaceutical Biology, Campus, Bldg. C 2 2, Saarland University, 66123 Saarbrücken,
Germany
* To whom correspondence should be addressed: [email protected]
Background: Inflammation is characterized by the production of inflammatory
mediators as well as a decrease in anti-inflammatory regulators. Glucocorticoids are well-established anti-inflammatory compounds, but exert distinct side
effects. The induction of the glucocorticoid-induced leucine zipper (GILZ,
TSC22D3) protein plays a pivotal role in the therapeutic actions of glucocorticoids, e.g. by inhibiting the inflammatory transcription factor NF-B, but is not
involved in glucocorticoids’ adverse actions [1]. We therefore sought for GILZinducing compounds, which do not act via the glucocorticoid receptor.
Results: Macrophages obtained from GILZ knockout mice displayed an
inflammatory phenotype associated with increased NF-B and ERK activity,
confirming an induction of GILZ as a promising therapeutic strategy against
inflammatory diseases. We observed that the natural product NF-B inhibitor
curcumin induces GILZ protein in a dose- and time-dependent fashion in
murine and human macrophages. Curcumin exerts its anti-inflammatory
actions via induction of GILZ: GILZ knockdown by specific siRNA antagonized
curcumin’s inhibitory action on lipopolysaccharide (LPS)-induced iNOS and
NF-B dependent luciferase activity. Consistently, curcumin failed to inhibit
TNF- production and ERK activation in GILZ knockout macrophages.
Activation of the glucocorticoid receptor does not contribute to increased GILZ
protein. We also observed neither increased GILZ mRNA nor protein stability.
We rather suggest a translational involvement of the RNA-binding protein HuR
(Elavl1) with HuR being translocated after curcumin treatment.
Conclusion: We provide evidence for a steroid-independent GILZ-mediated
anti-inflammatory action of curcumin, probably via posttranscriptional
regulation of GILZ.
Acknowledgments: This work was supported, in part, by the Deutsche Forschungsgemeinschaft (DFG, KI 702). We thank Indou Kepbane, Susanne Schütz and Oliver Wild for
technical support.
Reference:
1. Beaulieu, E.; Morand, E.F.: Nat. Rev. Rheumatol.2011, 7(6): 340-348.
AD.06
Termination of inflammatory processes in the endothelium by
inhibition of BMP2K
Bischoff, I.1; Dai, B.2; Strödke, B.3; Bracher, F.3; Fürst, R.1
1 Institute
of Pharmaceutical Biology, Goethe-University Frankfurt, Germany
Biology, Center for Drug Research, University of Munich, Germany
of Pharmacy - Center for Drug Research, University of Munich, Germany
2 Pharmaceutical
3 Department
During chronic inflammation in diseases such as Crohn’s disease or psoriasis,
angiogenesis accompanied by leukocyte infiltration is an ongoing process. The
termination of these processes, which normally occurs during the physiological
situation of an acute inflammation, is not or only inadequately initiated under
chronic conditions. Thus, the application of substances that induce the
termination of inflammation seems a promising approach. We hypothesized
the bone morphogenetic protein-2 (BMP-2)-inducible kinase (BMP2K/BIKE)
might be a novel target of such substances. Surprisingly, the role of BMP2K in
the endothelium has not been investigated up to now. During this study, C81, a
small molecule, was used to inhibit BMP2K in order to determine potential antiangiogenic and anti-inflammatory effects of the substance. In addition, C81 as
well as BMP2K gene silencing were used to elucidate the role of BMP2K in
these processes.
Initial experiments demonstrated that C81 concentrations up to 3 µM did not
exhibit any cytotoxic effect on a human microvascular endothelial cell line
(HMEC-1) or on primary human umbilical vein endothelial cells (HUVEC), The
performance of a migration assay revealed that increasing concentrations of
C81 reduced the migratory capacity of HMEC-1. The treatment with C81
reduced the TNFα-triggered expression of the cell adhesion molecules ICAM1, VCAM-1 and E-selectin on the endothelial cell surface with rising concentrations of C81 (flow cytometry). Similar results were detected in BMP2K-silenced
HUVECs (siRNA), as ICAM-1 and VCAM-1 exhibited to be markedly reduced
in BMP2K-silenced cells. Based on these findings, a cell adhesion assay using
the monocytic leukemia cell line THP-1 and HUVECs was performed.
Fluorescence-labelled THP-1 cells showed a significantly decreased adhesion
to TNFα- or LPS-activated HUVECs upon C81 treatment, as determined by
fluorescence measurements of cell lysates. Analysis of BMP-2-treated
HUVECs using quantitative real-time PCR revealed that BMP2K seems not to
be regulated by BMP-2 on the gene expression level.
Our results indicate that BMP2K might be involved in inflammatory processes
of the endothelium, Furthermore, these results suggest C81 as potential antiinflammatory compound in vitro and, moreover, as a promising tool to interrupt
BMP2K-mediated signalling events. Further studies are needed to elucidate
the precise role of BMP2K in inflammatory and angiogenic processes and to
clarify the underlying mechanisms.
AD.05
Omega-3 Fatty Acids (Omegaven) protect from Mitochondrial
Dysfunction in a MCAO mouse model of stroke
Berressem, D.; Koch, K.A.; Franke, N.; Klein, J.; Eckert, G.P.
Goethe Universität Frankfurt am Main, Germany
Recent investigations demonstrated efficacy of docosahexaenoic acid (DHA)
to reduce stroke size and severity in the transient middle cerebral arterial
occlusion (MCAO) model in rats when applied intravenously after reperfusion.
In this study we investigated the beneficial effect of OMEGAVEN (Fresenius
Kabi, Germany) a medical lipid emulsion for parenteral nutrition that contains
the long-chain omega-3 fatty acids eicosapentaenic acid (EPA) and DHA in a
model of transient stroke. Mice underwent transient MCAO and OMEGAVEN
was administered intravenously (5 ml/kg b.w.) after stroke (90 min) at
reperfusion that represents an early moment for potential intervention. The
degree of damage, mitochondrial function and neuroinflammation were
investigated. Treatment with OMEGAVEN significantly decreased the stroke
area by 21% and lowered the severity of stroke by 50%. OMEGAVEN
significantly improved mitochondrial membrane potential (MMP) and ATP
levels in the ischemic brain hemisphere. These findings are accompanied by
an enhanced mitochondrial function, e.g. improved respireation of the
complexes responsible for oxidative phosphorylation in mitochondria isolated
from the ischemic brain hemisphere. The inflammation markers COX-2 and
iNOS significantly decreased after treatment with OMEGAVEN. This pilot study
demonstrated that OMEGAVEN could represent a promising, approved lipid
emulsion for the early therapeutic intervention in ischemic stroke.
AD.07
Generic Cell-Permeable Probes of Eukaryotic and Bacterial Sialyl
Transferases Inhibit Cellular Sialylation: New Targets in Inflammation and Cancer?
Preidl, J.J.1; Gnanapragassam, V.S.2; Horstkorte, R.2; Rademann, J.1
Institut für Pharmazie, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin,
Germany,
2 Institut für Physiologische Chemie, Martin-Luther-Universität, Hollystr. 1, 06114 Halle,
Germany.
1
Oligosaccharides of the glycolipids and glycoproteins at the outer membranes
of human cells carry terminal neuraminic acids, which are responsible for
recognition events and adhesion between cells, with bacteria, and virus
particles. Synthesis of neuraminic acid-containing glycosides is accomplished
by intracellular sialyl transferases. Hypersialylation of cells is found in
inflammation and enables immune cells to intrude into infected tissue.
Moreover, strongly hypersialydated cells are capable to leave their primary
tissue environment, to migrate, and to form metastases in remote tissues.
Hypersialylation is, therefore, strongly indicative for a bad prognosis of
neoplasia and inhibition of this event could be an alternative therapeutic
strategy against cancer.
We have developed and applied the first nanomolar fluorescent inhibitors of
sialyl transferases. The obtained carbohydrate-nucleotide mimetics were found
to bind all four commercially available and tested eukaryotic and bacterial sialyl
transferases in a fluorescence polarization assay. Moreover, it was observed
123
that the anionic mimetics intruded rapidly and efficiently into cells in vesicles
translocating to cellular organelles surrounding the nucleus of CHO-cells. The
new compounds inhibit cellular sialylation in two cell lines and open new
perspectives for investigations of cellular sialylation. Finally, the established
binding assay enabled high-throughput screening of a chemical library of druglike molecules and small-molecule inhibitors of sialyl transferases were
identified.
17(R)-Resolvin D1 (17(R)-RvD1) via chiral LC-MS/MS analysis. In addition, we
have detected LX biosynthesis so far in a variety of in-vitro systems. Co-culture
experiments with PMNL/platelets, apoptotic PMNL/different macrophage
phenotypes (M1/M2) and Human Umbilical Vein Endothelial Cells (HUVEC)/PMNL with/without aspirin provided various native and 15-epi-LXs.
Incubations of macrophages alone failed to generate detectable LX via LCMS/MS. Potential lipoxin enhancing drug candidates are being tested in the
different cell-based systems.
NH 2
OH
OH
HO
R
O
P O
O O
H
N
O
HO
O
N
O
N
O
O
O
OH OH
NH 2
O
P O
O
O
(S)
HN
R
Neu-5-Ac-CMP
(R=Me)
P O
O O
N
O
N
O
OH OH
O
Generic Probe
for Sialyl Transferases
1: - meta or para
- (R) or (S)
- R=fluorophore
w/ or w/o spacer
Acknowledgements: Support is gratefully acknowledged from the DFG (FOR 806, SFB
765, TRR 67) and the Berlin School of Integrative Oncology.
Reference:
1. Preidl, J.J. et al.: Angew. Chem. 2014, 126: 5808-5813; Angew. Chem. 2014, 53: 57005705.
AD.08
For abstract see short lecture SL.07.
AD.09
Cellular Assay Methods for Detection of Compounds Enhancing
the Generation of Lipoxins
Lehmann, C.1; Homann, J.2; Ferreirόs, N.2; Parnham, M.J.1; Geisslinger, G.1,2;
Stark, H.3; Steinhilber, D.4; Kahnt, A.S.4
1 Fraunhofer
Institute for Molecular Biology and Applied Ecology IME, Project Group
Translational Medicine and Pharmacology, Theodor Stern Kai 7, 60596 Frankfurt/Main,
Germany
2 Institute of Clinical Pharmacology, Goethe University Hospital, Theodor Stern Kai 7,
60590 Frankfurt/Main, Germany
3 Heinrich Heine University, Institute of Pharmaceutical and Medicinal Chemistry,
Universitätsstraße 1, 40225 Düsseldorf, Germany
4 Goethe University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9,
Uncontrolled inflammation is a characteristic of chronic diseases such as
rheumatoid arthritis, diabetes and atherosclerosis. Recent findings indicate that
the resolution of inflammation is an active process controlled through
endogenous mediators and mechanisms that switch off acute inflammation by
suppression of pro-inflammatory gene expression and cell trafficking and
induce inflammatory cell apoptosis and phagocytosis [1]. Lipoxins (LX) are a
unique class of arachidonic acid (AA) derived lipid mediators displaying proresolving activities during the resolution phase of acute inflammatory reactions.
In exchange between distinct cell types such as neutrophils and endothelial
cells, neutrophils and platelets or even in single cells, AA undergoes a double
oxygenation by the sequential action of two different lipoxygenases (LO)
(either 15- / 5-LO or 5- / 12-LO) to form LX [2, 3, 4]. Besides LO triggered LX
synthesis, a series of epimeric 15-LX has been found. This group of ‘alternative’ LX is aspirin triggered [2]. Here, acetylation by aspirin of cyclooxygenase2 (COX-2) abolishes prostaglandin H2 (PGH2) synthesis while retaining the
oxygenase activity of the enzyme, leading to the production of 15(R)-hydroxy5Z,8Z,11Z,13E-eicosatetraenoic acid (15(R)-HETE) instead. Epimeric 15HETE is then further processed by 5-LO to give rise to 15-epi-LXs which share
many anti-inflammatory properties with the regular LX [2,5].
Diverse cell-based, in-vitro co-culture systems have been described in
literature [2, 3, 4]. Some of them we were able to establish. First of all, we
spiked freshly isolated 5-LO positive, peripheral blood mononuclear leukocytes
(PMNL) with 10 µM 15(S)-HETE, 15(R)-HETE and 17R-hydroxy4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17(R)-HDoHE) as a proof of
principle. We were able to detect different epi- and native lipoxins as well as
124
References:
1. Willoughby, D.A. and Gilroy, D.W.: Nature Reviews Immunology, 2002, 2: 787-795.
2. Serhan, C.N. et al.: Molecular Medicine, 1996, 2: 583-596.
3. Serhan, C.N. et al.: The Journal of Pharmacology and Experimental Therapeutics,
1998, 287: 779-790.
4. Serhan, C.N. and Sheppard, K.N.: Journal of Clinical Investigation, 1990, 85: 772-780.
5. Romano, M.: The Scientific World Journal, 2010, 10: 1048-1064.
AD.10
A smart cell-based screening system for inhibitors of leukotriene
biosynthesis
Garscha, U.; Gerstmeier, J.; Werz, O.
Chair of Pharmaceutical/ Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller
University Jena, Philosophenweg 14, 07743 Jena, Germany
Leukotrienes (LT) are potent lipid mediators released during chronic inflammation, cancer, allergy, and cardiovascular diseases [1]. During the initial step of
the LT biosynthesis, 5-Lipoxygenase (5-LO) transforms liberated arachidonic
acid (AA) to 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and subsequently to leukotriene A4 (LTA4). The latter is further converted by LTA4
hydrolase to the pro-inflammatory mediator LTB4, or conjugated with reduced
glutathione by leukotriene C4 synthase (LTC4-S) to LTC4 [2]. Consequentially,
for many years, inhibition of 5-LO has played a pivotal strategy to reduce LT
biosynthesis as mean for pharmacological intervention with related diseases.
However, in intact cells 5-LO requires the nuclear membrane-bound 5lipoxygenase-activated protein (FLAP) that supplies 5-LO with AA and
facilitates the conversion of AA to 5-LO products [3]. Therefore, FLAP
represents an additional interesting target during inflammatory processes.
Unfortunately, so far a suitable screening system for new FLAP inhibitors is still
missing.
Here we present a smart cell-based model to evaluate putative 5-LO and FLAP
inhibitors. HEK293 cells were stably transfected with 5-LO alone or together
with FLAP. Upon stimulation by Ca-ionophore and 3 µM AA, co-expression of
FLAP increased product formation by 5-LO due to enhancing LTA4formation.This FLAP-mediated effect could be significantly reduced by the
known FLAP inhibitor MK886. On the other hand, MK886 was not able to
inhibit the LT biosynthesis in cells that expressed only 5-LO. Zileuton, a wellcharacterized direct 5-LO inhibitor, reduced 5-LO product formation with equal
potency, independently of FLAP expression.
Together, our test system allows evaluating the inhibitory potency of LT
synthesis inhibitors and, for the first time, enables to discriminate between
putative 5-LOX and/ or FLAP inhibitors. Furthermore, site-directed mutagenesis approaches will help to elucidate new potential sides that are targeted in
cellular LT biosynthesis.
References:
1. Peters-Golden, M. et al.: Leukotrienes. N. Engl. J. Med. 2007, 357(18): 1841-1854.
2. Panossian, A. et al.: FEBS Lett. 1982, 150(2): 511-513.
3. Dixon, R.A. et al.: Nature. 1990, 343(6255): 282-284.
AD.11
Characterization and cellular localization of protein isoforms of
human 5-lipoxygenase
Ball, A.-K.1; Saul, M.J.1,2; Hofmann, B.1; Steinhilber, D.1; Häfner, A.-K.1
1 Institute
of Pharmaceutical Chemistry, Max-von-Laue-Straße 9, 60438 Frankfurt am
Main, Germany
2 Institute of Biology, Schnittspahnstraße 10, 64287 Darmstadt, Germany
Human 5-lipoxygenase (5-LO) is the key enzyme in leukotriene (LT) biosynthesis which play an important role in many diseases like asthma bronchiale,
atherosclerosis and in many types of cancer. The 5-LO catalyzes two reaction
steps, first oxygenation of arachidonic acid to 5(S)-hydroperoxy-6-trans8,11,14-cis-eicosatetraenoic acid (5-HpETE). In a second step, 5-HpETE is
converted to the instable epoxide leukotriene A4 (LTA4) that can be further
metabolized by the LTA4 hydrolase to LTB4 or by the LTC4 synthase to the
cysteine containing leukotrienes LTC4, D4 and E4, causing chemotaxis,
vasoconstriction and tumor growth [1-3]. 5-LO is expressed in many cell types
of the human immune system like polymorphonuclear leukocytes, monocytes/macrophages and B-cells [4-6].
Recently, we were able to identify novel in-frame mRNA splice variants in Bcells and T-cells named delta 4 and delta p12. Co-transfection of delta 4 or
delta p12 with 5-LO wild type (WT) in HEK293 cells shows an influence of the
activity in contrast to transfection of WT alone. In crude cell lysates the effect
was less pronounced. Moreover, we made the observation that 5-LO is able to
form dimers [7]. Thus, we hypothesize that 5-LO and its isoforms can form
heterodimers, regulating its activity. By investigating the cellular localization of
WT and isoforms, we could determine that the 5-LO isoforms are only present
in the nuclear fraction whereas WT 5-LO can be found in both, nuclear and
non-nuclear fractions.
References:
1. Samuelsson, B. et al.: Science, 1987, 237: 1171-1176.
2. Funk, C.D.: Science, 2001, 294: 1871-1875.
3. Shimizu, T. et al.: PNAS, 1986, 83: 4175-4179.
4. Steinhilber, D.: Pharm. Acta Helv., 1994, 69: 3-14.
5. Spanbroek, R. et al.: PNAS, 1998, 95: 663-668.
6. Janssen-Timmen, U. et al.: PNAS, 1995, 92: 6966-6970.
7. Häfner, A.-K. et al.:Biol Chem, 2011, 392: 1097-1111.
AD.12
Synthesis and pharmalogical characterization of Nphenylbenzenesulfonamides as dual 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 inhibitors
Cheung, S.-Y.1; Hanke, T.1; Fischer, K.2; Listing, M.2; Werz, O.2; SchubertZsilavecz, M.1
1 Goethe
University of Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main,
Germany
2 Friedrich-Schiller University of Jena, Philosophenweg 14, 07743 Jena, Germany
Prostaglandins (PGs) and leukotrienes (LTs) are powerful bioactive lipid
mediators that have a large number of biological actions in the human body
[1,2]. The common precursor of PGs and LTs is arachidonic acid (AA). The 5lipoxygenase (5-LO) and the microsomal prostaglandin E2 synthase-1
(mPGES-1) are both enzymes which are involved in the arachidonic acid
cascade. The 5-LO is the initial enzyme which catalyzes AA to the corresponding LTs; whereas the mPGES-1 is responsible for the conversion of PGH2 into
PGE2 which is one of the most prominent mediator of inflammation, pain and
fever. A novel pharmacological approach for anti-inflammatory therapy is the
dual inhibition of 5-LO and mPGES-1. In contrast to the traditional NSAIDs the
dual inhibition of PGs and LTs might be superior over single interference with
PGs in terms of anti-inflammatory effectiveness as well as regarding reduced
side effects [3].
In this study we wanted to explore the structure-activity relationship of Nphenylbenzensulfonamide derivatives as dual 5-LO/mPGES-1 inhibitors. Lead
structure of this series was 4-(N-octyl-4-methylbenzenesulfonamido)benzoic
acid (compound 1, see Fig. below), which was originally identified by a virtual
screening approach by Waltenberger, B. et al. [4]. For this compound a facile
three-step synthesis was developed and structural optimization should be
carried out in three directions while maintaining the central Nphenylbenzenesulfonamide scaffold (see Fig. below).
References:
1. Funk, C.D.: Science 2001, 294(5548): 1871−1875.
2. Samuelsson, B., Morgenstern, R., Jakobsson, P.J.: Pharmacol Rev 2007, 59(3): 207–
224.
3. Koeberle, A., Werz, O.: Curr. Med. Chem. 2009, 16(32): 4274–4296.
4. Waltenberger, B. et al.: J. Med. Chem. 2011, 54(9): 3163–3174.
AD.13
Zafirlukast – a Dual Modulator of Soluble Epoxide Hydrolase and
Peroxisome Proliferator-Activated Receptor γ
Diehl, O.; Schader, T.; Wittmann, S.K.; Steri, R.; Schubert-Zsilavecz, M.;
Maier, T.J.; Steinhilber, D.; Proschak, E.; Kahnt, A.S.
Goethe University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438
Frankfurt/Main, Germany
Cardiovascular diseases are the major causes of death and disability in
diabetic patients due to micro- and macroangiopathic complications. The gold
standard in the treatment of type II diabetes are up to now thiazolidinediones
(TZDs) which are potent activators of PPARγ with robust insulin-sensitizing
activities. However, their clinical use is limited due to excessive weight gain,
fluid retention and increased osteoporosis risk in treated patients. Metaanalyses of clinical trials have implicated rosiglitazone in increasing the risk of
congestive heart failure, myocardial infarction, cardiovascular disease and allcause mortality leading to tightly restricted access in the United States and a
recommendation for market withdrawal in Europe. Troglitazone was withdrawn
from the market due to hepatotoxicity and pioglitazone seems to trigger
bladder cancer. Another drawback is the poor effect of TZDs on the occurrence of macrovascular events, although the equilibration of blood glucose
levels reduces microvascular complications. Therefore, there is an unmet
medical need for safer PPARγ modulating drugs and the combination of
PPARγ agonism with vasoprotective and anti-inflammatory properties in a
compound might have beneficial effects in the treatment of type II diabetes.
In previous studies we found that zafirlukast, a CysLT 1 receptor antagonist
frequently used as add-on therapy in asthma, displays anti-inflammatory
properties in the low micromolar range due to inhibition of the microsomal
prostaglandin E2 synthase. Further studies revealed that the compound also
displays PPARγ agonism (EC50 3.2 µM, max. 126%) in the same concentration
range. This was confirmed by reporter gene assays as well as by influence of
zafirlukast on mouse adipocyte differentiation. Here, zafirlukast (3 – 10 µM)
triggered triglyceride accumulation as well as target gene upregulation in a
concentration dependent manner. In addition, we found the compound to
inhibit soluble epoxide hydrolase (sEH) (IC50 1.9 µM), an enzyme which is
involved in atherosclerosis formation by depletion of endothelium-derived
hyperpolarizing factors (epoxyeicosatrienoic acids). Therefore, we postulate
that the combination of PPARγ agonism with sEH and mPGES-1 inhibition
represents a potent approach for the treatment of metabolic disease accompanied with type II diabetes mellitus. In vivo studies will answer this question in
the future.
Acknowledgements: The work has been supported by the Else Kröner-Fresenius
Foundation (EKFS), Research Training Group Translational Research Innovation –
Pharma (TRIP).
AD.14
Multi-dimensional optimization of N,4-diaryl-1,3-thiazol-2-amines
as potent 5-lipoxygenase inhibitors
Woltersdorf, S.1; Kretschmer, S.B.M.1; Rödl, C.B.1; Vogt, D.1; Steinhilber, D.1;
Stark, H.1,2; Hofmann, B.1
1 Institute
of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9,
D-60438 Frankfurt am Main, Germany; E-Mail: [email protected]
2 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University
Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany; E-Mail: [email protected]
Leukotrienes (LTs) are important lipid mediators derived from polyunsaturated
fatty acids which play an important role as regulators in immunity and the
inflammation process. These mediators have fundamental functions on
pathophysiological processes in a complex network of interactions. They play a
key role in the pathogenesis of inflammation as well as in acute and various
chronic diseases, e.g. asthma, allergic rhinitis, cardiovascular disease and
125
certain types of cancer [1]. The LT biosynthesis is initiated by the 5lipoxygenase enzyme (5-LO). It catalyzes the conversion of free arachidonic
acid to LTA4 which is subsequently converted into further LT subtypes. [2]
Up to date, there is only one approved direct 5-LO inhibitor in clinical use:
Zileuton. It acts by chelating the catalytic iron in the active site of the enzyme.
However, this drug exhibits a non-optimal pharmacodynamic and pharmacokinetic profile. Therefore, novel potent 5-LO inhibitors are of great interest for an
anti-inflammatory therapy. [3]
Starting from SKI-II, a known 5-LO inhibitor [4], we prepared a series of N,4diaryl-1,3-thiazol-2-amine based compounds with a heterogeneous substitution
pattern and investigated their influence on inhibition of human 5-LO activity.
The chemical structure of the thiazole-2-amine scaffold was further optimized
at positions R1, R2 and R3 with regard to the cytotoxicity profile, maintaining
high 5-LO inhibitory activity and selectivity.
OH
S
N
Cl
SKI-II
NH
R1
S
R2
N
R3
NH
characterized in detail regarding their binding sites in the 5-LO promoter
sequence. Three AP2 binding sites were found to be functional in the 5-LO
promoter. Interestingly, different effects were found for four different WT1
isoforms, which most likely act via the GC-boxes in the proximal 5-LO
promoter. 5-LO promoter regulation by WT1 was strictly dependent on the
presence of the amino acid stretch lysine-threonine-serine (KTS) in the WT1
protein.
Taken together, the results revealed novel aspects of 5-LO promoter activity
regulation. They may prove valuable for the understanding of 5-LO gene
(dys)regulation in pathogenesis and are part of the mechanistic picture
available for therapeutic intervention within the 5-LO pathway.
References:
1. Haeggström, J.Z., Funk, C.D.: Chem. Rev., 2011, 111: 5866-98.
2. Hoshiko, S., Rådmark, O., Samuelsson, B.: Proc. Natl. Acad. Sci. USA, 1990, 87: 90739077.
3. Silverman, E.S. et al.: Am. J. Respir. Cell. Mo.l Biol., 1998, 19: 316-323.
4. Klan, N.: Dissertation: Functional analysis of the human 5-LO promoter (Goethe
University, Frankfurt/Main) 2003.
5. Madden, S.L. et al.: Science, 1991, 253: 1550-1553.
Heterogenous substituens R1-R3
From this series a comprehensive structure-activity relationship analysis was
performed. With this, we could deduce the role of the chemical substitutes R1,
R2 and R3 on 5-LO inhibitory potency, selectivity and cytotoxicity. Among all
tested compounds, in vitro screening revealed that ST-1853 is the most potent
derivative of this series. It blocks the 5-LO activity with an IC50 value of 0.05
(0.039-0.066) M and demonstrates no signs of cytotoxicity.
AD.16
Multi-parameter optimimization of 1,3-thiazole-2-amine derivatives with potent 5-lipoxygenase inhibitory activity
Kretschmer, S.B.M.1; Rödl, C.B.1; Vogt, D.1; Woltersdorf, S.1; Stark, H.1,2;
Steinhilber, D.1; Hofmann, B.1
1 Goethe
References:
1. Gualde, N. et al.: Trends. Mol. Med. 2008, 14(10): 461-469.
2. Dennis, E.A. et al.: J. Lipid. Res. 2009, 50(6): 1015-1038.
3. Steinhilber, D., Hofmann, B.: Basic Clin. Pharmacol. Toxicol. 2014: 114(1): 70–77.
4. Suh, J. et al.: Chem. Biol. Drug. Des. 2012, 80(1): 89-98.
AD.15
Regulation of 5-lipoxygenase promoter activity by transcription
factors AP2, GATA-1, PU.1, Ets-1/2, and WT1
Fettel, J.; Wöbke, T.K.; Steinhilber, D.; Sorg, B.L.
Institut of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438
Frankfurt, Germany
Human 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of
leukotrienes, which are mediators of proinflammatory and immune modulatory
responses [1]. 5-LO is involved in the pathogenesis of atherosclerosis, asthma
and several types of cancer. In order to fully understand the pathomechanisms
underlying these conditions, which partly include 5-LO gene dysregulation, a
comprehensive understanding of 5-LO gene regulation is necessary. Furthermore, novel possibilities for therapeutic intervention might arise from the
complete mechanistic picture of 5-LO gene regulation. At this, the identification
of transcription factors involved in the transcriptional regulation of the 5-LO
gene is of particular interest.
The proximal 5-LO gene promoter is highly GC-rich and comprises nine GCboxes (GGGCGG) [2] which are targeted by the transcription factors Sp1 and
Egr-1 [3]. Besides these well characterized motifs the 5-LO promoter harbors
several other putative transcription factor binding sites although their distinct
functional significances are unknown. Among those are three putative binding
sites for the activation protein 2 (AP2) [2]. Moreover, in silico analysis
postulated putative binding sites for GATA-1, PU.1, and Ets-1/2 [4]. Additionally, the proximal 5-LO promoter contains a consensus motif for the transcription
factor Wilms' tumor 1 (WT1), which overlaps with the sequence recognized by
Egr-1 [5].
To investigate the direct effect of these transcription factors on 5-LO promoter
activity, reporter gene studies in HeLa cells were carried out with successive
promoter deletion constructs of the 5-LO gene. 5-LO promoter activity was
increased by the transcription factors AP2, GATA-1, PU.1, Ets-1/2, and WT1.
The effect of Ets-1 was proven by the use of a dominant-negative reporter
construct. Additionally, the effects accomplished by AP2 and WT1 were
126
University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438
Frankfurt am Main, Germany; E-Mail: [email protected]
2 Heinrich Heine University, Institute of Pharmaceutical and Medicinal Chemistry,
Universitätsstraße 1, 40225 Düsseldorf, Germany; E-Mail: [email protected]
5-Lipoxygenase (5-LO) mediated biosynthesis of leukotrienes (LTs) from
arachidonic acid plays a pivotal role in immunity and inflammation. LTs are
involved in the pathogenesis of asthma and allergic rhinitis, but may also play
a role in certain types of cancer and cardiovascular diseases [1]. Currently only
one 5-LO inhibitor (Zileuton, trade name Zyflo®) is approved by the FDA.
However its usage is limited due to its unfavourable pharmacokinetic profile
with a short half-life and safety issues concerning liver toxicity [2].
Therefore, there is an urge for novel 5-LO inhibitors with favourable pharmacodynamic and pharmacokinetic profiles. Substituted 1,3-thiazole-2-amines
represent a new class of inhibitors of 5-LO [3]. Yet only little is known about the
molecular mechanisms of 5-LO inhibition and other key factors in drug
development. In order to develop a successful, safe and efficacious drug
candidate a small set of 1,3-thiazole-2-amines with distinct substitution
patterns was synthesized and tested in various cell-based and cell-free
assays. Starting from N-4-hydroxyphenyl-4-(4-chloro-phenyl)-1,3-thiazole-2amine (ST1083) within this study certain derivatives were tested to investigate
the manner of inhibition as well as the dependency on different stimuli or
conditions, cytotoxicity and specificity with regard to the impact on other key
enzymes involved in eicosanoid metabolism.
OH
S
N
Cl
NH
ST1083
R1
S
R2
N
R3
N
R4
5-LO residual activity [% of control]
Acknowledgements: This work was supported by Else Kröner-Fresenius-Stiftung, TRIP,
LOEWE, OSF and Fonds der Chemischen Industrie.
H.S. and B.H. share senior authorship.
5-LO activity PMNL
ST1083
ST1853
150
100
50
0
0.0001 0.001
0.01
0.1
1
10
concentration [µM]
As a result from these studies a highly promising new lead ST-1853 is profiled,
displaying high potency in various 5-LO dependent in vitro assays. Together
with the non-cytotoxic profile as well as the selectivity this new lead establishes
the basis for further characterization of the molecular mode of action and
optimization for a novel anti-inflammatory drug approach.
Acknowledgements: This work was supported by Else Kröner-Fresenius-Stiftung, TRIP,
LOEWE, OSF and Fonds der Chemischen Industrie.
References:
1. Peters-Golden, M. and Henderson, W.R.: N. Engl. J. Med. 2007, 357(18): 1841-1854.
2. Steinhilber, D., Hofmann, B.: Basic Clin. Pharmacol. Toxicol. 2014, 114(1): 70–77.
3. Suh, J., et al.: Chem. Biol. Drug. Des. 2012, 80(1): 89-98.
100
AD.17
Development and evaluation of ST-1829 based on 5-benzylidene2-phenylthiazolones as promising agent for anti-leukotriene
therapy
Lill, A.P.1; Rödl, C.B.1; Steinhilber, D.1; Stark, H.1,2; Hofmann, B. 1
1 Institute
of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9,
D-60438 Frankfurt am Main, Germany; E-Mail: [email protected]
2 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University
Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany; E-Mail: [email protected]
Arachidonic acid released from cytoplasmic membrane upon external stimuli is
the source of one important group of inflammatory mediators: the leukotrienes
(LTs). The key enzyme of their biosynthesis is the iron-containing, heme-free
5-lipoxygenase (5-LO). LTs play a pivotal role in inflammation, allergic
disorders, asthma, cardiovascular diseases and cancer. Up to now, only one
LT biosynthesis interfering drug is marketed with limited success: the the iron
chelating 5-LO inhibitor Zileuton (Zyflo®). The need to discover new active
ligands for anti-leukotriene therapy is still urgent [1,2].
This study presents the synthesis and development of a potent and direct 5-LO
inhibitor based on the molecular pharmacologically characterized 5benzylidene-2-phenylthiazolone C06 whose further pharmacological investigation was precluded due to its low solubility [3,4]. Through optimization of C06,
extensive evaluation of the structure-activity relationships including profound
assessment of the thiazolone core and consideration of the solubility we
developed the 5-benzyl-2-phenyl-4-hydroxythiazoles as new and effective 5LO inhibitors. ST-1829, 5-(4-chlorobenzyl)-2-p-tolylthiazol-4-ol, showed an
improved 5-LO inhibitory activity in a cell-based (IC50 value 0.14 µM) and a
cell-free assay (IC50 value 0.03 µM) as well as a greatly enhanced solubility.
Furthermore, it keeps its promising inhibitory potency, even in the presence of
blood serum, excluding excessive binding to serum proteins. Together with a
non-cytotoxic profile, the thiazolone-based parent compound could successfully be optimized which thereby marks a major step towards an effective antiinflammatory therapy.
Acknowledgements: This work was supported by Fonds der Chemischen Industrie,
Deutsche Forschungsgemeinschaft DFG (SFB 1039), by the EU COST Actions BM0806,
BM1007, CM1103, and CM1207 as well as the Hesse LOEWE Schwerpunkte Fh-TMP,
OSF and NEFF, the Else-Kröner-Stiftung, TRIP and the Deutsches Konsortium für
Translationale Krebsforschung, DKTK (HS).
A.P.L. and C.B.R contributed equally to this work. H.S. and B.H. share senior authorship.
References:
1. Peters-Golden, M., Henderson, W.: N. Engl. J. Med. 2007, 357(18): 1841-1854.
2. Werz, O., Steinhilber, D.: Pharmacol. Ther. 2006, 112(3): 701-718.
3. Hofmann, B. et al.: J. Med. Chem. 2011, 54(6): 1943-1947.
4. Hofmann, B. et al.: Br. J. Pharmacol. 2012, 165(7): 2304-2313.
AD.18
AD.19
127
ANTICANCER DRUGS AND EPIGENETICS (ACE01-ACE33)
ACE.01
ACE.03
A fluorescence polarisation based phospho-Tyr Myt1 kinase
activity assay
Bicyclic acetals as potential inhibitors of Golgi alphamannosidase II
Platzer, C.1; Rohe, A.1; Schutkowski, M.2; Sippl, W.1; Schmidt, M.1
Borek, C.1; Irsheid, L.1; Weickert, A.2; Seibel, J.2; Engels, B.2; Stauber, R.3;
Schirmeister, T.1
1 Institute
of Pharmacy, Department of Medicinal Chemistry, Martin-Luther-UniversityHalle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
2 Institute of Biochemistry and Biotechnology, Department of Enzymology, Martin-LutherUniversity-Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany
The human Myt1 kinase is a negative regulator of Cdk1/Cyclin B complex,
hence, important for the G2/M transition in the cell cycle especially in cancer
cells. It may act as a drug target in anti-cancer therapy [1], but an activity
assay for assessing potential inhibitors is lacking so far. Because the natural
human Myt1 substrate Cdk1 is unsuitable for assay development [2] 2304
short chain peptides were investigated with Myt1 on a microarraychip. 11
peptides were recognized by Myt1 and EFS_HUMAN_302 was identified to be
the best Myt1 substrate. With this substrate an activity assay could be
developed. Here the steps of development of a fluorescence polarisation
based phospho-Tyr assay is presented. Therefore the kinase reaction of Myt1
full length was optimized by adaption of reaction time, temperature, buffer
system and other reaction conditions. So the pH optimum of Myt1 was
identified at 7.5 in HEPES buffer. The assay relies on the immunodetection of
the phosphorylated substrate using a phospho-tyrosin antibody and the
fluorescence labeld pentapeptide (6-FAM-) KI(pY)VV. This assay extends the
spectrum of methods to investigate the Myt1 kinase, which consist of two
binding assays so far. So it allows the exactly characterisation of Myt1
inhibitors.
References:
1. Chow, J.P., Poon R.Y.C.: Oncogene 2013, 32(10): 4778-4788.
2. Rohe, A., et al: Bioorg. Med Chem. Lett. 2012, 22(2): 1219-1223.
1 Institute
3 Ear,
Golgi α- mannosidase II (GMII) plays a crucial role in the N-glycosylation
pathway. In various tumor cell lines, the distribution of the N-linked sugars on
the cell surface is modified and correlates with the progression of tumor
metastasis [1]. GMII is therefore a molecular target for anticancer agents and
its inhibition has shown tumor repression [2]. GMII, a member of the family 38
glycoside hydrolases, cleaves two mannose units (α-(1,3) and α-(1,6)) of the
intermediate GlcNAcMan5(GlcNAc)2.The active site of the enzyme consists of
two aspartate residues and a zinc cation. GMII acts as a retaining glycosidase
and cleaves the sugars in a two-step-SN2-mechanism in which a covalent
glycosyl-enzyme complex is formed. The mechanism preserves the configuration of the anomeric C-atom [3,4]. Several natural product-based or synthetic
inhibitors have been investigated. However, the clinical use of the known
potent inhibitor swainsonine is restricted due to the side effects resulting from
inhibition of lysosomal α-mannosidases [5]. The aim of our work is the
computational design and syntheses of selective, covalent-reversible GMIIinhibitors. QM/MM calculations and molecular docking have shown that bicyclic
acetals are favorable candidates, in terms of both, high affinity to the target
enzyme and reaction kinetics. Based on L-gulose, we synthesize 1-6 bridged
derivatives which are promising lead structures.
References:
1. Fujita, T. et al.: Org. Lett. 2004, 6 (5): 827-830.
2. Van den Elsen, Y.M.H., Kuntz, D.A., Rose, D.R.: The EMBO Journal 2001, 20 (12):
3008-3017.
3. Petersen, L. et al.: J. Am. Chem.Soc. 2010, 132: 8291-8300.
4. Zhong, W. et al.: J. Am. Chem.Soc. 2008, 130: 8975-8983.
5. Cheng,T-J.R. et al: Chem. Asian J. 2013, 8: 2600-2604.
ACE.02
Evaluation of the receptor tyrosine kinase RON as therapeutic
target in pediatric sarcoma
C.1,2,
Schleithoff,
Lechtape,
Dirksen, U.1, Potratz, J.1
B.1,
Tillmanns,
A.1,
Schaefer,
C.1,
Hempel,
ACE.04
G.2,
1 Universitätsklinikum
Münster, Albert-Schweitzer-Campus A1, 48149, Germany
für Pharmazeutische und Medizinische Chemie, Westfälische WilhelmsUniversität Münster, Corrensstaße 48, 48149, Germany
2 Institut
The receptor tyrosine kinase (RTK) RON (recepteur d‘origine nantais) is a cellsurface receptor, involved in the regulation of cellular migration and with
potential relevance to cancer cell metastasis. Previous data suggested a
cross-talk of RON with the insulin-like growth factor receptor (IGF1R) in Ewing
sarcoma. Therapeutic inhibition of IGF1R has shown clinical efficacy, but
resistance is being observed, possibly due to alternative RTK activations. This
led us to investigate the role of RON in pediatric sarcoma, alone and in crosstalk with IGF1R.
RON is expressed (mRNA and protein) and constitutively activated, i.e.
phosphorylated, as were downstream signals ERK and AKT in Ewing sarcoma
and rhabdomyosarcoma cell lines. In tumor samples RON mRNA expression
was significantly higher in 6 Ewing sarcoma patients with metastases than in
15 patients with localized disease. shRNA knockdown of RON reduced cellular
migration in wound healing and Transwell assays. An N-terminal RON blocking
antibody was tested, but did not show any effect on cell proliferation or
migration, neither alone nor in combination with an IGF1R blocking antibody.
These data encouraged us to investigate RON variants such as short form (sf)
RON that lacks the N-terminal antibody-binding domain but sustains tyrosine
kinase activity. A small molecule inhibitor binding and blocking this tyrosine
kinase domain showed some activity. A second sfRON isoform was detected
in patient samples. Additional RON variants are currently being investigated.
RON is expressed and activated in Ewing sarcomas with a role in sarcoma cell
migration. RON variants were detected in cell lines and tumor samples with
potential impact on therapeutic targeting strategies.
Acknowledgements:
The project is supported by the Deutsche Krebshilfe.
128
of Pharmacy and Biochemistry, University of Mainz, GERMANY
of Physical and Theoretical Chemistry, University of Würzburg, GERMANY
Nose, Throatclinic and Policlinic, University of Mainz, GERMANY
2 Institute
The impact of latent heparanase on integrin-mediated adhesion
and migration of melanoma cells in metastatic spread – A novel
target for therapeutic interference?
Hoß, S.G.1; Gerber, U.1; Schlesinger, M.1; Naggi, A.M.2; Ilan, N.3; Vlodavsky,
I.3; Bendas, G.1
1 University
Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn, Germany
Ronzoni Institute for Chemical and Biochemical Research, Via G. Colombo 81, 20133
Milan, Italy
3 Rappaport Faculty of Medicine, Technion, P.O.B. 9649, 31096 Haifa, Israel
2 G.
Background: Heparanase is an endoglycosidase that cleaves glycosidic
chains of cell surface heparan sulfate proteoglycans (HSPG), which is
important in cancer progression, metastasis development and inflammatory
diseases. The latent, non-enzymatically active precursor form of heparanase
mediates signaling functions e.g. activation of Akt, Src or Rac via cell surface
HSPGs, which can result in stronger adhesion and migration, but the molecular
mechanisms remain elusive. Integrins appear as promising candidates for
upregulated functionality by heparanase, since integrin signaling overlaps in
part with HSPG pathways especially with those of syndecan-4 (SDC-4). In light
of our recent findings on the dominant contribution of VLA-4 to melanoma
metastasis and interference by heparin [1], latent heparanase might be a
potential target for heparin in this tumor entity.
Aim/objectives: This study aims to investigate i) whether and how latent
heparanase affects VLA-4 functionality on melanoma cell lines, and ii) whether
heparin can interfere with this activity.
Methods: A MV3 cell clone with reduced SDC-4 expression was generated
and binding of VLA-4 to VCAM-1 was determined by flow cytometry in
presence/absence of latent heparanase (2 µg/mL). Integrin-mediated cell
migration was investigated using a modified scratch assay. For a better
understanding of invasive aspects of metastasis a transmigration assay using
a modified Boyden chamber was performed. Interference of heparanase
actitvity by different heparins was investigated and mainly confirmed by
surface acoustic wave (SAW) biosensor technology, different chemical entities
were used to interfere with steps of intracellular signaling in a selective
manner.
Results: Latent heparanase affects VLA-4 activity in binding the cellular
ligands and in melanoma cell migration/transmigration via a SDC-4 dependent
pathway. An insight into the underlying mechanisms will be provided. Heparin
can interfere with the heparanase activity. To differentiate heparin effects
directly related to VLA-4 from those affecting heparanase we employed
different heparin derivatives that either bind solely latent heparanase, or bind
both heparanase and VLA-4 directly using SAW technology. Comparing MV3
wt and SDC-4 knock-down cells we could show that blockade of heparanase is
a vital part in heparin activity for reducing VLA-4 function and thus a potential
novel target in the antimetastatic approaches using low molecular weight
heparin.
Conclusions: We identified latent heparanase as a novel target of heparin in
tumor progression of melanoma cells and thereupon support the motivation to
use heparins as anti-metastatic drugs in clinics. Nonetheless, the clarification
of intracellular underlying mechanisms and involved pathways need further
investigation.
Reference:
1. Schlesinger, M. et al.: Thromb. Res. 2014, 133: 855-862.
ACE.05
Tryptophan-Induced Quenching for the Detection of Allosteric
Akt Inhibitors – Development of an Inter-Domain Interaction
Sensing System
Weisner, J.1; Fang, Z.1; Rauh, D.1
1 Technical
University Dortmund, Department of Chemistry and Chemical Biology, OttoHahn-Straße 6, 44227 Dortmund, Germany
The survival kinase Akt (Protein Kinase B/PKB) plays a pivotal role in many
cellular signal transduction pathways that are responsible for regulation of e.g.
cell proliferation, cell growth and apoptosis[1]. Dysregulation of this multidomain enzyme is directly associated with neoplastic transformation,
malignant progression as well as increased resistance to chemo- and
radiotherapy in a variety of solid tumors[2].
Novel allosteric ligands, first identified and characterized in 2005, were shown
to exclusively bind to the protein in the presence of the regulatory PH
domain[3]. These small molecules target a unique pocket that is formed at the
interface of the kinase and the PH domain in the inactive closed conformation
which allows for a specific modulation of kinase localization and activity and
thus offering great potential in medical therapy[4].
In order to allow for the selective detection of such allosteric inhibitors in highthroughput screenings an inter-domain interaction sensing system was
developed based on Tryptophan-Induced Quenching (TrIQ)[5] and the
previously established FLiK and iFliK technologies[6,7]. Therefore, artificial
tryptophans were individually introduced to the kinase domain being located in
close proximity to a fluorescent probe coupled to the PH domain in the inactive
state. The newly generated constructs were assessed via steady-state
fluorescence measurements resulting in the identification of a suitable
combination of fluorophore and artificial tryptophan.
This novel TrIQ-FLiK assay (Tryptophan-Induced Quenching of Fluorescent
Labels in Kinases) readily distinguishes between ATP-competitive and
allosteric inter-domain Akt inhibitors.
6. Simard, J.R. et al.: J. Am. Chem. Soc. 2009, 131(37): 13286-13296.
7. Fang. Z. et al.: ACS Chem. Biol. 2014, in press.
ACE.06
Lysophospholipid induced antimetastatic effects - Insights into
the underlying mechanisms
Ross, T.1; Jakubzig, B.1; Schlesinger, M.1; Raynor, A.2; Jantscheff, P.2;
Gorzelanny, C.3; Massing, U.2; Bendas, G.1
University of Bonn, Pharmaceutical Chemistry II, An der Immenburg 4, 53121 Bonn,
Germany
2 Tumor Biology Center, Freiburg, Clinical Research, Breisacher Str. 117, 79106 Freiburg,
Germany
3 Experimental Dermatology, Medical Faculty Mannheim, Heidelberg University, TheodorKutzer-Ufer 1-3, 68167 Mannheim, Germany
1
Based on the findings that empty liposomes, applied to tumor bearing mice,
possess antimetastatic effects [1] lysophosphatidylcholine (LysoPC) was
regarded as the active agent resulting from the liposomal phospholipid
degradation within the tumor tissue. Thereupon we demonstrated that a
LysoPC pretreatment of human as well as mice melanoma cells strongly
attenuated their metastatic spread in a lung invasion model in mice [2]. In vitro
studies revealed attenuated integrin functions in cell adhesion as well as in cell
migration as the functional consequence of LysoPC for affecting metastasis.
Since LysoPC did not possess apoptotic or cytotoxic effects, the underlying
molecular mechanisms of reduced integrin functionality remained open and are
the aim of this study.
Using gas chromatography it becomes apparent that upon exposure to
LysoPC, melanoma cells massively incorporate LysoPC associated fatty acids
into the cell membrane, affecting the lipid composition of the membrane
dramatically, which goes along with morphological changes of the membrane
observed by electron microscopy.
We could confirm that the shift in lipid composition alters membrane properties,
in dependence on the LysoPC species employed (C18:0 /C18:1), examined by
fluorescence based methods as well as atomic force microscopy (AFM).
Using trimethylammoniumdiphenylhexatriene as fluorescence anisotropy
probe, we detected a significant rigidification of the membrane of MV3
melanoma cells by saturated LysoPC (C18:0). These finding could be
confirmed by a fluorescence recovery after photo bleaching (FRAP) technique.
The increased membrane rigidity induced by the saturated LysoPC corresponds with a diminished receptor-mediated migration capacity of the cells,
which is induced by an affected focal adhesion complex via the signaling
function of the proteoglycan syndecan 4 and different protein kinase C
isoforms.
Consequently we suppose that the reduced integrin functionality is a result of
membrane rigidification and affected lateral raft formation with strong
consequences for underlying signaling pathways.
Our data shed a new light on liposomal drug carrier approaches in cancer
therapy with respect to novel, yet not considered activities of phospholipid
degradation products.
References:
1. Graeser, R. et al.: Pancreas 2009, 38(3): 330-337.
2. Jantscheff, P. et al.: Mol Cancer Ther 2011, 10(1): 186-197.
ACE.07
Structural analysis of novel covalent inhibitors of the epidermal
growth factor receptor
Becker, C.; Engel, J.; Rauh, D.
Technische Universität Dortmund, Fakultät für Chemie und Chemische Biologie, OttoHahn-Str. 6, 44227 Dortmund
email to: [email protected]
References:
1. Manning, B.D., Cantley, L.C.: Cell 2007, 129(7): 1261-1274.
2. Rudner, J. et al.: Radiat. Oncol. 2010, 5(108).
3. Barnett, S.F. et al.: Biochem. J. 2005, 385(2): 399-408.
4. Wu, W.I. et al.: PLoS One 2010, 5(9), e12913.
5. Mansoor, S.E., Dewitt, M.A., Farrens, D.L.: Biochemistry 2010, 49(45): 9722-9731.
The epidermal growth factor receptor (EGFR, ERBB1) is one of the most
prominent members of the ERBB family of receptor tyrosine kinases and plays
an important role in the regulation of cell proliferation and cell survival. A
dysregulation of the receptor leads to the emergence of different cancer types
[1,2]. In 10 % of all cases, non-small cell lung cancer (NSCLC) is caused by a
single point mutation L858R within the kinase domain of EGFR [3]. NSCLC
patients harbouring EGFR L858R respond well to the small molecule kinase
inhibitors erlotinib and gefitinib, at least until these patients acquire a secondary point mutation at the gatekeeper amino acid (T790M), which causes a drug
129
resistance and relapse of the disease [4,5]. Recent developments proved
covalent inhibitors to have a great potential to overcome drug resistant
mutations in NSCLC [6,7].
Here we report our efforts to elucidate the structure and binding mode of novel
covalent, mutant selective EGFR inhibitors. We were able to crystallize these
compounds in complex with the drug-resistant gatekeeper mutant T790M and
clearly confirm their covalent binding mode by protein X-ray crystallography [8].
pre-mRNA degradation. We found that RNA-based Pim1 targeting reduced
tumor growth significantly without changing liver enzyme activities or inducing
unwanted immune responses.
In a next step, we combined RNAi with statin treatment to downregulate Pim1.
Statins are competitive HMG-CoA-Reductase inhibitors with pleiotropic
antitumor effects and importantly, we found that statins can downregulate Pim1
at low micromolar concentrations. Here we show that statin treatment in
combination with Pim1-specific siRNA or other Pim1 inhibitors improves statindependent effects on Pim1, thereby decreasing the statin concentrations for
Pim1 inhibition to the sub-micromolar range. We will now further evaluate
statin effects on Pim1 in mouse xenografts of colon carcinoma and glioblastoma.
References:
1. Thomas, M. et al.: Oncogene 2012, 31(7): 918-928.
2. Ibrahim, A.F. et al.: Cancer Research 2011, 71(15): 5214-5224.
3. Weirauch, U. et al.: Neoplasia 2013, 15(7): 783-794.
4. Weirauch, U. et al.: Nucleic Acids Therapeutics 2013, 23(4): 264-272.
ACE.09
Novel strategies to overcome the cisplatin resistance of tumor
cells – Liposomes and low molecular weight heparin as promising modulators
Pfankuchen, D.1; Stölting, D.P.1; Royer, H.D.2; Bendas, G.1
Pharmaceutical Department, University of Bonn, D-53121 Bonn, Germany
of Human Genetics & Anthropology, University of Düsseldorf, D-40225
Düsseldorf, Germany
1
2 Department
Figure: Crystal structure of EGFR T790M in complex with a covalent inhibitor
(only electron density is shown).
References:
1. Arteaga, C.L., et al.: Cancer Cell 2014, 25(3): 282-303.
2. Zandi, R., et al.: Cell Signaling 2007, 19(10): 2013-2023.
3. Sharma, S.V., et al.: Nat Rev Cancer 2007, 7(3): 169-181.
4. Wong, K.K.: Lung Cancer 2008, 60 Suppl. 2: S10-18.
5. Heuckmann J.M., et al.: J Clin Oncol 2012, 30(27): 3417-3420.
6. Walter, A.O., et al.: Cancer Discov 2013, 3(12): 1404-1415.
7. Zhou, W., et al.: Nature 2009, 462(7276): 1070-1074.
8. Stamos, J., et al.: J. Biol. Chem. 2002, 277: 46265-46272.
ACE.08
RNA-based therapeutic strategies for targeting the oncogenic
Pim1 kinase
Lange-Grünweller, K.1; Weißer, A.1; Weirauch, U.2; Aigner, A.2; Grünweller, A.1;
Hartmann, R.K.1
1 Institut
für Pharmazeutische Chemie, Pharmazie, 35037 Philipps-Universität Marburg,
Germany
2 Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Klinische Pharmakologie,
04107 Universität Leipzig, Germany
The serine/threonine kinase Pim1 is overexpressed in several aggressive solid
tumors and lymphomas with a bad prognosis for patients. Pim1 activates cell
proliferation and inhibits apoptosis, and it seems that the Pim kinase family
(Pim1-3) have no essential function in healthy cells and adult tissues. We have
recently shown that Pim1 is a target of miRNA regulation by miR-33a and miR15b [1-3] and that Pim1 together with c-Myc can regulate the expression of the
oncogenic miRNA-cluster miR-17-92 at the transcriptional level [4]. Therefore,
inhibition of Pim1 seems to be an interesting antitumor strategy.
Here, we used RNAi strategies to inhibit Pim1 in mouse xenograft tumor
models of colon carcinoma and glioblastoma. Delivery of miR-33a mimics or
Pim1-specific siRNAs into tumors was achieved by forming nanoplexes of RNA
with branched low molecular weight polyethylenimine (PEI). This approach
was also used to explore a new antisense strategy in vivo which is called U1Interference (U1i) [4]. Mechanistically, the abundant U1 snRNP, a splicing
subparticle, is recruited to the last exon of a pre-mRNA by a chemically
modified oligonucleotide with dual specificity. This U1 adaptor is able to
simultaneously bind the targeted pre-mRNA and the U1 snRNA. The recruitment of the U1 snRNP results in a blockage of polyadenylation, followed by
130
Cisplatin is a well-established cytostatic agent in the therapy of ovarian
carcinoma. However, the therapeutic application and benefit of cisplatin is
often restricted by the development of chemoresistance. While resistance
mechanisms are multifactorial and not fully understood, we recently reported
on strong differences in apoptotic pathways affected by free and liposomal
cisplatin [1]. Furthermore, we also found a chemosensitizing effect of LMWH
[2]. However, the molecular mechanisms of the latter findings remained open.
The aim of this study was to obtain an insight into chemoresistance mechanisms by using liposomal cisplatin as well as heparin to assess similarities or
differences in their mode of action as chemosensitizers.
Cisplatin liposomes with a POPC/Chol/m-PEG-PE (6.5/3/0.5) lipid composition
were prepared by hydration technique as described [1]. The LMWH tinzaparin
was used up to therapeutic threshold concentrations (50 µg/mL). Cytotoxicity
(MTT-assay), cellular platinum accumulation (fAAS), transporter expression
(SDS-PAGE/WB) and expression of apoptosis proteins (antibody array) were
investigated in A2780 ovarian cancer cells and the cisplatin resistant cell line
A2780cis. Intracellular pathways after treatment with liposomal cisplatin were
analyzed by gene array data (photometric, microarray scan) and subsequent
ranked with the GeneGO software pathway tool (n-p, t-t, Mann-Whitney test for
comparison, p < 0.05).
Based on the transporter status of A2780cis cells it became evident that the
superior cytotoxicity of cisplatin liposomes is not to explain by raising influx
dependent cytotoxicity or by bypassing native uptake mechanisms. . These
results refer to a more complex mode of action of liposomes. Gene array and
protein expression data reveal that liposomal cisplatin affects apoptotic
pathways differently from the free drug bringing up a different view on
liposomes as modulators of apoptosis more than simple carriers for a drug
payload.
In contrast, LMWH affected the chemoresistance in A2780cis cells by
alterations in transporter expression independent of the cytostatic drug(formulation), but did not act via a blockade of cell adhesion molecule mediated
drug resistance (CAM-DR), as the integrin status of resistant cells checked by
flow cytometry revealed. Also for LMWH a different expression profile of
apoptosis proteins was evident, suggesting alterations in apoptotic pathways to
be the missing link between extracellular tinzaparin concentrations and its
beneficial effect on cisplatin cytotoxicity in resistant cells. Future and ongoing
studies will provide an insight into the mechanistical basis of these surprising
findings.Altogether, our data underline chemoresistance as a matter of
pathway rather than mere intracellular cisplatin concentrations.
References:
1. Stölting, D.P. et al.: Anticancer Res. 2014, 34(1): 525-530.
2. Stölting, D.P. et al: Int. J. Clin. Pharmacol. Ther. 2013, 51(1): 70-73.
ACE.10
Structure-based design and synthesis of covalent TBK1Inhibitors
Kaitsiotou, H.; Basu, D.; Rauh, D.*
Department of Chemistry and Chemical Biology, Technical University of Dortmund, OttoHahn-Straße 6, 44227 Dortmund, Germany
Protein kinases regulate cellular processes such as proliferation, differentiation, apoptosis, or inflammatory and antiviral responses. The cytosolic Ser/Thr
kinase TANK-binding kinase 1 (TBK1) is an important mediator of antiviral and
inflammatory immune responses.[1] Besides its regulating role in the mediation
of antiviral responses, TBK1 has been proposed as a potential target in cancer
therapy.[2,3] However, its detailed biological function in tumor biology remains
unclear. Therefore, deciphering the complex regulation and activation
mechanism and associated signaling pathways of TBK1 was brought into
focus and encouraged the conduction of numerous studies to identify and
develop tool compounds as well as novel TBK1 inhibitors. The recently
reported crystal structure of TBK1 in complex with the fairly potent, but nonselective, type I inhibitor BX795 has provided the structural basis for medicinal
chemistry approaches.[4]
Here we present the structure-based design approach to generate covalent
modulators of TBK1 for deciphering its role in cancer biology. Covalent binding
probe-molecules represent promising auxiliaries to investigate the function of
TBK1 in regulating cellular signaling of the antiviral immune response, as well
as its role in cancer development and progression. Based on the scaffold of
BX795 we designed and synthesized analogues incorporating Michael
acceptors for covalent biding which may enhance inhibitor selectivity and
residence time and thereby may serve as molecular probes for further
chemical biology approaches.
References:
1. Fitzgerald, K.A. et al.: Nat Immunol 2003, 4: 491-496.
2. Barbie, D.A. et al.: Nature 2009, 462: 108-112.
3. Wei, C. et al.: Proc Natl Acad Sci U S A 2014, 111: E601-610.
4. Ma, X. et al.: Proc Natl Acad Sci U S A 2012, 109: 9378-9383
ACE.11
Design and Synthesis of covalent Inhibitors to overcome drug
resistance in EGFR
Lategahn, J.; Flaßhoff, M.; Engel, J.; Becker, C.; Rauh, D.
Technische Universität Dortmund, Department of Chemistry and Chemical Biology, OttoHahn-Straße 6, D-44227 Dortmund, Germany
eMail: [email protected]
The discovery of mutations in the epidermal growth factor receptor (EGFR) has
marked a dramatic change in the treatment of non-small cell lung cancer.
Patients with EGFR-mutant lung carcinoma receiving EGFR inhibitors have a
median overall survival of more than 2 years, contrasting with the survival of
unselected patients receiving chemotherapy.[1] Acquired resistance to these
targeted drugs is in 50% of the cases mediated by a secondary point mutation
in the kinase domain of EGFR, the gatekeeper position (T790M).[2,3] The
particular size and physicochemical properties of the amino acid found at this
position are critical determinants for kinase inhibitor affinity and selectivity.
Gatekeeper mutations affect the thermodynamic and kinetic binding characteristics of all 4-amino-quinazoline-based inhibitors (originally developed to target
wild-type EGFR).[4]
Multi targeted EGFR/VEGFR inhibitor AEE788 was developed by Novartis, but
failed to inhibit the T790M drug resistant mutant variant of EGFR.[5,6] Here we
present our efforts to develop novel EGFR inhibitors based on the pyrrolopyrimidine scaffold that overcome drug resistance by reduced spatial dimension
in proximity to the gatekeeper side chain. Moreover covalent alkylation of a
unique cysteine (C797) by accurate positioning of a reactive Michael acceptor
system is achieved. Biochemical characterization substantiates our approach
of targeting gatekeeper mutant EGFR by combining covalent binding and
spatially flattened compounds.
References:
1. Heuckmann, J.M. et al.: Journal of clinical oncology 2012, 30: 3417-3420.
2. Pao, W., et al.: PLoS medicine 2005, 2: e73.
3. Kosaka, T. et al.: Clinical cancer research 2006, 12: 5764-5769.
4. Sos, M.L. et al.: Cancer research 2010, 70: 868-874.
5.Traxler, P. et al.: Cancer research 2004, 64: 4931-4941.
6. Yun, C.H. et al.: PNAS 2008, 105: 2070-2075.
ACE.12
V-ATPase regulates epithelial-mesenchymal transition in breast
cancer cells
Merk, H.1; Müller, R.2; Vollmar, A.M.1; Liebl, J.1
Department of Pharmacy, Pharmaceutical Biology, University of Munich, Butenandtstrasse 5-13, 81377 Munich, Germany
2 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for
Infection Research and Department of Pharmaceutical Biotechnology, Saarland
University, PO 151150, 66041 Saarbrücken, Germany
1
Breast cancer represents one of the leading causes of cancer related death of
females and its incidence is growing. Despite the initial effectiveness of
chemotherapy, treatment resistance limits therapeutic success of breast
cancer treatment. The relatively high rate of relapse and metastases of
aggressive breast cancer is attributed to breast cancer stem cells (CSCs)
which have self-renewing and high tumor-initiating potential, are resistant to
standard therapy and cause establishment of metastases. CSC formation is
closely linked to epithelial-mesenchymal transition (EMT) which confers
mesenchymal properties on epithelial cells [1]. Therapeutic strategies that
target breast CSCs may substantially improve breast cancer treatment and
patient prognosis.
Vacuolar H+-ATPases (V-ATPases) are ubiquitous multimeric ATP-dependent
proton pumps that acidify intracellular compartments and translocate protons
across the plasma membrane. The enzyme comprises the cytoplasmic V 1
domain that carries our ATP hydrolysis and the membrane-bound V0 integral
subunit that is responsible for proton transport from the cytoplasm to the
endosomal/lysosomal lumen or the extracellular space. V-ATPase is essential
for endocytotic processes, receptor internalization and recycling, and
lysosomal degradation [2]. Of note, recent reports show that V-ATPase is
responsible for the degradation and recycling of epithelial adhesion proteins
e.g. E-cadherin and increased V-ATPase activity has been shown during EMT
[3, 4]. We hypothesized that the proton pump V-ATPase is such a target
affecting EMT and CSC formation.
For our study we used the V-ATPase inhibitor Archazolid A. Archazolid A is a
natural compound, first isolated from cultivated myxobacteria Archangium
gephyra and is also available by chemical synthesis [5]. Like the known VATPase inhibitors concanamycin or bafilomycin A1, it binds to the V0 subunit c
and inhibits V-ATPase activity. To investigate whether Archazolid A influences
EMT, we used immortalized human mammary epithelial cells (HMLEs) with
tamoxifen-inducible TWIST transcription factor overexpression. Activated
TWIST is a direct suppressor of E-cadherin and activates EMT markers like Ncadherin or vimentin and therefore promotes EMT. Archazolid A treatment
during EMT decreased migratory capability of HMLEs. Moreover, HMLEs
which have already undergone EMT and thus show mesenchymal properties
show decreased migration after Archazolid A treatment. To analyze a potential
implication of V-ATPase in breast CSCs, mammosphere assays were
performed. The mammosphere assay is based on the fact that only breast
CSC can survive in suspension culture and mature tumor cells die by anoikis.
In fact, Archazolid A decreased mammosphere formation of mesenchymal
HMLEs that have undergone EMT.
In summary, our results indicate that the V-ATPase inhibitor Archazolid A
inhibits migration of breast CSC and point to a function of V-ATPase in EMT
and breast CSC formation. Thus, V-ATPase inhibition by Archazolid A might
be investigated as potential new strategy for the treatment of invasive and
metastatic breast cancer.
Supported by DFG FOR 1406
131
References:
1. Sendurai, A. Mani et al.: Cell, 2008, 133(4):704-715.
2. Forgac, M.: Nat Rev Mol Cell Biol., 2007, 8(11):917-929.
3. Le, T.L.; Yap A.S.; Stow, J.L.: J Cell Biol, 1999, 146:219-232.
4. Cao, X. et al.: Am J Physiol Renal Physiol, 2012, 302:F1121-F1132.
5. Sasse, F. et al.: J Antibiot, 2003, 56(6):520-525.
ACE.14
V-ATPase inhibition affects iron metabolism: a novel therapeutic
option for breast cancer
Schneider, L.S.; von Schwarzenberg, K.; Vollmar, A.M.
ACE.13
Department of Pharmacy, Pharmaceutical Biology, University of Munich, Butenandtstraße
5-13, 81377 Munich, Germany
Synthesis and characterization of new substrate-analogue
matriptase inhibitors
Maiwald, A.; Hammami, M.; Wagner, S.; Heine, A.; Klebe, G.; Steinmetzer, T.
Institute of Pharmaceutical Chemistry, Marbacher Weg 6, Philipps University, 35032
Marburg, Germany
Matriptase is a type II transmembrane protein that contains an extracellular
trypsin-like serine protease domain at the C-terminus. In vitro experiments
revealed that matriptase can activate a number of substrates involved in tumor
progression and metastasis such as the proforms of hepatocyte growth
factor/scatter factor (HGF), urokinase-type plasminogen activator (uPA),
protease-activated receptor 2 (PAR-2), the cancer-related growth factor
macrophage-stimulating protein 1 (MSP-1), or the metalloproteases MMP-3
and MMP-1. Furthermore, matriptase is often upregulated in cancer, e.g. in
gastrointestinal tract, breast, ovary, prostate, cervix and lung tumors. Therefore, matriptase appears to be a promising target for cancer therapy. Moreover, recent studies suggest matriptase as a major PAR-2 activator in osteoarthritis leading to increased collagenase expression involved in cartilage
degradation.
Several groups have described substrate-analogue inhibitors containing a Cterminal 4-amidinobenzylamide in combination with an N-terminal sulfonyl
residue as inhibitors for various trypsin-like serine proteases such as thrombin,
factor Xa, factor VIIa, uPA, plasmin and plasma kallikrein. Therefore, we have
screened analogues of this type available from previous studies as matriptase
inhibitors. The most potent compound contains an N-terminal benzylsulfonyl
group in P4 position and a D-homoPhe-Pro moiety as P3-P2 segment.
However, this compound suffers from poor selectivity and is a stronger
thrombin and factor Xa inhibitor. Based on this result several new analogues
have been prepared by incorporation of substituted D-homophenylalanines in
P3 position, whereby the highest inhibitory potency was found for derivatives
containing D-homotyrosine. Further replacement of the P2 proline by alanine
and elimination of the N-terminal benzylsulfonyl group changed the selectivity
profile and provided a first substrate-analogue inhibitor (MI-470), which exhibits
a stronger affinity for matriptase compared to thrombin and factor Xa.
NH2
O
H2N
N
H
H
N
O
NH
MI 470
K i Matriptase = 26 nM
K i Thrombin = 302 nM
K i Factor Xa = 570 nM
OH
Due to the insufficient amount of available matriptase we used trypsin for
crystallization in complex with inhibitor MI-470. The refined structure of the
trypsin/MI-470 complex was superimposed with the known crystal structure of
matriptase. The obtained model reveals that the D-homoTyr side chains binds
into a well defined binding pocket above Trp215 of matriptase, which is
surrounded by Phe99 on the right and Gln175 on the left side.
132
Within the last decade evidence is increasing that the vacuolar H+-ATPase (VATPase), a heteromultimeric proton pump, plays a vital role in the survival of
tumor cells. However, the precise mode of action still awaits molecular
explanation. To investigate V-ATPase mediated cytotoxicity we used the
myxobacterial derived compound archazolid, a highly potent V-ATPase
inhibitor. Here we show that V-ATPase inhibition clearly induces apoptosis in
breast cancer cells in vitro. Remarkably, these findings were recapitulated in
vivo using a 4T1 mammary mouse model which showed reduced tumor growth
upon archazolid treatment.
With regard to the molecular mechanisms of V-ATPase related cell death we
found that archazolid stabilizes the HIF1α protein which is associated with the
apoptosis induction in p53 wild type tumor cells. We unveil that the stabilization
of HIF1α is due to iron depletion in the cytosol and associate this with
disrupted transferrin/transferrin-R internalization upon V-ATPase inhibition. As
a consequence, activity of the iron dependent enzyme ribonucleotide
reductase is diminished leading to S-phase block and double strand breaks.
Finally, we connect the HIF1α expression as well as the occurrence of double
strand breaks with the stabilization of the tumor suppressor protein p53. This
eventually connects V-ATPase inhibition to fundamental cellular processes
such as DNA synthesis, DNA repair and apoptosis. Hence, our study reveals a
novel mode of action for V-ATPase induced cell death in tumor cells and
suggests V-ATPase inhibition as a promising and viable strategy for breast
cancer therapy.
Supported by DFG FOR 1406
Acknowledgments: We thank Prof. Dr. Dirk Trauner (University of Munich) for synthesizing
archazolid, Prof. Dr. Dirk Menche (University of Bonn) for isolating archazolid and Dr.
Rebekka Kubisch and Prof. Dr. Ernst Wagner (University of Munich) for performing the in
vivo studies.
ACE.15
CDK5 regulates angiogenesis via stabilizing HIF-1α in endothelial
and liver tumor cells: a novel signaling mechanism with potential
importance for HCC therapy
Herzog, J.1; Ehrlich, S.M.1; Liebl, J.1; Fröhlich, T.2; Mikulits, W.3; Vollmar, A.M.1;
Zahler, S.1
1 Department
of Pharmacy, Pharmaceutical Biology, University of Munich, Butenandtstr. 513, 81377 Munich, Germany
2 Gene Center Munich, University of Munich, Feodor-Lynen-Strasse 25, 81377 Munich,
Germany
3 Department of Medicine I, Division: Institute of Cancer Research, Comprehensive
Cancer Center Vienna, Medical University of Vienna, Spitalgasse 23,1090 Vienna, Austria
The cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase which has
recently been shown to regulate angiogenesis [1]. However, the underlying
signaling mechanisms are still unclear. We hypothesized that CDK5 might be
involved in hypoxic signaling. Hypoxia is often a key feature of tumor progression, promoting the expression of angiogenic factors by hypoxia inducible
factors (HIFs) [2]. The aim of this study was to characterize the link between
CDK5 and HIF in endothelial cells (EC) and liver tumor cells (HUH7) in vitro
and in vivo, since hepatocellular carcinoma (HCC) is one of the most vascularized solid tumors [3].
We could show that pharmacological inhibition of CDK5 with roscovitine as
well as transient and stable knockdown lead to a significantly reduced protein
level of HIF-1α in EC and HUH7 cells. Additionally, the transcription of HIF
target genes involved in the regulation of angiogenesis such as VEGF A or
VEGFR1 was decreased. Of note, immunhistochemistry of HIF-1α in murine
liver cancer tissue sections confirmed a reduced protein level of the transcription factor by CDK5 inhibition.
As treatment of CDK5 knockdown cells with a proteasomal inhibitor (MG132)
results in an increase of HIF-1α, we assume that CDK5 is involved in the
stabilization of the transcription factor. Our data showing that CDK5 coimmunoprecipitates with HIF-1α and CDK5 phosphorylates HIF-1α in vitro
underline the hypothesis that both proteins directly interact. In mass spectrometry analysis, Serine 687 could be identified as a CDK5 phosphorylation site on
HIF-1α putatively involved in the regulation of the HIF-1α level. Remarkably,
mutation studies could confirm the importance of this phosphorylation site for
HIF-1α stability.
As in vivo proof of our concept, CD31 staining of tissue sections from xenograft
and orthotopic liver cancer tumor models revealed a decreased number of
microvessels for CDK5 knockdown tumors and tumors from mice treated with
roscovitine or its derivatives.
In summary our data indicate a direct phosphorylation of HIF-1α by CDK5 at
Serine 687, resulting in a stabilization of the transcription factor thereby
promoting angiogenesis. Since the expression of HIFs in HCC patients is
associated with a poor prognosis [3], CDK5 might be a promising target for
therapy.
Acknowledgements: This study was supported by the German Research
Council (DFG) grant ZA 186/7-1.
References:
1. Liebl, J. et al.: J. Biol. Chem. 2010, 285(46): 35932-35943.
2. Liao, D., Johnson, R.S: Cancer Metastasis Rev 2007, 26: 281-290.
3. Li, S. et al.: Hepat Mon. 2011, 11(10): 821-828.
ACE.16
ACE.17
Pim-1 dependent transcriptional regulation of the oncogenic
miR-17-92 Cluster
Schulte, F.W.1; Seidler, S.1; Thomas, M.1; Lange-Grünweller, K.1; Weirauch,
U.2; Aigner, A.2; Grünweller, A.1; Hartmann, R.K.1
1 Institute
of Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-University Marburg,
Marburg, Germany
2 Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology,
University of Leipzig, Leipzig, Germany
MicroRNAs (miRNAs) are small non-coding RNAs, important for the posttranscriptional regulation of gene expression. In general, miRNAs are derived
from RNA polymerase II primary transcripts (pri-miRNA) that are further
processed to ~70 nt precursors (pre-miRNA) and after nuclear export to
mature miRNAs by the activity of two endonucleases. miRNAs are incorporated into the miRNA-induced silencing complex (miRISC) and act as
repressors of translation by imperfect base-pairing to their target sites in
mRNAs. The majority of miRNAs is encoded in intronic regions, either
individually or as miRNA clusters that are cotranscribed. Several miRNAs are
involved in tumourigenesis, accounting for their designation as tumoursuppressing or as oncogenic miRNAs. Such miRNAs can downregulate targets
involved in the regulation of apoptosis or cell cycle progression. Thereby they
are interesting targets for cancer therapies.
Chemical-modified miRNA inhibitors as high-potential anticancer
molecules
Harloff, M.; Lange-Grünweller, K.; Hartmann, R.K.; Grünweller, A.
Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-University Marburg,
Marbacher Weg 6, 35032 Marburg, Germany
1
MicroRNAs (miRNAs) are small (21-23 nt) non-coding RNAs with important
regulatory functions, which inhibit gene expression at the post-transcriptional
level via RNA-Interference (RNAi). Approximately 30 % of the protein-coding
genome is regulated by miRNAs. Thus miRNAs are involved in critical
pathogenic processes in human diseases. Several studies have shown that
small regulatory RNAs are deregulated in cancer cells, thus miRNA replacement or miRNA inhibition are interesting new anticancer strategies. In the case
of up-regulated miRNAs, complementary antisense oligonucleotides (ASO), so
called antimiRs, are used to block miRNAs sterically through Watson-Crick
base-pairing. Given that RNA is generally not stable in biological fluids, a lot of
different chemical modifications and delivery strategies have been developed
to improve the properties of ASOs, e.g. 2’- O-methylations in combination with
a phosphorothioate backbone are often used in antisense designs. Another
well-known strategy is the incorporation of Locked Nucleic Acids (LNA) into
ASOs, which leads to substantially increased nuclease stability. In our group
especially short all-LNA antimiRs are used to block miRNAs. These miRNA
inhibitors, which we termed LNA-antiseeds, target the highly conserved seed
region of miRNAs, making this a valuable strategy to inhibit entire miRNA
families. We already have shown that LNA (PO) 14-mer antiseeds against
ongogenic miR-17-5p and miR-20a efficiently derepress the p21 tumor
suppressor. Moreover, we showed functional delivery of LNA-antiseeds into
cancer cells upon complexation with the branched cationic polymer polyethylenimine (PEI F25-LMW) [1]. With longer antimiRs, however, it is possible to
block specifically single miRNAs. We are currently performing studies with
different ASO designs against the oncogenic miRNAs miR-19a, miR-21 and
miR-155 to test their inhibitory potential in a glioblastoma model. We have
analyzed the effects of oncogenic miRNA inhibition on cell proliferation and on
the derepression of some miRNA targets with the goal to identify the most
efficient ASOs for further exploring their anticancer effects in vivo. Another goal
is to optimize antisense strategies by developing miRNA inhibitors with novel
chemical modifications. In this project, we like to improve the efficiency of
miRNA inhibitors by coupling artificial chemical nucleases (guanidine-analogs).
Moreover, specificity of ASO-dependent inhibition is another important issue.
The coupling of photocaging groups to miRNAs or miRNA inhibitors to activate
them with a light signal in a temporally and spatially controlled manner is a
promising new approach that we try to establish for specific RNA targeting.
Especially the human miRNA cluster miR-17-92, which encodes six miRNAs,
is overexpressed in several solid tumours and some hematopoietic malignancies. Because of numerous targets of its individual miRNAs, the miR-17-92
locus exerts pleiotropic functions during development, proliferation, apoptosis
and angiogenesis. To understand transcriptional regulation of oncogenic
miRNAs is an important issue for cancer therapy; unfortunately, the most
miRNA promoters have not been characterized or even identified yet. In the
case of the miR-17-92 cluster, expression is in part controlled by a host gene
promoter, which is regulated by the transcription factor E2F. Interestingly, the
AT-rich region between the host gene promotor and the miRNA coding region
reveals a host gene promoter independent transcriptional activity. Deletion
analysis shows a strong reliance on the binding of c-Myc to a functional c-Myc
binding site (E3 site), a conserved E-Box element about 1.5 kb upstream of the
miRNA coding region. Furthermore, the proto-oncogenic kinase, Pim-1, its
phosphorylation target HP1γ and c-Myc colocalize to this E3 region, as inferred
from chromatin immunoprecipitation. Analysis of pri-miR-17-92 expression
revealed that siRNA-mediated knockdown of E2F3, c-Myc or Pim-1 negatively
affects cluster expression, with a synergistic effect caused by c-Myc/Pim-1
double knockdown [1]. We have recently shown that Pim-1 is also a target for
miRNA regulation by miR-33a [2]. Interestingly, a miR-33a mimic reduces
transcriptional activity of the A/T-rich sequence as well.
In some preliminary experiments we observed an influence of statins (HMGCoA-Reductase inhibitors) on the expression levels of single miRNAs like miR33a. As a consequence of these results we want to analyze the effects of
statins on miR-17-92 cluster expression levels by quantitative PCR (qRT-PCR)
and further on global miRNA levels via high-throughput sequencing. With these
approaches we hope to get a deeper insight into the observed anticancer
effects of statins.
References:
1. Thomas, M. et al.: Int. J. Mol. Sci. 2013, 14: 12273-12296.
2. Thomas et al.: Oncogene 2012, 31(7): 918-928.
References:
1. Thomas, M. et al.: RNA Biol. 2012, 9(8): 1088–1098.
133
ACE.18
Bacterial Tumor Cell Targeting using the Autodisplay Technology
Weckenbrock, W.V.; Blaßhofer, F.; Jose, J.
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische
Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany
In the last decades several approaches have been made towards using
bacterial infection in the treatment of cancer diseases. In the 1940´s it was
already shown that attenuated strains of Clostridium tetani accumulated in
cancer tissue lacking oxygen, thus predominantly affecting malignant cells and
sparing healthy tissue [1]. Similar experiments using attenuated Salmonella
strains have even reached phase I clinical trials [2].
Autodisplay is a well-established system for surface display of proteins on
gram-negative bacteria [3]. The surface expression of functionally active
antibody fragments directed against a tumor factor could allow to use
genetically modified cells as bacterial drugs e.g. in gastroenterologic cancer
therapy. The antibody fragment 425 directed against the Epidermal Growth
Factor Receptor (EGFR) is a suitable candidate for targeting strategies, since it
is known for its specificity and high affinity towards EGFR.
In this study the surface display of a functional single chain variable fragment
(scFv) of anti-EGFR antibody 425 on the surface of Escherichia coli was
performed. The affinity of E. coli cells displaying the antibody fragment towards
tumor cells overexpressing EGFR was demonstrated in FACS and fluorescence microscopic assays. Specificity of binding to the EGF-receptor was
proven in experiments using siRNA down-regulation of this receptor.
Apart from usage for directed infection of malignant tissues and drug targeting
applications, autodisplay of antibody fragments provides the possibility of a
simple and rapid screening tool for new antibodies against pre-given antigens,
for example via fluorescence activated cell sorting.
References:
1. Parker, R.C. et al.: Proc Soc Exp Biol Med 1947, 66: 461-467.
2. Toso, J.F. et al.: Journal of Clinical Oncology 2002, 20: 142-152.
3. Jose, J.: Appl Microbiol Biotechnol 2006, 69: 607-614.
ACE.19
Cisplatin resistance is associated with altered signalling in
NSCLC cells
Sarin, N.1; Engel, F.2; Kalayda, G.V.1; Roberto, S.1; Frötschl, R.2; Cinatl jr., J.3;
Rothweiler, F.3; Michaelis, M.4; Jaehde, U.1
Institute of Pharmacy, Clinical Pharmacy, University of Bonn, An der Immenburg 4,
53121 Bonn, Germany
2 Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175
Bonn, Germany
3 Institute of Medical Virology, Goethe University Hospital Frankfurt,Frankfurt/Main,
Germany
4 Centre for Molecular Processing and School of Biosciences, University of Kent,
Canterbury, UK
1
Background: Platinum complexes are still widely used in the treatment of
several cancer entities, among them non-small cell lung cancer (NSCLC). The
treatment outcome is often limited by the development of resistance, with the
exact mechanisms still posing many questions. There are hints that altered
signalling plays a role in the development of cisplatin resistance.
Aim: This projects aims at revealing signalling differences between cisplatinsensitive and cisplatin-resistant NSCLC cells.
Methods: Cisplatin-sensitive A549 and resistant A549Pt NSCLC cells were
characterized for cisplatin cytotoxicity (MTT assay) and drug uptake. Additionally, the activation of the ERK signalling was analyzed on the proteome level.
Gene expression was measured with a whole genome array. Cells were
treated with 10 µM cisplatin for 24 h after 4 h of serum starvation.
Results: Cisplatin cytotoxicity is markedly reduced in resistant cells (pEC 50
4.141 ± 0.013; mean ± SD, n=3) as compared to the sensitive cell line (pEC50
= 4.332 ± 0.048; n=4). Intracellular platinum accumulation in resistant cells
was reduced to 0.363 ± 0.116 ng platinum/µg protein (n=8), compared to
sensitive cells exhibiting 0.939 ± 0.151 ng platinum/µg protein (n=9). Although
basal ERK activation and expression were higher in cisplatin-resistant cells,
134
cisplatin treatment led to ERK activation in both cell lines. Gene expression
analysis exhibits similarities as well as differences of activated signalling
pathways between sensitive and resistant cell lines and between treatment
conditions.
Conclusions: Although cisplatin-resistant NSCLC cells exhibit a reduced
cellular uptake and a higher basal ERK expression, ERK activation was not
different from the sensitive cells. The preliminary pathway analysis suggests
that sensitive cells treated with cisplatin regulate the same pathways which are
altered in resistant cells.
ACE.20
Effect of GRP78 knockdown on cisplatin cytotoxicity in ovarian
cancer cells
Kullmann, M.1; Kotz, S.2; Hellwig, M.1; Metzger, S.2; Kalayda, G.V.1; Jaehde,
U.1
1 Institute
of Pharmacy, Department of Clinical Pharmacy, University of Bonn, An der
Immenburg 4, 53121 Bonn
2 Cologne Biocenter, University of Cologne, Zülpicher Straße 47b, 50674 Cologne
Cisplatin is an effective treatment for different cancer entities. Besides toxic
side effects, acquired resistance occurs frequently and compromises therapy
outcome. An increased intracellular formation of biologically inactive adducts
with proteins or peptides seems to be a relevant factor contributing to
resistance.
This project aimed at identifying intracellular binding partners of CFDA-cisplatin
(CFDA-Pt), a fluorescent cisplatin analogue. [1] The effect of these proteins on
cisplatin cytotoxicity was examined by siRNA-mediated knockdown.
The ovarian cancer cell line A2780 and its cisplatin-resistant subline A2780cis
were exposed to 25 µM CFDA-Pt for 2 h. Fractionated cell lysates were
separated by 2D gel electrophoresis. Protein spots were digested and
analysed by high-resolution mass-spectrometry (ESI-MS/MS). Amongst others,
we investigated glucose-regulated protein 78 (GRP78) as binding partner of
CFDA-Pt. We performed densitometric Western Blot analysis of GRP78
following cisplatin treatment. SiRNA directed against GRP78 was transfected
into A2780 and A2780cis cells with the K2® transfection system. Cisplatin
cytotoxicity was compared in non-transfected and transfected cells using an
MTT-based assay.
Following treatment with CFDA-Pt and fractionation of cell lysates several
intracellular protein-adducts were identified, including adducts with GRP78.
Knockdown of GRP78 was achieved with a relative protein expression of 59 ±
9 % and 37 ± 8 % in A2780 and A2780cis cells, respectively, each compared
to untreated controls. No significant change of cisplatin cytotoxicity after
GRP78 knockdown was observed (pEC50A2780 = 5.356 ± 0.07 vs.
pEC50A2780+GRP78kd = 5.369 ± 0.03 and pEC50A2780cis = 4.745 ± 0.06 vs.
pEC50A2780cis+GRP78kd = 4.625 ± 0.05).
In conclusion, we established a method for identification of intracellular CFDAPt-protein adducts. There appears no significant influence of GRP78 knockdown on cisplatin cytotoxicity. Further Pt binding partners will be assessed to
identify proteins contributing to cisplatin resistance.
This project is supported by the Deutsche Forschungsgemeinschaft (JA 817/41).
Reference:
1. Molenaar, C. et al.: J. Biol. Inorg. Chem. 2000, 5: 655–665.
ACE.21
Tumor-specific Ligands for Targeted Delivery of Polymer-based
Nucleic Acid Complexes
Kietz, A.1; Vornicescu, D.2; Keusgen, M.2; Aigner, A.1; Höbel, S.1
1 Rudolf-Boehm-Institute
for Pharmacology and Toxicology, Clinical Pharmacology,
Faculty of Medicine, University of Leipzig
2 Institute for Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-University Marburg
The specific knockdown of disease-related genes by induction of RNAInterference (RNAi) or micro-RNA replacement are highly attractive intervention strategies for the treatment of cancer. One of the major hurdles to be
overcome in gene therapy approaches is the efficient delivery of therapeutically active nucleic acids. Upon systemic administration in vivo, targeted delivery
systems are an essential prerequisite to enhance therapeutic effects and to
reduce unwanted actions.
Within the group of non-viral delivery systems, several compounds based on
cationic polymers and lipids are under intense investigation. Among those
compounds, polyethylenimine (PEI) takes a prominent position due to the socalled “proton-sponge-effect”. The molecular structure of PEI and its ability to
form complexes with nucleic acids (polyplexes) provide the basis for the
generation of targeted nanoparticles by modification with cell-specific ligands
(see Figure 1).
Complexation
Endocytosis
Cetuximab-modified
PEI/nucleic acid polyplexes
Nucleus
Ago2
Transport to
the Nucleus
Activation
of RISC
Endosome
Cl-
H+
Cl-
H+
Rupture
H2O
H2O
H2O
H2O
Cl-
H+
Cytoplasm
Figure 1. Mode of action of Tumor-specific
polyplexes.
Laser
SPR-Detetctor
Figure 2. SPR-Meaasurements of TumorSpecific Polyplexes.
Here, we present a tumor-specific delivery system based on covalent coupling
of the antibody cetuximab to our low molecular weight PEI F25-LMW [1].
Exhibiting high biocompatibility and biological activity this PEI proved to be an
efficient platform for the delivery of DNA and small RNAs in vitro and in vivo
[2,3]. Ligand-modification of PEI was performed via a PEG-spacer to reduce
non-specific interactions, which is of high interest especially for systemic
administration of the polyplexes. This coupling procedure yielded a targetspecific gene carrier as demonstrated by surface plasmon resonance (SPR)
measurements [4]. Using this method, we were able to real-time investigate
the specific binding of cetuximab-modified PEG-PEI as well as cetuximabmodified PEI/siRNA complexes to immobilized epidermal growth factor
receptor (EGFR) without labelling (see Figure 2). Furthermore, ligandmediated uptake of the polyplexes by EGFR-overexpressing cells was shown
by flow cytometry experiments and by carrier-mediated transfection of a
reporter gene.
In addition, we report on the generation of a novel tumor-specific protein (TSP)
as targeting ligand. Employing a bacterial expression system, a recombinant
peptide is produced that binds to cell surface structures highly abundant on
various tumor cells. Although quantitative amounts of TSP are expressed, only
a very small portion of the protein is part of the soluble fraction. Due to an
optimized purification procedure relying on denaturing conditions and
subsequent column-based refolding of the protein, sufficient amounts are
obtained. As demonstrated by flow cytometry measurements, TSP is biologically active and ready to use for coupling to PEI.
Taken together, we established coupling of the antibody cetuximab to PEI via a
PEG-spacer resulting in a targeted drug delivery system that is a very
promising platform for therapeutic knockdown strategies in vivo. Moreover, we
present the development of a tumor-specific protein for the generation of novel
tumor-targeted polyplexes.
Acknowledgments: Deutsche Krebshilfe (A.A.), DFG Forschergruppe
Nanohale (A.A.), Junior Research Grant from the Medical Faculty, University of
Leipzig (S.H.)
References:
1. Werth, S. et al.: J. Control. Release. 2006, 112(2): 257-270.
2. Höbel, S. et al.: Eur. J. Pharm. Biopharm. 2008, 70(1): 29-41.
3. Höbel, S. et al.: J. Gene. Med. 2010, 12(3): 287-300.
4. Höbel, S., Vornicescu, D. et al.: Anal. Chem. 2013, Nov 20.
ACE.22
Halogenated Gold(I) NHC Complexes and their Antiproliferative
Effects on Tumor Cells and Bacteria
Schmidt, C.1; Sergeev, G.2; Franke, R.2; Brönstrup, M.2; Ott, I.1
Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstr. 55, 38106 Braunschweig, Germany;
2 Department of Chemical Biology, Helmholtz Centre for Infection Research GmbH,
Inhoffenstr. 7, 38124 Braunschweig, Germany
1
Thioredoxin reductase (TrxR) is an example for a relevant enzyme, which
protects cells against oxidative stress and apoptosis. It is upregulated in
carcinoma cells and represents a possible target in cancer chemotherapy.
Especially gold(I) containing compounds inhibit TrxR in vitro, due to the high
affinity of gold to selenocysteine moieties. A well-known drug is auranofin,
which is established in the current therapy of rheumatoid arthritis. It shows
antiproliferative effects and triggers a remarkableTrxR inhibition.
N-heterocyclic carbene (NHC) metal complexes have already shown antiproliferative effects on tumor cell lines as well as bacteria and have proven their
potential as anticancer and antibacterial agents. [1-6]
New halogenated mono- and bis-NHC-gold(I)-complexes of the imidazole-,
benzimidazole- or phenylimidazole-type were synthesized, purified and
characterized (see figure 1). To study the effects on tumor cell growth, they
were tested against tumorigenic HT-29 colon carcinoma cells, MCF-7 breast
carcinoma cells, MDA-MB-231 breast carcinoma cells and non-tumorigenic
RC-124 human kidney cells. All complexes showed cell growth inhibition with
IC50 values in the low micromolar range. In particular the activities of the bisNHC-complexes were comparable to auranofin.
The potency of TrxR inhibition of the gold(I) derivatives was determined with
IC50 values in the submicromolar range. Cellular uptake studies were performed using high resolution continuum source atomic absorption spectroscopy to quantify the intracellular concentration.
Assays for antibacterial activities also proved the antiproliferative activities of
the new halogenated gold(I) NHC complexes on different gram positive as well
as gram negative bacterial strains. Agar diffusion tests were performed and the
minimum inhibitory concentrations (MIC) were calculated. For some bacterial
strains the gold(I) complexes showed lower MIC values than the established
antibiotics ciprofloxacin and vancomycin, which were used as references.
In conclusion, halogenated mono- and bis-NHC-gold(I)-complexes trigger
strong antiproliferative effects in tumor cells as well as bacteria. Their mode of
action involves the inhibition of thioredoxin reductase and related enzymes.
References:
1. Hickey, J.L. et al.; J Am Chem Soc. 2008, 130: (38):12570-12571.
2. Oehninger, L. et al.; Dalton Trans. 2013, 42, (10):3269-3284.
3. Liu, W. et al.; Chem. Soc. Rev. 2013, 42: 755-773.
4. Hackenberg, F. et al.; Organometallics 2013, 32: 5551−5560.
5. Öznur, D. et al.; MonatshChem. 2013, 144: 313–319.
6. Fernández, G.A. et al.; J. Inorg. Biochem. 2014, 135: 54–57.
ACE.23
Synthesis and biological studies of new alkynylgold(I)(NHC)complexes as anticancer agents
Prochnicka, A.; Ott, I.
Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstraße 55, D-38106 Braunschweig,Germany
Current research activities deal with a great variety of metal-complexes,
containing gold, ruthenium or platinum. Cisplatin and Carboplatin, for example,
are used as anticancer agents and Auranofin is established in the therapy of
rheumatoid arthritis.
Organometallic gold(I) complexes are a new potent group of drugs, that show
activity against tumor-relevant enzymes like thioredoxin reductase (TrxR) and
have good antiproliferative activity in-vitro. [1] They are part of the attempt to
develop new anticancer agents, which particularly show fewer side effects, a
good tumor cell selectivity and no drug resistance. N-heterocyclic carbenes
(NHC), phosphanes or alkynyls as ligands of gold(I) offer a high potential for
the design of new anticancer agents. [2] Cellular uptake studies indicated, that
such gold(I) complexes can accumulate in cancer cells. Many of these gold(I)
complexes also showed selective activity against TrxR, which is one of the
overexpressed enzymes in cancer cells. [3] A decrease in respiration
suggested mitochondria as a further possible target. The group of alkynylgold(I)(phosphane)-complexes triggered anti-angiogenic effects, as shown in
former work with zebrafish embryos. [4]
The choice of stably coordinated ligands is critical in the design of new gold
metallodrugs. Based on previous results we chose alkynyl and NHC ligands for
coordination to gold(I) (see figure 1). The resulting alkynyl-gold(I)(NHC)complexes offer a system with high stability under physiological conditions.
The structural versatility of the organic ligands allows a broad variety of
structural modifications and the preparation of compound libraries.
135
In this pilot study alkynylgold(I)(NHC)-complexes, containing different NHC
ligands derived from imidazole and benzimidazole and 1-ethynyl-4methoxybenzene, were synthesized, purified and analyzed by nuclear
magnetic resonance spectroscopy (NMR), mass spectroscopy and elemental
analysis. Cytotoxic and antiproliferative effects were evaluated in three tumor
cell lines (MDA-MB-231 breast carcinoma, MCF-7 breast carcinoma and HT29 colon carcinoma) as well as in healthy human kidney cell line (RC-124). To
get more information about the biological properties of the new compounds,
the activity against TrxR was measured and the cellular uptake was quantified
using high resolution continuum source atomic absorption spectroscopy.
Conclusion: Model 2, including a saturable drug concentration-effect
relationship on kprol with an additional elimination rate constant of proliferating
cells, was able to successfully characterise the data. Given the more physiological approach, this model might be able to use in vitro measurements of
cytotoxicity assays to predict the outcome of new drugs prior to treatment.
Nevertheless, further experiments are needed in order to assess the validity of
this model for different scenarios.
References:
1. Friberg, L.E. et al.: J. Clin. Oncol. 2002, 20(24): 4713–4721.
2. Léger, F. et al.: Clin. Pharmacol. Ther. 2004, 76(6): 567-578.
ACE.25
Heterogeneous antibody based activity assay for lysine specific
demethylase 1 (LSD1) on a histone peptide substrate
Schulz-Fincke, J.1,2; Schmitt, M.1; Ladwein, K.I.1; Carlino, L.3; Willmann, D.4;
Metzger, E.4; Schilcher, P.5; Imhof, A.5; Schüle, R.4; Sippl, W.3 ; Jung, M.1
1 University
of Freiburg, Institute of Pharmaceutical Sciences
Cancer Consortium, (DKTK); German Cancer Research Center, (DKFZ)
3 Martin-Luther-University of Halle-Wittenberg, Institute of Pharmacy
4 University of Freiburg Medical Center, Department of Urology/Women´s Hospital and
Center for Clinical Research
5 Ludwig Maximilians University of Munich, Adolf-Butenandt Institute and Munich Center of
Integrated protein science (CIPS)
2 German
Fig.1 Design of alkynylgold(I)(NHC)-complexes
References:
1. Ott, I.: Coord. Chem. Rev. 2009, 253: 1670-1681.
2. Oehninger, L., Rubbiani, R., Ott I.: Dalton Trans. 2013, 42: 3269-3284.
3. Rubbiani, R. et al.: Med. Chem. Commun. 2013, 4: 942-948.
4. Meyer, A. et al.: Angew. Chem. 2012, 51: 8895-8899.
ACE.24
Comparison of three pharmacokinetic/toxicity models describing
neutropenia caused by topotecan in cancer patients
Henrich, A.1,2; Parra-Guillen, Z.1; Kloft, C.1
Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,
Kelchstr. 31, 12169 Berlin, Germany
2 and Graduate Research Training program PharMetrX
Posttranslational modifications of histone tails are very important for epigenetic
gene regulation. The lysine specific demethylase LSD1 (KDM1A/AOF2)
demethylates in vitro predominantly mono- and dimethylated lysine 4 on
histone 3 (H3K4) and is a promising target for drug discovery. We report a
heterogeneous antibody based assay, using dissociation-enhanced lanthanide
fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used
a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine
4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody
mediated quantitation of the demethylated product. We have successfully used
the assay to measure the potency of reference inhibitors. Currently, work on
Medicinal Chemistry and testing of new inhibitors is ongoing.
1 Dept.
Objectives: One of the most important dose-limiting toxicities in common
anticancer therapies is myelosuppression leading e.g. to neutropenia. The
pharmacokinetic/pharmacodynamic (PK/PD) model proposed by Friberg et al
[1] describes the time-course of this life-threatening adverse event by
assuming a linear, non-saturable effect of the drug concentration on the
proliferation rate constant (kprol) of progenitor cells in the bone marrow.
Another, probably more physiological approach is the maximum effect model
(Emax), which would facilitate the use of parameters obtained in vitro such as
the EC50 value of myelotoxicity assays. Thus, the objective of this work was to
develop Emax models to link PK and PD of topotecan, an anticancer agent.
These models shall be compared with the linear model concerning the ability to
predict neutropenia.
Methods: Concentration-time data for neutrophils were included from 71
patients pooled from three different trials receiving topotecan monotherapy [2].
Individual topotecan concentration-time profiles were simulated and a linear
(model 1) and two different Emax models (model 2 and 3) for neutropenia were
developed using NONMEM 7.2. Model 2 was a saturable Emax model with Emax
= 1 and an additional elimination rate constant from the compartment with
proliferating cells (kel), and model 3 was an Emax model with an estimated
maximum effect. Both Emax models were compared with a linear model 1.
Model evaluation was done using precision of parameter estimates, goodness
of fit plots and visual predictive checks utilising PsN 3.5.3 and Xpose4 4.4.0.
Results and Discussion: Adequate description of the time course of the
neutrophil data, including nadir and time to recovery was possible with all three
PK/PD models. As expected, different values for kprol were estimated for model
2 in comparison to model 1 and 3, given that this parameter in those models is
a mixture of proliferation and elimination. The three PK/PD models were able
to provide precise estimates for system-related parameters. Both Emax models,
especially model 3 did not lead to precise drug-related parameter estimates
(EC50 and Emax), probably caused by the cytotoxic drug effect (Edrug), which
might be still in the linear part of the PK/PD relationship.
136
We thank DKFZ and DKTK for funding of research of synthesis and assay
development of new LSD1 inhibitors.
References:
1. Spannhoff, A. et al.: ChemMedChem 2009, 4: 1568.
2. Arrowsmith, C.H. et al.: Nat. Rev. Drug Discov. 2012, 11: 384.
3. Willmann, D. et al.: Int. J. Cancer 2012, 131: 2704–2709.
4. Schmitt, M.L. et al.: J. Biomol. Screen. 2014, March [Epub ahead of print]
ACE.26
Carbamate inhibitors of NAD+-dependent histone deacetylases
(Sirtuins)
Swyter, S.1; Beese, K.2; Link, A.2; Jung, M.1
1 Institute
of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, 79104
Freiburg, Germany
2 Institute of Pharmaceutical Sciences, Ernst-Moritz-Arndt-University Greifswald, 17489
Greifswald, Germany
Introduction:
There are seven different human Sirtuins (Sirt 1-7), which are all NAD+dependent enzymes [1]. Originally, Sirtuins have been classified as class III
Histone deacetylases (HDACs). But it was shown that other non-histone
targets like p53 or α-tubulin exist [2], as well as other enzymatic activities like
deacylation of fatty acids [3].
Sirtuins are potential drug targets in age dependent diseases e.g. cancer or
diabetes. The inhibition of Sirtuin-mediated p53-deacetylation is an example
for a possible approach in the treatment of cancer [2].
Starting from lactone containing inhibitors like splitomicin [4], we studied other
analogues with carbamate or thiocarbamate structures. Here we present
screening results on Sirtuin 1-3.
Assay screening:
The screening is performed by a trypsin-coupled homogeneous assay, using
the low-moleculare substrate ZMAL [5]. All compounds were pretested on
auto-fluorescence, quenching effects and trypsin inhibition.
Results:
We were able to find some selective inhibitors with an IC50 in the low µM
range, e.g. Benzoxazinones:
Fig. 1
Name
KBE 5a2
KBE 5a8
R
-CH3
-CH2-CH3
Sirt1
n.i.*
n.i.*
IC50 Sirt2
2.6 ± 0,3 µM
2.6 ± 0,4 µM
Sirt3
n.i.*
n.i.*
the start sites of transcriptionally engaged genes3. H3K4 methylation is
associated with transcriptional activation. These H3K4-modified regions are
largely constrained to the transcription start site regions of genes that are
transcriptionally active.
The fusion partner AF4 is part of a nuclear machinery that activates RNAPolymerase II transcriptional elongation by P-TEFb and mediates coordinated
chromatin remodeling4 but exerts also chromatin modifying activity (H3K79,
H3K36, etc). First results with affinity-purified AF4-MLL fusion protein complex
showed that the purified complex exhibits properties of both MLL and AF4
wildtype proteins. It exhibits H3K4 and H3K79 histone methyltransferase
activity and is able to activate P-TEFb kinase which leads to an strong
activation of the RNA-Pol II and thus influences the elongation process5. In
contrast the MLL-AF4 fusion protein lacks the C-terminal SET-domain, but is
still capable of enhancing gene expression by recruitment of the endogenous
AF4 complex, thereby causing extended H3K79 signatures6. Therefore we
want to further investigate the mechanism that causes the disease by studying
genome-wide alterations of epigenetic signatures in the presence of MLL-AF4,
AF4-MLL or the combination of both reciprocal fusion proteins.
This work is funded by the DFG grant Ma 1876/10-1 to RM.
References:
1. Scholz, B. and Marschalek R.: Br. J. Haematol. 2012, 158(3): 307-322.
2. Hess, J. L.: Trends Mol Med. 2004, 10(10): 500-507.
3. Dou, Y. et al.: Nat. Struct. Mol. Biol. 2006, 13(8): 713-719.
4. Bitoun, E., Oliver P.L., Davies K.E.: Hum. Mol. Genet. 2007, 16(1): 92-106.
5. Benedikt A. et al.: Leukemia 2011, 25(1): 135-144.
6. Krivtsov A. V. et al.: Cancer Cell 2008, 14(5): 355-368.
Fig. 2
1R
Name
KBE 5b13
-H
KBE 5b2
-CH3
*n.i.:no inhibition
2R
-Br
-H
Sirt1
n.i.*
n.i.*
IC50 Sirt2
10.6 ± 2,5 µM
7.1 ± 1,0 µM
Sirt3
n.i.*
n.i.*
Acknowledgement: We thank the DFG for funding (RTG 1976)
References:
1. Trapp, J., Jung, M.: Curr. Drug Targets 2006, 7(11), 1553-1560.
2. Hoffmann, G. et al.: J Biol Chem. 2014, 289(8), 5208-5216.
3. Feldman, J.L., Baeza, J., Denu JM.: J Biol Chem. 2013, 288(43), 31350-31356.
4. Neugebauer, R.C. et al: J. Med. Chem. 2008, 51(5), 1203-1213.
5. Heltweg, B., Trapp, J., Jung, M.: Methods. 2005, 36(4), 332-337.
ACE.27
Investigation of genome-wide alterations of epigenetic signatures in the presence of the leukaemia-initiating MLL-AF4 and
AF4-MLL complexes
Löscher, D.; Kühn, A.; Kowarz, E.; Marschalek, R.
Institute of Pharmaceutical Biology, Biocentre, Goethe-University, Max-von- Laue-Str. 9,
It has been convincingly demonstrated that aberrant epigenetic modifications
are a driving force in many cancers. Epigenetics is based on DNA methylation,
and moreover, on covalent post-translational modifications of histone core
proteins. Based on these findings, changes in the pattern of DNA or histone
modifications are e.g. associated with genetic instability and are correlated with
the rapid development of pre-cancerous cells. In some human diseases, such
as MLL- rearranged leukaemia, these epigenetic changes seem to be sufficient
for disease onset, without the requirement of further mutations1.
Chromosomal translocations that involve the MLL (Mixed lineage leukaemia)
gene result in the production of novel MLL fusion proteins which initiate critical
steps of malignant transformation and lead either to the development of acute
lyphoblastic leukemia (ALL) or acute myeloid leukemias (AML)2.
The t(4;11) chromosomal translocation is one of the most frequent MLL
translocations known today and is a major cause of infant acute lymphoplastic
leukemia (ALL). Infant ALL is an aggressive disease with very poor outcome.
t(4;11)(q21;q23) chromosome translocations fuse MLL in-frame with the AF4
gene and produce both the MLL-AF4 and AF4-MLL fusion proteins.
In mammals the MLL protein is an example of a developmentally important
protein that controls the epigenetic maintenance of gene activation. MLL
possesses a C-terminal SET-domain that methylates histone H3 on Lysine 4 at
ACE.28
Investigating the interplay between the Iroquois homeoprotein
family and the MLL-AF4 complex
Kühn, A.; Löscher, D. ; Kowarz, E.; Marschalek, R.
Pharmazeutische Biologie, Max-von-Laue Straße 9, Biozentrum, Frankfurt Campus
Riedberg
The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is the most
frequent target gene of chromosomal translocations and rearrangements in
childhood leukemia and therapy related leukemia. Until now 80 different
translocation partners of the MLL-gene have been cloned, all of which causing
either acute myeloid (AML) or acute lymphoblastic (ALL) leukemia [1]. In our
working group we focus on the t(4;11) leukemia, where the MLL gene of
chromosome 11 is fused to the AF4 gene of chromosome 4 leading to the 2
chimeric proteins MLL-AF4 and AF4-MLL, respectively. Within all ALL-patients,
the t(4;11)-mediated leukemia is the most common form of MLLrearrangements. Especially pediatric leukemic patients bear this form of
translocation and mostly having a poor outcome with an overall survival of only
about 25-45%, due to a high risk of relapse despite the treatment with high risk
protocols.
Until 2009 it was generally accepted that MLL-fusion genes were recognized
as transcriptional deregulators of HOXA genes claiming that all MLL fusion
proteins work by a similar concept: they increase and maintain high level
transcription of MEIS1 and HOXA gene family members [2]. In contrast to
these findings Trentin et al. could show that only about one half of the
examined t(4;11)-patient cohort exhibits the expected overexpression of
HOXA-gene [3]. The other part showed a decreased HOXA level. An idiosyncratic upregulated gene in this “HOXA-low” patient group was the transcription
factor Iroquois 1 (IRX1). Further work confirmed the presence and importance
of IRX1 in t(4;11) patients [4,5].
IRX1 is a member of the homeoprotein family and binds to the promotor
regions of target genes in a sequence specific manner [6]. Therefore it is
classified as a transcription factor being important for pattern formation,
especially for lung, brain and heart development [7] [8]. Furthermore it acts as
a tumor suppressor gene in gastric cancers [9].
Our focus stands now on the investigation of the interplay between the Iroquois
family members and MLL and its derivatives. At first we performed microarray
analysis to explore which genes were up- or down-regulated in the presence of
ectopically expressed IRX1. During our work we were able to demonstrate that
IRX1 itself causes the down-regulation of HOXA-genes in a dominant fashion,
explaining the observed downregulation of HOXA genes in the “HOXA-low”
patient group. Furthermore we were able to demonstrate that Iroquois proteins
(IRX1 and IRX2) seem to counteract the molecular actions deriving from the
MLL-AF4 protein.
137
This work is funded by the DFG grant MA1876/10-1 to RM.
References:
1. Meyer, C. et al.: Leukemia. 2013, 27(11):2165-2176.
2. Ayton, P.M. and Cleary, M.L.: Genes Dev. 2003, 17(18): 2298-2307.
3. Trentin, L. et al.: Eur. J. Haematol. 2009, 83(5): 406-419.
4. Stam, R.W. et al.: Blood. 2010, 115(14): 2835-2844.
5. Kang, H. et al.: Blood. 2012, 119(8): 1872-1881.
6. Bilioni, A. et al.: Proc. Natl. Acad. Sci. U.S.A. 2005, 102(41): 14671-14676.
7. Doi, T. et al.: J. Pediatr. Surg. 2011, 46(1): 62-66.
8. Gómez-Skarmeta, J.L. and Modolell, J.: Curr. Opin. Genet. Dev. 2002, 12(4): 403-408.
9. Guo, X. et al.: Oncogene. 2010, 29(27): 3908-3920.
ACE.29
Inhibition of class I HDACs abrogates the dominant effect of
MLL-AF4 at the 5-lipoxygenase promoter by activation of MLL
Ahmad, K.1; Katryniok, C.1; Scholz, B.2; Merkens, J.2; Marschalek, R.2;
Steinhilber, D.1
1 Goethe
University Frankfurt, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9,
D-60438 Frankfurt am Main, Germany
2 Goethe University Frankfurt, Institute of Pharmaceutical Biology, Max-von-Laue-Str. 9, D60438 Frankfurt am Main, Germany
5-Lipoxygenase (5-LO), which is encoded by the ALOX5 gene and mainly
expressed in leukocytes, is an enzyme that catalyzes the first two steps in the
biosynthesis of leukotrienes derived from arachidonic acid. Physiologically,
leukotrienes are a part of the innate immune system. In the pathophysiological
context, leukotrienes are associated with inflammatory, allergic and cardiovascular diseases as well as certain types of cancer [1]. Thus, there is a reasonable interest to understand the regulation of ALOX5 gene expression.
Previously, it was shown that the ALOX5 promoter is activated by the panhistone deacetylase (HDAC) inhibitor TSA leading to enhanced transcript
initiation [2-3]. However, the molecular mechanism behind the induction of 5LO transcript initiation by HDAC inhibitors remained unknown. By chromatin
immunoprecipitation, we observed that induction of 5-LO mRNA expression by
HDAC inhibition correlates with histone H3 lysine 4 trimethylation (H3K4me3).
The SET domain of the MLL (mixed lineage leukemia) protein catalyzes the
formation of H3K4me3 [4,5]. In order to study the role of MLL on 5-LO
promoter activity, an ALOX5 promoter construct was cotransfected with
expression constructs for MLL or the leukemogenic fusion proteins MLL-AF4
(der11) and AF4-MLL (der4) [6,7]. The constitutively active MLL derivative
MLL-AF4 stimulated 5-LO promoter activity by more than 50-fold whereas
wildtype MLL was inactive. Addition of class I HDAC inhibitors (which lead to
MLL activation) induced 5-LO promoter activity in an MLL-dependent fashion.
Interestingly, in the presence of constitutively active MLL-AF4, addition of
HDAC inhibitors attenuated its activity in an MLL-dependent manner.
Thus, these results reveal that HDAC class I inhibitors can attenuate the
oncogenic effects of MLL-AF4 by activation of wildtype MLL, suggesting that
these compounds might be of considerable therapeutic interest for the
treatment of leukemias with constitutively active oncogenic MLL fusion
proteins. In subsequent experiments, we found that class I HDAC inhibitors are
indeed very effective inhibitors of cell growth of a leukemia cell line that carry
the respective t(4;11) translocation leading to these MLL fusion proteins.
Acknowledgments:
We are grateful to Else Kröner-Fresenius-Stiftung (Dr. Hans-KrönerGraduiertenkolleg) and CEF for financial support.
References:
1. Rådmark, O. et al.: Trends Biochem. Sci. 2007, 32(7): 332-341.
2. Klan, N. et al.: Biol. Chem. 2003, 384(5): 777-785.
3. Schnur, N. et al.: Biochim. Biophys. Acta. 2007, 1771(10): 1271-1282.
4. Milne, T.A. et al.: Mol. Cell. 2002, 10(5): 1107-1117.
5. Wang, P. et al.: Mol. Cell. Biol. 2009, 29(22): 6074-6085.
6. Marschalek, R.: FEBS J. 2010, 277(8): 1822-1831.
7. Meyer, C. et al.: Leukemia 2013, 27(11): 2165-2176.
Scholl, F.1; Basavarajappa, D.1; Weigert, A.2; Suess, B.3; Steinhilber, D.4;
Rådmark, O.1
1 Department
of Medical Biochemistry and Biophysics (Kemi II), Karolinska Institutet,
Scheeles väg 2, 17177 Stockholm, Sweden
2 Institute of Biochemistry I, Goethe University Frankfurt, Theodor-Stern-Kai 7, building 74,
60590 Frankfurt, Germany
3 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-LaueStrasse 9, 60438 Frankfurt, Germany
4 Biology Department, TU Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt,
Germany
Leukotrienes (LTs) are inflammatory mediators also known to be involved in
cancer. They are synthesized by 5-Lipoxygenase (5-LO)[1]. In vitro studies
revealed that 5-LO can interact with Dicer, an enzyme responsible for the
microRNA (miRNA) maturation [2]. MicroRNAs are transcribed as primary
miRNAs (pri-miRNA) and further processed by Drosha to intermediates (premiRNA). These intermediates then undergo a final cleavage to mature miRNAs
by Dicer [3].
The aim of this study is to determine if 5-LO has any impact on biosynthesis of
miRNAs in the monocytic cell line Mono Mac 6. Possibly, interaction of 5-LO
with Dicer in the cell could modulate Dicer activity.
We first performed a microarray to compare the miRNA expression in 5-LO
knockdown cells (Δ 5-LO) and cells treated with a control shRNA[4]. The
strongest effect appeared for hsa-miR-99b-5p and hsa-miR-125a-5p.
Interestingly, these miRNAs are organized in a common pri-miRNA and
enclose a third miRNA, hsa-let-7e-5p. However, an approximately 2-fold
decrease of miR-125a-5p and miR-99b-5p in Δ 5-LO could be confirmed
whereas let-7e-5p did not show a significant change. Since the pri-miRNA level
are unaffected by Δ 5-LO, the data suggest that the regulation occurs at the
level of miRNA processing.
Further on, we investigated whether the downregulation of the mature miRNAs
could be mimicked by blocking leukotriene biosynthesis. But neither the
inhibition of 5-LO led to a decrease in miRNA production nor led the additional
treatment with leukotrienes to an upregulation. This showed that the 5-LO
protein and not its products is necessary for maturation of specific miRNAs.
MiR-125a and miR-99b are already known to be involved in inflammatory
processes [5, 6] . We could show that interleukin 6 is increased in the absence
of miR-125a and miR-99b and especially tumornecrosis-factor-α (TNF-α) is
significantly elevated in cells lacking both miRNAs. Interestingly Woo et al.
showed that TNF- α has an impact on 5-LO activity [7].
To summarize, our findings suggest that 5-LO can directly interact with Dicer to
fine-tune inflammatory responses. This occurs by a negative feedback
mechanism with miRNAs and TNF- α as linker.
We thank the Else Kröner-Fresenius-Stiftung (Dr. Hans Kröner Graduiertenkolleg) for financial support.
References:
1. Radmark, O. and Samuelsson, B.: J. Intern. Med. 2010, 268(1): 5-14.
2. Dincbas-Renqvist, V. et al.: Biochim. Biophys. Acta 2009, 1789(2): 99-108.
3. Ha, M. and Kim, V.N.: Nat. Rev. Mol. Cell Biol. 2014.
4. Basavarajappa, D. et al.: Proc. Natl. Acad. Sci. USA 2014.
5. Banerjee, S. et al.: J. Biol. Chem. 2013, 288(49): 35428-35436.
6. Singh, Y. et al.: J. Biol. Chem. 2013, 288(7): 5056-5061.
7. Woo, C.H. et al.: J. Biol. Chem. 2000, 275(41): 32357-32362.
ACE.31
Compound C causes resistance to tubulin inhibition independent
of AMPK – possible role of c-Myc ubiquitination?
Scherzberg, M.-C.1, Steinhilber, D.1, Ulrich-Rückert, S.1
ACE.30
Modulation of Dicer-mediated microRNA processing by 5lipoxygenase - roles in inflammation & cancer
138
1 Institute
of Pharmaceutical Chemistry Goethe-University Frankfurt, Max-von-Laue-Straße
9, 60438 Frankfurt a.M., Germany
Tubulin inhibitors belong to the most frequently used anti-cancer drugs. In
recent years it became evident, that beyond affecting the mitotic spindle
apparatus, also anti-mitotic activities influencing crucial cellular functions seem
to underlie the established efficacy [1]. C-Myc is a transcription factor, which
expression levels closely correlate with the proliferation of mammalian cells [2].
Therefore its degradation is tightly controlled by the ubiquitin-proteasome
system [3]. Compound C is a widely used kinase inhibitor to investigate
biological responses mediated by AMPK although recent studies propose that
Compound C exerts effects which cannot be attributed to AMPK inhibition
[4,5].
The inhibition of tubulin polymerisation or –depolymerisation with various
tubulin inhibitors significantly reduces cell growth of HT-29 cells (***p<0.001)
determined by crystal violet staining. Interestingly, combination with the AMPK
inhibitor Compound C significantly counteracts this effect (***p<0.001) without
actually affecting the AMPK signalling pathway, which was confirmed by RNAi
experiments. FACS analysis revealed that Compound C considerably
attenuates tubulin inhibitor induced G2/M arrest and increases the number of
cells in G0/G1 phase (e.g. 17% for vinblastine, *p<0.05).
Western Blot experiments showed multiple bands for c-Myc after tubulin
inhibition for 8-16h followed by complete disappearance after 24h, which
suggests an intensive posttranslational modification of the c-Myc protein
leading to subsequent degradation. Combination with Compound C partly
prevents this occurrence of higher molecular bands for c-Myc. To evaluate if
these multiple bands for c-Myc might be due to ubiquitination and proteasomal
degradation cells were treated with proteasome inhibitor MG132 and blotted
for c-Myc protein. Interestingly, the same pattern of multiple bands also
observed with tubulin inhibitors was evident. Furthermore, similar to Compound C, also MG132 was able to significantly reverse tubulin inhibitor induced
growth arrest (***p<0,001).
We should take into consideration that Compound C induced resistance to
microtubule interfering agents in human colon cancer cells might be due to an
altered process of c-Myc ubiquitination and degradation, but the exact
mechanism remains to be elucidated.
References:
1. Fürst, R., Vollmar, A.: Pharmazie 2013, 68(7): 478-83.
2. Schuhmacher, M., Eick, D.: Transcription 2013, 4(4): ahead of print.
3. Farrell, A., Sears, R.: Cold Spring Harb Perspect Med 2014, 4(3).
4. Vucivecic, L. et al.: Autophagy 2011, 7(1): 40-50.
5. Kim, Y. et al.: Atherosclerosis 2011, 219(1): 57-64.
ACE.32
Ionizing radiation-induced glioblastoma cell migration in vivo
Butz, L.1,2; Stegen, B.2; Zips, D.2; Buschauer, A.3; Huber, S.M.2; Ruth, P.1
1 Department
of Pharmacology, Toxicology and Clinical Pharmacy, University of Tübingen,
Auf der Morgenstelle 8, 72076 Tübingen
2 Department of Radiation Oncology, University of Tübingen, Hoppe-Seyler-Str. 3, 72076
Tübingen
3 Department of Pharmaceutical/Medicinal Chemistry II, University of Regensburg, 93040
Regensburg
Invasion of the brain by glioblastoma cells reportedly requires Ca2+-activated IK
and BK K+ channels in the plasma membrane. These channels are involved in
both, the generation of Ca2+ signals that program cell migration/invasion and
cell volume changes that motorize migration. Moreover, ionizing radiation (IR,
2 Gy) has been shown to stimulate glioblastoma cell migration in vitro by
increasing K+ channel activity. Importantly, pharmacological targeting of these
channels abolishes IR-induced migration in vitro. The present project aims to
monitor in an orthotopic mouse model the migration of glioblastoma cells
during fractionated radiation and to define the role of K+ channels herein.
Human U87MG Kat glioblastoma cells (30,000) which express the Katushka
fluorescence protein and which have high copy numbers of functional IK and
BK K+ channels were injected stereotactically into the right striatum of
immunocompromised NSG (NOD scid gamma) mice and grown for 7 days to
solid glioblastomas with a volume of about 1 mm 3.On days 7 until 11 after
tumor challenge, mice received daily fractions of 0 (control group) or 2 Gy Xray to a total dose of 10 Gy (irradiated group) delivered to an 5 x 3 mm2 area of
the tumor bearing right hemisphere by the use of a 6 MV linear accelerator.
The mouse torso and the mouse head outside the target-volume were shielded
by the multi leaf collimator and an 8 cm thick on-body lead block, respectively.
Film dosimetry using a mouse head phantom suggested a steep dose gradient
at the field margin with a residual dose of below 2% at the shielded brain area.
On day 21, mice were sacrificed, dissected brains were fixed for 24 h with 2%
paraformaldehyde in PBS, postincubated for 24 h in 30% sucrose in PBS,
frozen, sliced into 20 µm thick sections and the Katushka fluorescence of the
glioblastoma cells was analyzed by fluorescence microscopy.
As a result, tumor challenge resulted in reproducible formation of U87MG Kat
tumors. In addition, mice tolerated the 5 fractions of partial head irradiation
very well. Importantly, fractionated migration increased number of emigrated
cells (437 ± 51 vs. 253 ± 30, n= 5-6 mice, p < 0.02) suggesting radiationinduced hypermigration in vivo. This hypermigration was paralleled by a
radiation-induced increase in tumor-associated SDF-1 protein abundance as
demonstrated by immunofluorescence microscopy. Moreover, preliminary data
suggest that the BK K+ channel inhibitor paxilline (8 mg/kg BW i.p.) applied 6 h
prior to and 6 h after each radiation fraction abolishes the radiation-induced
hypermigration while having no effect on tumor spreading of un-irradiated
glioblastomas.
In conclusion, the orthotopic U87MG Kat mouse model seems to be well suited
to study hypermigration of glioblastoma cells during fractionated radiation in
vivo and to disclose potential effects of fractionated radiation and K + channel
targeting.
Acknowledgements: This work has been supported by the Wilhelm-SanderStiftung (Grant: 2011.0831.1)
ACE.33
Characterization of 5-lipoxygenase overexpression in tumor cell
lines
Dos Santos Capelo, R.; Brüggerhoff, A.; George, S.; Steinhilber, D.; Kahnt,
A.S.
Goethe University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9,
60438 Frankfurt/Main, Germany.
Accumulating evidence from laboratory and epidemiological studies suggests
that aberrant 5-lipoxygenase (5-LO) activity can promote carcinogenesis. In
contrast to healthy tissues the enzyme is frequently over expressed in solid
malignancies of the prostate, colon and lung, to name a few. There is a
correlation between 5-LO overexpression and cancer cell formation, proliferation and metastasis. Nevertheless, the enzyme’s direct role in the formation of
solid tumours remains elusive so far. Profound understanding of the enzyme’s
role in carcinogenesis would help to predict if combination of leukotriene
inhibitors with chemotherapy regimens constitutes a promising approach to
successfully treat 5-LO overexpressing tumors.
We screened different tumour cell lines for 5-LO expression. Out of these we
chose 2 over expressing cell lines (Capan-2, HT-29) and checked them for the
presence of other enzymes of the leukotriene machinery (5-LO-activating
protein (FLAP) and cytosolic phospholipase A2(cPLA2)) by western blot
analysis. FLAP and cPLA2were expressed in both cell lines, pointing to
functional leukotriene generation in these cell lines. Next, intact cells were
treated with different stimuli to trigger leukotriene formation. Interestingly, no
enzyme activity was found. In contrast, broken cell preparations showed robust
5-LO activity leading to the conclusion that the enzyme was functional but not
activated in these cells due to an unknown inhibitory mechanism. 5-LO activity
is tightly regulated by different factors such as glutathione peroxidases
controlling the intracellular redox tone and activating and inactivating phosphorylations. We could show that the enzyme is phosporylated on Ser523, a
PKA-dependent modification known to inhibit enzymatic activity in both cell
lines. Nevertheless, preincubation of HT-29 cells with different PKA inhibitors
prior to stimulation did not reconstitute enzyme activity. Also incubation with
diamide, to impair GPx activity and enhance oxidative stress had no impact on
leukotriene formation. Therefore, further studies are needed to elucidate the
role of 5-lipoxygenase in tumor progression and survival.
139
BIOTECHNOLOGY (BT01-BT16)
BT.02
BT.01
Detection of Extracellular Glyosylphosphatidylinositol-Anchored
Proteins Within Phospholipid Complexes Using a Biosensor for
the Prediction and Stratification of Type 2 Diabetes
One substrate – seven products with different prenylation
positions in one-step reactions by using fungal prenyltransferases
Müller, G.; Tschöp, M.
Fan, A.; Li, S.-M.*
Helmholtz Center Munich, Institute for Diabetes and Obesity, Am Parkring 13, 85478
Garching-Hochbrück, Germany
Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg,
Deutschhausstrasse 17A, 35037 Marburg, Germany
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) have been shown to
exhibit high susceptibility for release in ECGAPP from the surface of mammalian cells in vitro and in vivo in response to cellular and metabolic stress, such
as high glucose, fatty acids and reactive oxygen species [1,2], that is prevalent
during the pathogenesis of type 2 diabetes (T2D). The underlying non-classical
secretory mechanism is commonly thought to rely on the anchorage of GPIAPs in the outer leaflet of the plasma membrane phospholipid bilayer by the
glycosylphosphatidylinositol moiety, exclusively, that is covalently attached to
the carboxyl-terminus of the polypeptide chain. However, the presence of
ECGAPP in the plasma of T2D patients, that are faced with elevated blood
glucose and free fatty acids levels, has not been studied so far. This is
presumably due to conceptual restrictions (i.e. reductionistic and holistic
thinking) and technological challenges. To overcome these hurdles, a novel
type of chip-based biosensor will be used for the specific detection and
biophysical characterization of ECGAPP. Its principle relies on the generation
of horizontal surface acoustic waves (SAW) of defined frequency and
amplitude within the gold surface of a microfluidic four-channel chip. Any
interaction of (macro)molecules with the gold surface will result in corresponding changes in the shape of the SAW, altering both their frequency and
amplitude. These alterations reflect mass loading (i.e. binding of ECGAPP) to
and biophysical properties (i.e. size and shape depending on the ECGAPP
protein composition as well as viscoelasticity and rigidity depending on the
ECGAPP phospholipid composition, cholesterol content and open/emptyclosed/filled configuration) at the chip surface. The major advantages of the
SAW vs. the commonly used surface plasmon resonance biosensor rely on the
possibility of measurement of large (lipid-containing) macromolecules even in
the presence of serum [3] as well as on the potential high sensitivity towards
alterations in the composition (proteins, phospholipids) and structure (micelles,
nanodiscs, vesicles, particles) of the ECGAPP. Albeit SAW biosensing per se
does not enable the delineation of the type of ECGAPP contained in a given
sample, the SAW signatures will be characteristic for the overall contents of all
ECGAPP, either as summation signals or as 1D-/2D-signatures of high
informative value.
Prenylated indole alkaloids are widely distributed in nature and show diverse
biological and pharmacological activities, usually distinct from their nonprenylated precursors. Prenyltransferases catalyze the transfer reactions of
prenyl moieties onto indole nucleus and contribute largely to the structural
diversity of these compounds. Based on their sequences and biochemical
properties, prenyltransferases can be divided into different subgroups.[1;2] One
of the most investigated subgroup is the dimethylallyl tryptophan synthase
(DMATS) superfamily. So far, more than 30 such prenyltransferases have
been identified and characterised biochemically[3-5]. The majority of the DMATS
superfamily can be classified into tryptophan and tryptophan-containing cyclic
dipeptide prenyltransferases according to their substrates. In this study, we
demonstrate the acceptance of cyclo-L-homotryptophan-D-valine, an unnatural
cyclic dipeptide, by three tryptophan prenyltransferases and five cyclic
dipeptide prenyltransferases. Seven products with one prenyl moiety at each
position of the indole nucleus and one diprenylated derivative were isolated
from enzyme assays of cyclo-L-homotryptophan-D-valine with dimethylallyl
diphosphate as prenyl donor. Our results presented here expand potential
usage of these enzymes in the production of prenylated derivatives.
Acknowledgments: This work was supported by the Deutsche Forschungsgemeinschaft
(to S.-M. Li). A. Fan is a recipient of a fellowship from China Scholarship Council.
References:
1. Li, S.-M.: Appl.Microbiol.Biotechnol. 2009, 84; 631-639.
2. Heide, L.: Curr.Opin.Chem.Biol. 2009, 13: 171-179.
3. Winkelblech, J.; Li, S.-M.: Chembiochem. 2014, 15:1030-1039.
4. Yu, X.; Li, S.-M.: Methods Enzymol. 2012, 516: 259-278.
5. Miyamoto, K. et al.:, Bioorg.Med.Chem. 2014, 22: 2517-2528.
BT.03
References:
1. Müller, G. et al.: Br. J. Pharmacol. 2009, 158: 749-770.
2. Müller, G. et al.: Cell. Signalling 2009, 21: 324-338.
3. Gronewold, T.: Anal. Chim. Acta 2007, 603: 119-128.
Zebrafish as model organism for pharmacological research on
soluble guanylyl cyclase
Dittmar, F.1; Bähre, H.1,2; Kaever, V.1,2; Seyfried, S.3; Seifert, R.1
Institute of Pharmacology, Hannover Medical School, 30625 Hannover, Germany
Research Core Unit Metabolomics, Hannover Medical School, 30625 Hannover,
Germany
3 Max-Delbrück-Centrum für molekulare Medizin (MDC), 13125 Berlin, Germany
1
2
The zebrafish Danio rerio has become an important model organism for a wide
range of scientific research questions regarding vertebrates [1]. Current
studies are mainly focused on development, genetics and disease [1]. The
beneficial features of the zebrafish include its small size, rapid development,
140
References:
1. Lawrence, C.: Aquaculture 2007, 269: 1–20.
2. Langheinrich, U.: BioEssays 2003, 25: 904–912.
BT.04
Surface modifications of polyethylene sinter bodies for serological diagnosis of borreliosis
Alasel, M.; Dassinger, N.; Vornicescu, D.; Keusgen, M.
1.00
0.95
Transmittance (%)
short generation time, optical transparency of embryos and larvae as well as
conservation in functional domains [1]. Furthermore, application of drugs is
simple as zebrafish absorb compounds from their surrounding media [2].
The aim of our study was to examine the impact of various activators and
stimulators on the soluble guanylyl cyclase (sGC) in Danio rerio. Therefore,
embryos were treated with these compounds from one day post fertilization
(dpf) to five dpf.
First of all, we determined if cyclic nucleotides occur naturally in several
developmental stages and organs of this vertebrate. The well-established
purine nucleotides cAMP and cGMP as well as the little-known pyrimidine
nucleotides cCMP and cUMP were detected in embryos, larvae and some
organs of adult zebrafish via high-performance liquid chromatography tandem
mass spectrometry (HPLC-MS/MS). Treatment of embryos with the NOsynergistic sGC stimulator 3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (Bay 41-2272) increased cGMP
concentrations.
Current studies comprise similar treatment with the NO-independent sGC
activator cinaciguat (Bay 58-2667). Future research will focus on sGC
expression pattern in different tissues and the entire organism as well as
treatment of redox-mutants with the abovementioned sGC drugs. Analysis of
the NO-sGC system in Danio rerio will provide valuable information on drugs
for cardiovascular diseases.
-OH
0.90
-CO
0.85
Unmodified
Modified
0.80
500
1000
1500
2000
2500
3000
3500
4000
-1
Wavenumber (cm )
Figure 1: FTIR spectra of unmodified (―) and modified ( )
polyethylene
Conclusions
Functionalization of the 3D-polyethylene sinter bodies has been successfully
achieved using photografting technique. The obtained hydroxyl groups could
be utilized for further immobilizations. The final coating by the polysaccharide
mannan allows fixation of genetically designed fusion proteins with a ConA
moiety by self-organization. The latter step is a rather smooth procedure,
which does not influence the biological functionality of the fusion protein in a
negative manner.
Acknowledgements: I would like to thank Yousef Jameel scholarship fund for funding me
during my research.
References:
1. Bandopadhay, D. et al.: Journal of Applied Polymer Science 2004, 92: 3046–3051.
Institute of Pharmaceutical Chemistry, Marbacher Weg 6-10, D-35032, Marburg, Germany
Abstract: Allyl alcohol was utilized as a monomer to introduce hydroxyl groups
on the surface of 3D-polyethylene sinter bodies. Using UV photografting
technique, allyl alcohol could be polymerized on the surface of the polyethylene providing active hydroxyl groups that can be linked via (3aminopropyl)triethoxy silane (APTES) to polysaccharides like mannan. In the
next step, a fusion protein consisting of the lectin binding domain ConA and a
Borrelia surface antigene has been immobilized by self-organization.
Keywords: UV photografting, monomer, allyl alcohol, diagnostic immunoassay.
Introduction
For the immobilization of biomolecules on solid polymer surfaces, a primary
functionalization of these materials is necessary. Generally functionalization of
polyethylene takes place using wet chemistry, dry chemistry like plasma
activation or photochemistry. One of the easiest methods in photochemistry is
by ultraviolet light (UV) in order to activate chemical bounds. This technique
can be also applied to polyethylene polymers, which later were utilized in
serological diagnosis of Lyme Borreliosis disease [1].
Results and Discussion
Functionalization of the 3D-polyethylene surface has been successfully
performed utilizing the radical reaction of allyl alcohol with the polymer after
their exposure to UV light in the presence of an initiator (benzophenone).
Surface modification has been confirmed by Fourier transform infrared
spectroscopy (FTIR) (Figure1) and surface electron microscopy (SEM)
measurements.
After this primary surface modifications, hydroxyl groups were linked to self
assembled mono layers of (3-aminopropyl)triethoxy silane (APTES) to provide
the surface with amine groups. For amine coupling reaction, mannan was first
activated using N,N-disuccinimidyl carbonate (DSC) followed by kovalent
immobilization via amide bounds.
Using a fusion protein with one lectin (Concanavalin A; ConA) part and another
antigen (lyme antigen) part, serological diagnosis of Lyme Borreliosis was
possible. Test could be successfully performed with different Borrelia + and –
sera.
BT.05
Crystal Structure of Blood Coagulation Factor XIII: Template for
the Design of a Novel Anticoagulant
Stieler, M.1; Weber, J.2; Hils, M.2; Kolb, P.1; Heine, A.1; Büchold, C.2; Pasternack, R.2; Klebe, G.1
1 Institut
of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6,
35037 Marburg, Germany
2 Zedira GmbH, Roesslerstrasse 83, 64293 Darmstadt, Germany
Blood coagulation factor XIII (FXIII) is the last enzyme of the blood coagulation
cascade and represents one of the most promising target for the development
of safer alternatives to presently administered anticoagulants such as
heparins, vitamin K antagonists or direct acting thrombin and factor Xa
inhibitors. All the latter drugs affect the level of thrombin, which activates not
only fibrin assembly for coagulation but also stimulates platelets, a prerequisite
for primary clot formation. Thus, undesirable and even life-threatening bleeding
episodes can result. While other enzymes of the cascade are serine proteases,
FXIII is a plasma transglutaminase catalyzing isopeptide bond formation. It
acts downstream of thrombin, effectively determining mechanical stability, halflife and lysis rate of clots. FXIII has been discussed as ideal target to interfere
with coagulation, but no inhibiting drug candidates are available to explore its
pharmacological potential. This is mainly due to the fact that a protein structure
representing the relevant active state is unknown. Here we report the first highresolution crystal structure (1.98 Å) of FXIII in the active state complexed with
an irreversible inhibitor. Only crystal structures of inactive, homodimeric FXIII
have been reported so far where the active site is completely buried and any
access to the catalytic center is obscured. Such structures are unsuitable for
drug design. In the present structure, three calcium ions recruit polar functional
groups of the protein and establish local metal ion coordination sites which
induce rearrangements of two domains along with local adaptations of the
catalytic domain to expose the enzyme in its active state. The observed
transformations establish the substrate and co-substrate binding sites for
isopeptide bond formation and suggest involvement of a catalytic triad and a
diad in the enzyme mechanism which is considered valid for the entire
transglutaminase family.
141
therefore studied the conformational preference of α-aminoxy peptoids in
established peptoid model systems. A comprehensive analysis of model
peptoids by 1D and 2D NMR spectroscopy, X-ray analysis (Fig. 1, B), and
computational conformational analysis (Fig. 1, C) revealed that α-aminoxy
peptoids prefer a cis-configuration even in the presence of side chains that
usually are giving inhomogeneous mixtures of cis- and trans-configured
amides. Thus, α-aminoxy peptoids represent an interesting novel peptidomimetic backbone architecture featuring a distinct folding pattern.
References:
1. Miller, S.M. et al.: Bioorg. Med. Chem. Lett. 1994, 4(22): 2657–2662.
2. Kwon, Y.-U.; Kodadek, T.: J. Am. Chem. Soc. 2007, 129(6): 1508–1509.
3. Reddy, M.M.; Bachhawat-Sikder, K.: Kodadek T. Chem. Biol. 2004, 11(8): 1127–1137.
4. Suwal, S.; Kodadek, T.: Org. Biomol. Chem. 2013, 11(13): 2088–2092.
5. Hodges, J.A.; Raines, R.T.: Org. Lett. 2006, 8(21): 4695-4697.
6. Yoo, B.; Kirshenbaum, K.: Curr. Opin. Chem. Biol. 2008, 12(6): 714–721.
7. Shin, I.; Park, K.: Org. Lett. 2002, 4(6): 869–872.
We acknowledge the beamline support of Bessy II in Berlin for practical help and the HZB
for travel grants. Furthermore we acknowledge the BMBF for financial support.
References:
1. Stieler, M. et al: Angewandte Chemie (Int. Edition) 2013, 52(45): 11930-11934.
2. Shebuski, R. J. et al: Blood 1990, 75(7): 1455-1459.
BT.06
α-Aminoxy Peptoids: A Unique Peptoid Backbone with a Preference for Cis Amide Bonds
Syntschewsk, V.1; Ciglia, E.1; de Sousa Amadeu, N.2; Vasylyeva, V.2; Janiak,
C.2; Kurz, T.1; Gohlke, H.1; Hansen, F.K.1
Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität
Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
2 Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine-Universität
1
α-Peptoids (Fig. 1, A) are oligomers of N-substituted glycine and feature
several advantages over peptides as potential bioactive compounds, among
them proteolytic stability and increased cell permeability. [1,2] Peptoid libraries
have been utilized as protein binding agents and as inhibitors of protein-protein
interactions, [3] although primary screening hits identified from peptoid libraries
have usually not displayed high activity or potency. [4]
Figure 1: A) Selected peptoid backbones, B) X-ray structure of an α-aminoxy
tripeptoid, C) helical arrangement of a hexamer derived from MD simulations.
Due to the absence of internal hydrogen bonding, peptoids are less conformationally restrained than peptides. This may adversely affect their binding affinity
to proteins. Thus, it is important to study the preferred conformation of the
peptoid backbone.
Peptides possess mostly trans-amide bonds, apart from proline, whereas a
high degree of cis-amide bonds is observed in α-peptoids. [5] Based on the
fact that cis- and trans-amide conformations are almost isoenergetic for N-alkyl
α-peptoid monomers, studies have shown that the nature of the side chain can
significantly modulate the ratio of cis-/trans-amide bond conformers. [6] Hence
stable, helical secondary structures in α-peptoids can be achieved by an
optimally controlled cis-/trans isomerism. This tactical incorporation of specific
cis- or trans-directing side chains enables the control of the folding properties
of α- and β-peptoids. However, this comes at the expense of side chain
diversity. A preferable peptoid backbone should therefore not only predispose
a peptoid to adopt a specific conformation but also allow the introduction of
different substituents. Thus, it is worthwhile to address this limitation and to
design peptoid derivatives with conformationally constrained backbones.
In 2002, Shin and Park reported the preparation of α-aminoxy peptoid
pentamers. However, the folding properties were not investigated. [7] Learning
from the backbone-controlled secondary structures of α-aminoxy peptides, we
reasoned that α-aminoxy peptoids can form stable secondary structures due to
an increased energetic difference between cis and trans conformers. We
142
BT.07
Thrombin Revisited: Using a well established Protein to gain new
insights on Protein-Ligand Interactions
Collins, C.; Weimer, D.; Biela, A.; Heine, A.; Klebe, G.
Institute for Pharmaceutical Chemistry, Phillips-University Marburg, Marbacher Weg 6,
35032, Marburg, Germany
Thrombin is an important drug target in the prevention of blood clotting making
it a well studied protein. Due to the large prevalence of Cardiovascular
Disease throughout the world population, research in this field is immense.
Anticoagulants such as thrombin inhibitors play a key role in preventing heart
attacks and strokes. One of the newest being Dabigatran, an oral thrombin
inhibitor. Researchers are able to discover and characterize these new drugs
with the help of methods such as protein crystallography, Isothermal Titration
Calorimetry (ITC) and Surface Plasmon Resonance (SPR). Using crystallography, structures of the protein with the inhibitor can be determined and the
exact position of binding can be elucidated. ITC is a method which uses the
change in Enthalpy ΔH (the heat of an interaction) and the Gibbs Free Energy
ΔG to determine the thermodynamic profile. With this thermodynamic data the
binding modes of compounds can be compared which aids in the process of
drug development and allows a basis to rank the various compounds. This
method can measure the data without the requirement of labelling, immobilization or any other chemical modification.[1] To further study the interaction, SPR
can be utilized to study the binding kinetics.
In order to improve the process of drug development it is important to fully
understand the interactions between proteins and the molecules they bind
with. It is now understood that so many factors weigh in on the binding
process. The binding affinity is not only dependent on the non-covalent
interactions of the protein and its partner but also on the interaction of both
components with their solvent.[2] This makes the prediction of an interaction
much more complex than it was first imagined. Water molecules, for example,
play a crucial role in this process. They intercede the binding partners,
enhancing the binding by forming excess hydrogen bonds and expanding the
binding surface.[3] We are just now starting to learn about the circumstances in
which these water molecules can positively affect the thermodynamics of
binding.
By turning to Thrombin, a familiar protein, we are able to use the established
techniques to concentrate on these small but vital interactions which occur in
the binding processes of all proteins to their ligands. Only if the sum of these
interactions is understood will we someday be able to make predictions more
efficiently. This understanding also allows for the utilization of this knowledge
to develop new leads. These could then precisely trigger certain positive
effects such as the inclusion of water to improve the thermodynamics of an
interaction. For the study a series of ligands was synthesized to attempt to
alter the binding of waters in the binding pocket making it possible to evaluate
the importance of these waters in the protein ligand binding process. The
inhibitors consist of a tripeptide-like D-Phe-Pro-XXX scaffold which varies only
in the P1 side chain (the moiety which binds in the S1 pocket of Thrombin). [3]
Previous studies on inhibitors of this kind have shown that, depending on the
type of P1 substituent, a well-defined water molecule can be observed in the
active site. By coordinating with four neighbouring atoms, it can be assumed
that this water mediates hydrogen bonds necessary for the binding process. In
the next series inhibitors were especially designed to try to change the
interaction of this tetra-coordinated water. [3] By studying the structures and
thermodynamic properties of these ligands bound to the protein, taking the
involved waters into consideration, we are able to broaden our understanding
on the intricate requirements necessary for optimal interactions.
References:
1. Ladbury, J. et al: Nature Reviews Drug Discovery 2010, 9: 23-27.
2. Baum, B. et al: JMB 2010, 397: 1042-1054.
3. Biela, A. et al: JMB 2012, 418(5): 350–366.
2. Friesen, N. et al.: Aliso 2006, 22: 372-395.
3. Stoll, A. and Seebeck, E.: Helvet Chim Acta. 1949, 32(1): 197-205.
4. Nock, L.P. And Mazelis, M.: Plant Physiol. 1987, 85(4):1079-1083.
5. Kuettner, E.B. et al.: J Biol Chem. 2002, 277(48): 46402-46407.
6. Kubec, R. et al.: J Agric and Food Chem. 2011, 59(10): 5763-5770.
7. O’Donnell, G. et al.: J Nat Prod. 2009, 72(3): 360-365.
8. Gonzáles-Ballester, D. et al.: Anal Biochem. 2005, 340(2): 330-335.
BT.09
BT.08
Determination of the DNA sequence of the alliinase of Allium
stipitatum REGEL
Diedrich, D.1; Ciglia, E.1; Rüther, A.2; Kurz, T.1, Lüdeke, S.2; Gohlke, H.1;
Hansen, F.K.1
Mielke, M.G.; Keusgen, M.
Philipps-Universität Marburg, Marbacher Weg 6, 35037 Marburg, Germany
1 Institute
The genus Allium consists of more than 800 species worldwide and therefore
belongs to one of the largest genera in the plant kingdom [1,2]. This genus is
divided into 15 subgenera with Melanocrommyum being the second largest [1].
A characteristic attribute of Allium species is the occurrence of a C-S-lyase,
also called alliinase, and cysteine sulphoxides. This enzyme cleaves these
cysteine sulphoxides after cell disruption leading to the formation of bioactive
aroma compounds [3]. In Europe, common species like Allium sativum (garlic)
and Allium cepa (onion) are well characterised in respect to their cysteine
sulphoxides, but also to their alliinases [4,5]. Allium stipitatum, however, plays
an important role in Central Asian cuisine, but research on its alliinase has
hardly been done, yet. Furthermore, A. stipitatum contains a pyridinyl cysteine
N-oxide that is also subjected to alliinase-reaction (see fig.) [6]. The resulting
reaction products show high antimycobacterial activity, inducing an increasing
interest in alliinase-reaction chemistry of that species [7].
One of the predominant proteins in a protein extract of A. sativum is the
alliinase with a monomeric molecular weight of about 50 kDa. A protein extract
of A. stipitatum however, only shows a faint protein spot in the alliinase region
(see fig.), but alliinase-reaction can be easily detected in such a protein
extract. Other protein spots with molecular weights estimated at 21 and 28 kDa
are prevalent. To get information on the alliinase of A. stipitatum, already
known alliinase sequences were aligned and highly conserved regions were
1
2
α-Aminoxy Peptides as α-Helix Mimetics: Solid-Phase Synthesis
and Conformational Investigation
M [kDa]
170
130
100
70
55
40
of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität
Düsseldorf, 40225 Düsseldorf, Germany
2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104
Freiburg, Germany
Over the last few years much effort has been devoted to the investigation of
peptidomimetics consisting of unnatural amino acids to adopt defined
secondary structures such as helices or turns.[1,2] Recently, α-aminoxy
peptides, analogs of β-peptides in which the β-carbon is replaced by an
oxygen atom, were found to be promising candidates for foldamers.[2-4] It was
shown that even short α-aminoxy peptides can form stable folded structures
composed of strong intramolecular hydrogen bonds between the C=Oi and the
N-Hi+2 proton, so called N-O turns.[3] For example, 1.88 helices were observed
for D-α-aminoxy peptides containing only three residues and the backbone of
these peptides is more rigid than that of natural peptides.[3] Notably, the
aminoxy amide bond is resistant to enzymatic degradation. Altogether, these
properties make α-aminoxy peptides promising peptidomimetic foldamers.
As part of our research towards the development of novel α-helix mimetics for
the modulation of protein-protein interactions (PPIs),[5,6] we became
interested in α-aminoxy peptides as peptidomimetics. We reasoned that the
mimicry of three residues located on one α-helical face requires peptides
constructed from at least six homochiral α-aminoxy acids. Unfortunately, the
synthesis of longer α-aminoxy peptide sequences under solution phase
conditions is relatively cumbersome. Herein, we present an improved synthetic
access to α-aminoxy oligopeptides based on a straightforward solid-phase
supported approach using dimeric building blocks to synthesize a mini library
of oligomers. The conformational properties of selected oligomers were studied
by CD spectroscopy and MD simulations in order to get a more profound
understanding of the folding properties of α-aminoxy peptides.
35
25
15
10
M: Marker
Protein extract of:
1: A. sativum
2: A. stipitatum
used to design specific primers for alliinase DNA. Thus it was possible to
obtain an interior part of the gene of interest. The gene was subsequently
extended versus 5’ and 3’ ends using the method of restriction enzyme sitedirected amplification [8] leading to the whole DNA sequence. Amplification of
the gene from cDNA, derived of mRNA, not only verified the gene expression
in bulbs of A. stipitatum, but also provided valuable information about length
and position of introns. The alliinase is consisting of five exons and therefore
four introns. Despite the difference of the relative amounts of alliinase in
protein extracts, alignment of the amino acid sequences showed 83 % of
conformity with a difference in length of four amino acids.
O
N
+
O
O
alliinase
S
OH
NH2
S-(2-pyridyl)cysteine N-oxide
N
+
S
S
N
+
O
di(2-pyridyl) disulphide N,N'-dioxide
Acknowledgments: Leibnitz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Dr.
Reinhard M. Fritsch
References:
1. Fritsch, R.M. et al.: Phyton (Horn, Austria) 2010, 49(2): 145-220.
References:
1. Martinek, T.A., Fülüp, F.: Chem. Soc. Rev. 2012, 41(2): 687–702.
2. Li, X., Yang, D.: Chem. Commun. 2006, 32: 3367–3379.
3. Li, X., Wu, Y.-D., Yang, D.: Acc. Chem. Res. 2008, 41(10): 1428–1438.
4. Draghici, B., Hansen, F.K. et al.: RSC Adv. 2011, 1(4): 602–606.
5. Spanier, L., Ciglia, E. et al.: J. Org. Chem. 2014, 79(4): 1582–1593.
6. Ciglia, E., Vergin, J. et al.: PLoS ONE 2014, 9(4): e96031.
BT.10
Heterologous expression of the thiopeptide antibiotic GE2270
from Planobispora rosea in Streptomyces coelicolor requires
deletion of ribosomal genes from the cluster
Flinspach, K.1; Kapitzke, C.1; Tocchetti, A.2; Sosio, M.2; Apel, A.K.1*
1 Pharmaceutical
Biology, Eberhard Karls University, Auf der Morgenstelle 8, 72076
Tübingen, Germany. Email: [email protected]
2 KtedoGen Srl, Via G. Fantoli 16/15, 20138 Milan, Italy
The thiopeptide antibiotics comprise about one hundred natural compounds,
including micrococcin, thiostrepton, thiomuracin or berninamycin. They are
biosynthesized from ribosomally formed preproteins which undergo extensive
posttranslational modifications (1). They are active especially against Gram-
143
positive bacteria including MRSA and against malaria parasites, and they have
antiproliferative activity against human cancer cells (2). This makes them
promising drug candidates. However, their low water solubility and their poor
pharmacokinetics have so far prevented their clinical use. Structural modification by genetic engineering may provide a strategy to make these compounds
available for medical use.
The rare actinomycete Planobispora rosea produces the thiopeptide antibiotic
GE2270 which is the precursor of NAI-ACNE, an antibiotic against Propionibacterium acnes that has completed phase 1 clinical trials. In order to facilitate
genetic experiments for the generation of new GE2270 derivatives, the
GE2270 biosynthetic gene cluster together with an adjacent set of 22 genes
coding for ribosomal proteins was cloned into a SuperCos3-based cosmid.
Repeated attempts to introduce this cosmid into the heterologous expression
host Streptomyces coelicolor M1146 remained unsuccessful. However,
exconjugants could finally be obtained after the ribosomal genes were deleted
from the cosmid, and GE2270 was thereupon formed in the heterologous host.
Production of this antibiotic was further increased when the constitutive ermE*
promoter was introduced into the cluster upstream of the GE2270 resistance
gene.
Expression of ribosomal genes from Planobispora rosea may have a toxic
effect on Streptomyces coelicolor, possibly due to the rather large phylogenetic
distance between of P. rosea and S. coelicolor. In contrast, conjugation of the
entire cosmid into Nonomuraea sp. ATCC39727, which is more closely related
to P. rosea, was possible without removal of the ribosomal genes.
References:
1. Bagley, M.C. et al.: Chem. Rev. 2005, 105: 685-714.
2. Young, T.S. and Walsh C.T.: Proc. Natl. Acad. Sci. U S A 2011, 108: 13053-13058.
BT.11
Discovery and Structural Optimization of Peptides Inhibiting E.
coli RNA Polymerase
Kamal, A.; Haupenthal, J.; Hüsecken, K.; Negri, M.; Fruth, M.; Hartmann,
R.W.; Empting, M.
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Drug
Design and Optimization and Pharmaceutical and Medicinal Chemistry, Saarland
University, Campus C2.3, D-66123 Saarbrücken, Germany
The recent rise in antibiotic resistance by bacterial pathogens has rendered
several antibiotics ineffective, threatening our ability to cure infectious
diseases. Therefore, the search for new antibiotics with novel targets and
modes of action is of great importance.(1,2)
As such, peptides targeting RNA polymerase (RNAP) has been designed
interfering with RNA polymerase function via inhibiting protein-proteininteraction between the β' subunit of RNAP and the σ70 subunit interaction
and thus Holo-enzyme formation, resulting in inhibition of transcription
initiation. The interface between core RNAP and σ70 represents a promising
binding site. Nevertheless, detailed studies investigating its druggability are
rare.(3,4)
Techniques such as, ELISA, an abortive transcription assay, molecular
dynamics simulations, Circular dichroism (CD) spectroscopy, rational amino
acid replacement study and side-chain-to-side-chain macrocyclization revealed
several peptides with the ability to impede transcription initiation via binding to
the coiled-coil region in β′ and that its flexible N-terminus inhibits the enzyme
by interaction with the β′ lid-rudder-system (LRS). This work revisits the β′
coiled-coil as a hot spot for the protein−protein interaction inhibition and
expands it by introduction of the LRS as target site.(5,6)
References:
1. Chopra, I. et al.: Curr. Opin. Invest., Drugs 2007, 8: 600−607.
2. Hüsecken, K. et al.: ACS Chemical Biology, 2013, 8(4): 758-766
3. Sharp, M et al.: Genes Dev., 1999, 13: 3015−3026.
4. Lesley, S. et al.: Biochemistry, 1989, 28: 7728−7734.
5. Greenfield, N.J., Nature Protocols, 2007, 1: 2876 - 2890
6. Bergendahl, V. et al.: Appl. Environ. Microbiol., 2003, 69: 1492−1498.
144
BT.12
Expression, purification and crystallisation of MKK7
Wolle, P.; Mayer-Wrangowski, S.; Rauh, D.
Technische Universität Dortmund, Otto-Hahn-Str. 6, 44227 Dortmund, Germany
The mitogen-activated protein kinase kinase 7 (MKK7) is a dual-specific
protein kinase and a member of the c-Jun N-terminal protein kinase (JNK)
signalling pathway, which is involved in the regulation of numerous physiological processes during cellular development and in response to stress [1]. MKK7
is phosphorylated and thereby activated by MAP3K and phosphorylates JNK
together with MKK4 on specific Tyr and Thr residues in the activation loop [2].
For a better understanding of the cellular function of MKK7 in disease states,
we focus on the development of potent and selective inhibitors. As a starting
point, we set out to solve the crystal structure of MKK7 in complex with classic
ATP competitive inhibitors. For this, we cloned various constructs of MKK7,
which differ in mutations in the activation loop and modifications at the Nterminus. All constructs were expressed in E. coli and purified by affinity
chromatography, ion exchange chromatography and size exclusion chromatography. Suitable crystallisation conditions were selected by screening all
constructs against 386 different conditions at 4 °C and 20 °C. With these
conditions, we could obtain the first complex structure of MKK7. This complex
gave key insights into the pharmacological perturbation of MKK7 and now
serves as starting point for further compound development.
References:
1. Asaoka, Y.; Nishina, H.: J. Biochem. 2010, 148(4): 393-401.
2. Haeusgen, W.; Herdegen, T.; Waetzig,V.: European Journal of Cell Biology, 2011 90:
536-544.
BT.13
Interactions of differently phospholylated ERK1 with various
metal ions studied by affinity capillary electrophoresis
Mozafari, M.1; Nachbar, M.1; Redweik, S.1; Alhazmi, H.A.1; Albishri, H.M.2; ElHady, D.A.2,3; El Deeb, S.1,4; Wätzig, H.1
1 Institute
of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55, 38106 Brunswick, Germany.
2 Chemistry Department, Faculty of Science, King Abdulaziz University, 80203 Jeddah,
Saudi Arabia.
3 Chemistry Department, Faculty of Science, Assiut University, 71516-Assiut, Egypt
4 Department of Pharmaceutical Chemistry, Al-Azhar University-Gaza, Gaza, Palestine
Extracellular signal-regulated kinase (ERK) is a key regulatory enzyme in the
widely used signalling cascade of phosphorylation-dephosphorylation cycles
and plays a pivotal role in many aspects of biological processes such as
proliferation, differentiation and cell cycle progression. The ERK family belongs
to the mitogen-activated kinases (MAPKs) and in accordance with their
modified groups belongs to the serine/threonine kinases [1]. Secondary
structure transitions such as protein phosphorylations are integral modulators
of signal transduction. Many cellular processes are regulated by phosphorylation reaction inducing conformational changes and activates of proteins [2].
Due to the complexity of molecular interactions, simplified peptide models have
emerged as a useful tool for investigating the molecular interaction phenomenon. Phosphorylated residues can induce structural changes through metal
binding [3].
We investigated a peptide from the activation loop of the signal protein ERK1,
consisting of 19 amino acids using affinity capillary electrophoresis (ACE).
Affinity capillary electrophoresis has become a powerful method for separation
of peptides and proteins, so does for investigation of interactions between
various ligands and macromolecules. ACE identifies changes in the electrophoretic mobility of proteins and peptides due to changes in mass and charge
through binding or interaction with the ligand [4].
The determination of the mobility changes was carried out by using mobility
ratios of EOF-marker and the peptide to avoid the migration time shifts which
are not related to interactions.
The difference of the mobility ratio of the peptide with the ligand (Ri) and
without the ligand (Rf) was normalised to Rf (ΔR/Rf) [4].
The interaction of the unphosphorylated form of the above-mentioned peptide
with various metal ions e.g. Ba2+, Ca2+, Mg2+, Mn2+, Cu2+, Ni2+ was investigated.
Furthermore the results of the interactions of mono- and diphosphorylated form
with the same metal ions were compared.
Among all the investigated metal ions, we found strong interactions of Ni2+ and
Cu2+ with the mono- and diphosphorylated peptide which changed the overall
charge of the peptide positively and negatively respectively. Possibly Ba 2+-ion
shows significant interactions as well.
These different changes are probable due to the conformation changes of
peptide after binding to metal ions and the further interactions of binding metal
ions with the ions of surrounding solution.
Only a weak interaction with other metal ions could be found.
References:
1. Kolch, W. et al.: Biochemical Society 2000, 351: 289-305.
2. Nam, H-S. et al.: Journal of Chromatography A 2002, 976: 79-85.
3. Broncel, M. et al.: Organic & Biomolecular Chemistry 2010, 8(10): 2575-2579.
4. Redweik, S. et al.: Electrophoresis 2012, 33(22):3316-3322.
BT.14
Borreliosis Immunoassay using Reflectometric Interference
Spectroscopy (RIfS)
Acknowledgements:
This work was supported by „Arbeitsgemeinschaft Industrieller Forschungsvereinigungen“
(AiF)
References:
1. Merkl, S. et al.: Phys. Status Solidi A. 2014, DOI: 10.1002/pssa.201330436
2. Chandra, A. et al.: Clinical Immunology 2011, 141(1) : 103-110.
3. Liang, F.T. et al.: J. Clin Microbiol 2000, 38 (11) : 4160-4166.
BT.15
New Biochemical Features of Indole Prenyltransferases from
Streptomyces
Winkelblech, J.1,2
Philipps-Universität Marburg, Institut für Pharmazeutische Biologie und Biotechnologie,
Deutschhausstrasse 17A, 35037 Marburg (Germany), E-mail: [email protected]]
² Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg
Hans-Meerwein-Strasse, 35032 Marburg (Germany)]
1
Dassinger, N.; Vornicescu, D.; Keusgen, M.
Philipps Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6-10,
35037 Marburg
An improved assay for the detection of Borrelia infections in animals and
humans based on Reflectometric Interference Spectroscopy (RIfS) has been
developed. A recombinant fusion protein was used as recognition element on
the sensor surface. One moiety of the protein consists of the Borrelia antigene
VlsE (Variable major protein sequence Expressed) sequence and the other is
the lectine Concanavalin A (ConA). It was possible to immobilize the fusion
protein on sugar coated biochip surfaces via the ConA-part. With this assay, a
rapid detection of anti-Borrelia antibodies in serum will be possible.
Introduction
In previous investigations it has been shown that Reflectrometric Interference
Spectroscopy (RIfS) is suitable for the detection of bacteria [1]. Based on these
investigations, an assay for the detection of Borrelia in humans and animals
has been designed. If Borrelia enters the human body, antibodies against the
VlsE (Variable major protein sequence Expressed) antigene occur within days.
[2]. These antibodies are a valid marker for borreliosis.
The VlsE protein consists of invariable domains at the amino and the carboxyl
termini and a variable domain in the centre. The variable domain contains 6
variable regions named VRI to VRIV as well as 6 invariable regions named IR1
to IR6 [3]. For the here presented experiments, the IR 6 epitope of the Borrelia
garinii clone P7-1 was chosen. In order to bind the VlsE protein to the biochip
surface loaded with mannan, the genetic sequence of VlsE was fused to the
gene of the binding domain of the lectin Concanavalin A (ConA).
The C6-ConA protein was successfully expressed in Escherichia coli. After
inclusion body purification and refolding of the protein, the functionality was
tested on a mannan coated RIfS-chip surface. C6-ConA binds to the mannan
surface via the ConA part and the VlsE epitope is able to bind the borreliosis
specific antibodies in a positive serum sample (see fig. 2).
Prenyltransferases contribute largely to the structural diversity of natural
products, which are important resources for drug discovery and development.
Attachment of prenyl moieties derived from prenyl diphosphate, usually
dimethylallyl diphosphate (DMAPP), to various aliphatic or aromatic acceptors
often increases the biological and pharmacological activity of the resulted
compounds. Indole prenyltransferases from the dimethylallyltryptophan
synthase (DMATS) superfamily represent one of the most investigated
enzymes for the prenyl transfer reactions. These enzymes show high flexibility
towards their aromatic substrates and high regioselectivity of the prenylation
position on the indole ring. In contrast, most of them have a restricted
substrate specificity towards prenyl donors and utilize solely DMAPP as
substrate (1).
Two indole prenyltransferase genes SAML0654 and Strvi8510 were identified
in Streptomyces ambofaciens and Streptomyces violaceusniger, respectively.
Their deduced proteins with a sequence identity of 63 % on the amino acid
level to each other were overproduce in E. coli and used for enzyme assays.
HPLC analysis of the incubation mixtures revealed that L- tryptophan and
derivatives including D-tryptophan, 4-, 5-, 6- and 7-methyl-DL-tryptophan were
well accepted by both enzymes in the presence of DMAPP. Structure
elucidation of the isolated enzyme products and determination of kinetic
parameters demonstrated that L-tryptophan was accepted as the best
substrate and both enzymes function as 6-dimethylallyltryptophan synthases
(6-DMATS). Detailed biochemical characterization of 6-DMATSSa from S.
ambofaciens and 6-DMATSSv from S. violaceusniger revealed a number of
new features for indole prenyltransferases. For example, these enzymes
represent the first examples of tryptophan prenyltransferases, which accepts
both DMAPP and geranyl diphosphate (GPP) as prenyl donors and catalyzed
the same regiospecific prenylation. Furthermore, the prenylation of some
hydroxynaphtalenes and the unusual prenylation position on their unsubstituted rings also represent a new feature for bacterial indole prenyltransferases
(2).
Acknowledgements:
We thank the Deutsche Forschungsgemeinschaft and LOEWE program of the State of
Figure 2: RIfS sensorgramm. Mannan coated chip with injection of blocking
solution after 300 sec. Application of C6-ConA (50µg/ml) after 1822 sec and
injection of positive serum sample after 4822 sec.
Conclusions
The fusion protein C6-ConA is a very promising protein for detection of
borreliosis antibodies in serum samples. In combination with RIfS, a rapid
detection of a Borrelia infection seems to be possible.
Hessen (SynMikro) for financial supports.
References:
1. Yu, X. and Li, S.-M.: Methods Enzym 2012, 516: 259-278.
2. Winkelblech, J. and Li, S.-M.: Chembiochem 2014, 15: 1030-1039.
145
BT.16
DMAT synthases catalyze the formation of alkylated indoles by
using unnatural allyl and aryl diphosphates
Liebhold, M.1; Xie, X.2; Li, S.-M.1
1 Institut
für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg,
Deutschhausstrasse 17a, 35037 Marburg, Germany
2 Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032
Marburg, Germany
Prenylated indole alkaloids such as ergot alkaloids exhibit diverse pharmacological and biological effects [1,2], distinct from respective unprenylated
precursors [3]. The transfer reaction of the prenyl residues onto the aromatic
nucleus is catalyzed by prenyltransferases in nature [4]. The members of the
dimethylallyl tryptophan synthase (DMATS) superfamily belong to the recently
most investigated enzymes from this group and show high flexibility towards
aromatic substrates. They attach the prenyl moiety not only on simple indoles
or cyclic dipeptides, but also on flavonoids and hydroxynaphthalenes [5]. On
the other hand, these enzymes mostly accept DMAPP as prenyl diphosphate
[5,6,7,8].
The presented studies (Figure) show the acceptance of unnatural βunsaturated allyl diphosphates such as monomethylallyl and 2-pentenyl
diphosphate by L-tryptophan and tryptophan-containing cyclic dipeptide
prenyltransferases. In the first case (Figure A), the natural prenylation position
was partly or completely shifted, which depends on the available DMAPP
analogue. As shown in Figure B, in contrast to the natural reaction of the used
enzymes, a mixture of C2- and C3-reversely alkylated diastereomeres was
detected. Furthermore, the flexibility of several DMATS prenyltransferases
towards more space-demanding alkyl donors was demonstrated by using
benzyl diphosphate. Since FgaPT2 yielded the highest amount of products,
diverse L-tryptophan derivatives were subsequently incubated in the presence
of this enzyme. Structure elucidation proved the formation of C5-benzylated
indole derivatives (Figure C).
References:
1. Williams, R.M.; Stocking, E.M.; Sanz-Cervera, J.F.: Topics Curr. Chem. 2000, 209: 97173.
2. Li, Y.-X. et al.: Mar. Drugs 2013, 11(12): 5063-5086.
3. Botta, B. et al.: Curr. Med. Chem. 2005, 12(6): 717-739.
4. Li, S.-M.: Nat. Prod. Rep. 2010, 27(1): 57-78.
5. Yu, X.; Li, S.-M.: Methods Enzymol. 2012, 516: 259-278.
6. Chooi, Y.H. et al.: J. Am. Chem. Soc. 2012, 134(22): 9428-9437.
7. Pockrandt, D. et al.: Appl. Microbiol. Biotechnol. 2014, 98(11): 4987-4994.
8. Tarcz, S.; Xie, X.; Li, S.-M.: RCS Adv. 2014, 4(35): 17986-17992.
146
CLINICAL PHARMACY (CP01-CP15)
CP.01
CP.02
A target-mediated drug disposition model to characterize the
pharmacokinetics of bosentan after intravenous administration
in healthy subjects
Prevention of type 2 diabetes by participation in the pharmacybased lifestyle intervention program GLICEMIA: a multicenter,
randomized, controlled trial
Volz, A.-K.1; Krause, A.2; Markert, C.3; Haefeli, W.E.3; Dingemanse, J.2; Lehr,
T.1
Schmiedel, K.1; Schlager, H.1; Friedland, K.2
1 Saarland
University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany
2 Actelion Pharmaceuticals Ltd, Department of Clinical Pharmacology, Gewerbestrasse
16, Allschwil, 4123, Switzerland
3 Heidelberg University, Department of Clinical Pharmacology and Pharmacoepidemiology, Im Neuenheimer Feld 410, Heidelberg, 69120, Germany
Background and Objectives
The endothelin receptor antagonist bosentan (TracleerTM) is one of the most
frequently used drugs in the therapy of pulmonary arterial hypertension. Its
pharmacokinetic (PK) behaviour is influenced by interaction with different
transporters and metabolic enzymes [1].
The aim of this analysis was the development of a population PK model to
characterize the PK of bosentan after single intravenous application of different
doses in healthy male subjects.
Methods
Model development was performed using intravenous data from a single
ascending dose study (10, 50, 250, 500, and 750 mg) of bosentan [2]. Several
structural models were evaluated using NONMEM 7.2. Model selection was
performed based on statistical and graphical procedures. A covariate analysis
was applied using a stepwise forward inclusion (p=0.05) and backward
elimination (p=0.001) procedure.
Results
Overall, 706 plasma concentration–time data from 54 healthy subjects were
available. The PK of bosentan were best described by a 2-compartment targetmediated drug disposition (TMDD) model with internalization of the bosentantarget-complex and a turnover model for the target production- and degradation rate (Fig. 1). Interindividual variability for clearance, volumes of distribution
and kint was moderate ( 38 %CV). None of the covariates investigated
influenced the PK of bosentan systematically. A sine function on the production
rate of the target (ksyn) mimicked a circadian rhythm and improved the model
significantly (p<0.001).
Conclusion
A TMDD model was successfully developed describing the plasma concentration-time profiles of bosentan. The model revealed that binding of bosentan to
a target, presumably endothelin receptors, influences the PK significantly. In
addition, a new hypothesis on the circadian fluctuation of the free target was
generated. The model presented is a first step towards unravelling the PK
characteristics of bosentan and will serve as a valuable tool for future model
development following oral and multiple-dose administration.
1 Scientific
Institute for Prevention in Health Care (WIPIG), Maria-Theresia-Str. 28, 81675
Munich, Germany
2 Friedrich-Alexander-University, Department of Chemistry and Pharmacy, Molecular and
Clinical Pharmacy, Cauerstr. 4, 91058 Erlangen, Germany
Lifestyle intervention can be effective in decreasing risk for type 2 diabetes. A
recent meta-analysis has shown that diabetes prevention programs in
outpatient settings can be effective [1]. However, conveniently accessible
opportunities to prevent type 2 diabetes are still not introduced in Germany.
The purpose of this study was to assess the efficacy of a prevention program
carried out in community pharmacies in reducing the risk for diabetes.
Community pharmacies in Bavaria, South Germany, were randomly assigned
to the intervention (n = 21) or to the control group (n = 21). Eligible participants
had an increased risk for diabetes (FINDRISC score ≥ 7) and were at least 35
years old. All participants received written information about diabetes
prevention. In the intervention group (n = 565) the GLICEMIA program
combined three appointments with individual counselling and five educational
group sessions. The control group (n = 575) obtained only information about
their health status in three assessments. Primary outcome was the change of
the risk for diabetes indicated by the FINDRISC score after 12 months.
In the intention-to-treat population (n = 1,092), 68.6 % of the participants were
female and the median age was 57.5 years (IQR: 49.2 - 65.5). Highly prevalent
risk factors were overweight and sedentary lifestyle (81.9 % and 72.7 %,
respectively). The primary endpoint, a reduction of the FINDRISC score, was
achieved by a significantly higher proportion in the intervention group than in
the control group (39.1 % versus 21.0 %, p < 0.001). Statistically significant
differences between the groups were also demonstrated in the secondary
endpoints weight reduction, physical activity and physical quality of life. The
goal of a minimum weight reduction by 5 % was achieved by 21.6 % in the
intervention group and 8.3 % in the control group (p < 0.001). Moreover, the
blood pressure decreased and the mental quality of life increased in both
groups.
Pharmacists can help patients to reduce their risk for diabetes. The prevention
program GLICEMIA can be successfully conducted in community pharmacies
and is effective decreasing the risk for type 2 diabetes in the high-risk
population in Germany.
Acknowledgments: This study received funding through the Dr. August and Dr. AnniLesmüller Foundation, the Bavarian State association of Corporate Health Insurers (BKK),
the funding initiative for prevention (Förderinitiative Prävention e. V.) and the Bavarian
health promotion initiative “Gesund.Leben.Bayern.” by the Bavarian State Ministry of
Public Health and Care Services.
Reference:
1. Dunkley, A.J. et al.: Diabetes Care. 2014, 37(4): 922−933.
CP.03
03the impact of oral anticoagulants on the human
Predicting
coagulation pathway using a comprehensive systems pharmacology model
Fig.1. TMDD model of bosentan
References:
1. Dingemanse, J. and van Giersbergen, P.L.M.:Clinical Pharmacokinetics 2004, 43 (15):
1089–1115.
2. Weber, C. et al. :Clinical Pharmacoogy & Therapeutics 1996, 60: 124–137.
Dings, C.; Schäftlein, A.; Lehr, T.
Clinical Pharmacy, Saarland University, Campus C2 2, 66123 Saarbrücken, Germany
Background and Objectives: The introduction of the oral anticoagulation
drugs (OAC) warfarin, dabigatran, rivaroxaban, edoxaban and apixaban
changed the landscape of the treatment of thrombotic diseases substantially
[1]. The pharmacokinetics (PK) and pharmacodynamics (PD) of the OAC are
well known. Nevertheless, a mathematical coagulation model in humans,
which summarizes the influence of different OAC on clotting times such as e.g.
activated partial prothrombin time (aPTT) or prothrombin time (PT), is not
available yet.
The aim of this work was to extend a previously published human coagulation
model [2] by the effects of the OAC warfarin, dabigatran, rivaroxaban,
147
edoxaban and apixaban on the coagulation pathway and further by the
inclusion of the Ecarin clotting time as an important clinical coagulation marker.
Materials and Methods: A recently published coagulation pathway model [2]
was rebuilt in Matlab version R2013a. Published population PK models of
warfarin [3], dabigatran [4], rivaroxaban [5] and edoxaban [6] as well as a selfdeveloped PK model of apixaban based on digitized concentration-time
profiles [7] were included. The link between the PK and the PD of the drugs
was recognized using an Emax inhibition rate model on vitamin K, thrombin or
factor Xa. The effects of the OAC on the coagulation system were visualized
using Matlab and SAS version 9.3 and compared to the observed clotting
times after administration of the respective investigated drug [4-7]. Model
development of the apixaban PK model was performed using NONMEM 7.2.
Results: A linear one compartment model with first order absorption for
warfarin and rivaroxaban and a linear two compartment model for apixaban,
edoxaban and dabigatran were found to be adequate. The human coagulation
cascade was described by 47 ordinary differential equations (ODE) and 158
parameters. 13 ODE and 51 parameters connected the PK of the OAC to the
coagulation network. Overall, the model successfully predicted observed
concentration-time profiles of the OAC as well as the impact of the drugs on
clotting times like aPTT or PT. In addition, the Ecarin clotting time (ECT) was
implemented in the model and showed an adequate predictive performance.
Conclusion: The developed OAC-coagulation model is a powerful tool for a
wide range of applications. For example, the model enables a comparison of
different OAC and support the clinical important prediction of switching
algorithm between the OAC. Besides, timing of antidote dosing to reduce
major bleedings (e.g. Vitamin K for warfarin) or pharmacodynamic interactions
between the OAC on every coagulation factor of interest can be investigated.
References:
1. Kitslaar, D.B. et al.: Curr Treat Options Cardiovasc Med. 2014, 16(8): 326.
2. Wajima, T. et al.: Clincial pharmacology & Therapeutics 2009, 86(3): 291.
3. Lane, S. et al.: Br J Clin Pharmacol. 2012, 73(1): 66.
4. Liesenfeld, K.H.: et al. J Thromb Haemost 2011, 9: 2168.
5. Girgis, I.G. et al.: The Journal of Clinical Pharmacology 2014.
6. Song, S.H. et al.: The Journal of Clinical Pharmacology 2014.
7. Frost, C. et al.: Br J Clin Pharmacol. 2013, 75(2): 476.
CP.04
03 in an Afghan hospital: boon or bane?
Pharmaceuticals
Mielke, M.G.*; Neumann, S.*; Keusgen, M.
*equally contributing authors
Philipps-Universität Marburg, Institute for Pharm. Chemistry, Marbacher Weg 6-10, D35032 Marburg, Germany
Falsifications of pharmaceuticals are an increasing problem worldwide as was
recently demonstrated by operation Pangea VII, where 111 countries
participated in seizing about 9.4 million counterfeit medicines [1]. Although the
European market seems to be quite safe, if not ordering medicines by internet
other countries in Africa, Asia and South America have more problems
regarding drug falsifications [2]. Afghanistan can be considered as developing
country and the health care system is still to be built up. The hospital in Mazare Sharif is being helped to establish an up-to-date treatment of the population
in the region by the GIZ [3]. Nevertheless, pharmaceuticals in this hospital
seem to be incredibly cheap (0.20 € up to 2.63 €), so falsifications with no
active agent or a concentration, that is too low, could be assumed.
To examine if this is the case, the active compounds of some medicines of the
hospital in Mazar-e Sharif were analysed (see fig.). These pharmaceuticals
were produced in different countries nearby Afghanistan. Until now, some pain
killers of the NSAID category and several antibiotics were analysed based on
the European Pharmacopoeia and USP. Extraction methods were validated
by examining the content of some pharmaceuticals produced for the German
market. All Afghan pharmaceuticals investigated so far fulfilled the Pharmacopoeias’ requirements regarding identity and content.
Acknowledgements: Civil Hospital Mazar-e Sharif, Dr. Matthias Körner
References:
1. Interpol: http://www.interpol.int/Crime-areas/Pharmaceutical-crime/Operations
(25.06.2014)
2. WHO: http://www.who.int/mediacentre/factsheets/fs275/en/ (27.06.2014)
3. Deutsche Gesellschaft für Internationale Zusammenarbeit:
http://www.giz.de/de/weltweit/14695.html (25.06.2014)
CP.05
03 development and validation for the determination
HPLC method
of anidulafungin in microdialysis samples of healthy volunteers
and intensive care patients
Weiser, C.1; Kauzor, D.1; Brosig, H.1; Kees, F.2; Zeitlinger, M.3; Kloft, C.1
1 Dept.
of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet
Berlin, Kelchstr.31, 12169 Berlin, Germany
2 Dept. of Pharmacology, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany
3 Dept. of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20,
1090 Vienna, Austria
Objectives:
Anidulafungin (ANI), a new echinocandin, has been approved for the treatment
of invasive Candida infections. Up to now, knowledge about pharmacokinetics
for ANI is very rare particular in special patient populations and at the target
site. For the analysis of several upcoming pharmacokinetic microdialysis
studies e.g. in healthy volunteers and intensive care patients, which will be
performed at the University Hospital in Vienna, a suitable HPLC assay for the
determination of ANI had to be developed and validated. A low quantification
limit of the assay was required in order to reliably quantify also low tissue fluid
concentrations of ANI. In addition, unspecific adsorption of the drug shall be
avoided by appropriate sample preparation.
Methods:
A Thermo Scientific Dionex Ultimate 3000 HPLC with a DAD 3000 detector
was chosen for the quantification of ANI. Different ways of sample preparation
and adsorption prevention measures were investigated using additives
(methanol, ethanol and human serum albumin (HSA)) to prevent adsorption
and different volumes of methanol for subsequent protein precipitation. The
mobile phase, based on [1], and consisting of methanol and ammonium
dihydrogen phosphate, was investigated regarding the composition and flow
rate. Method development was also important for the column (Accucore
Phenyl-Hexyl column (50 x 4.6 mm, 2.6 µm) vs. Merck LiChrospher 100 RP-18
column (125 x 4 mm, 5 µm), both with a guard column (RP 18, 5 µm)) and
column temperature (25 - 40°C). After the assay development, validation was
performed in accordance to the EMA guideline for bioanalytical method
validation [2].
Results and Conclusions:
The successful HPLC assay development comprised a LiChrospher RP18
column (125 x 4 mm, 5 µm), a C18 guard column, isocratic mode with
methanol and ammonium dihydrogen phosphate 85:15 (v/v) at a flow rate of
1.0 mL/min, autosampler and column temperature at 10° C and 40° C,
respectively, detection at 310 nm, injection volume of 20 µL and fulfilled all
predefined prerequisites. The total run time was 5 minutes with a retention time
for ANI of 2.5 minutes. Adsorption of ANI was prevented by addition of HAS
(0.5%) and subsequent protein precipitation using methanol. Validation was
successfully performed according to the criteria of the EMA guideline for
bioanalytical method validation for the concentration range of 0.1 – 20 µg/mL.
148
The developed and validated assay allows quantification of ANI from in vitro
microdialysate samples as crucial precondition allow in vivo μD investigations
in healthy volunteers and patients. As next step the in vitro µD experiments will
be performed to decide settings for the clinical studies.
References:
1. Sutherland, C.A. et al.: J Chromatogr Sci 2011, 49 (5): 397-400.
2. European Medicines Agency (EMA): Guideline for bioanalytical method validation 2012
CP.06
Quality 03
assurance for pharmaceutical laboratories in developing
countries
Maul, K.J.1; Diergardt, T.2; Feldmann, D.3; Wätzig, H.1
1 Technische
Universität Braunschweig – Institute of Medicinal and Pharmaceutical
Chemistry, Beethovenstraße 55, 38106, Germany
2 Physikalisch-Technische Bundesanstalt – Braunschweig, Bundesallee 100, 38116,
Germany
3 Bausch+Lomb, Brunsbütteler Damm 165-173, 13581, Germany
Quality control is a major issue in the development, production and testing of
medicine. This should be practiced in all laboratories which deal with this
highly valuable good. Quality infrastructure in the East African Community
(EAC) is discussed if the requirements of the pharmaceutical sector are met.
At present the pharmaceutical industry can only produce for their own
domestic market. They are not allowed to sell their products anywhere else
due to the lack of quality standards assurance.
In this project initiated by the Physikalisch-Technische Bundesanstalt called
“Establishment of a regional Quality Infrastructure for the pharmaceutical
sector in the EAC“ proficiency tests, a tool for quality assurance, were
conducted. The first in October 2013 was about analyzing Amoxicillin 500 mg
tablets. Among the 15 participants were laboratories from five different
countries. According to the USP monograph assay and dissolution were
performed. The requirements of the USP are 90-120 % for the assay and at
least 75 % for the dissolution. The interlaboratory standard deviation for the
assay is 12.7 mg/tablet (RSD%=2.62) and for the dissolution 4.87 %
(RSD%=5.28). As assigned value the mean of all laboratories was used and
the z-score for each laboratory was calculated (Figure 1). The result for the
assay is very satisfying the overall average of 97.1 % is the exact same result
the manufacture of the tablets states in its release certificate.
Acknowledgments:
This work was supported by Bayerische Akademie für Klinische Pharmazie/Dr. August
und Dr. Anni Lesmüller-Stiftung, Munich, Germany.
The authors are grateful for the expert advice of Dr. Annette Freidank, Pharmacy
Department, Fulda Hospital, Germany and PD Dr. Wolfgang Frobenius, MME, Department
of Gynacology and Obstetrics, Erlangen University Hospital, Germany.
References:
1. Konsensuspapier der AG zur Ausbildung im Fach Klinische Pharmazie: 1. Teil:
Rahmenbedingungen und Organisation. http://www.klinische-pharmazie.org/. Assessed
June 10, 2014.
2. Smee, S.: BMJ 2003, 326(7391): 703-706.
Assay
z-score
Following the implementation of “Clinical Pharmacy” into the pharmacy course
curriculum (AAppO) in 2001, the German Pharmaceutical Society (DPhG)
published recommendations for its structure in a paper of consent [1] including
the involvement of a teacher practitioner to ensure a patient-centered
education. To systematically evaluate the benefits of bed-side teaching of
fourth year pharmacy students in a German university hospital setting a
randomized teaching and learning study was carried out.
A course was created consisting of 2x1,5h of class-room teaching and 2x4h of
practical teaching on a psychiatric ward in small student groups. Learning aims
concentrated on patient and interprofessional communication techniques and
included: taking medication histories, identification and handling of medicationbased problems and pharmaceutical counseling of psychiatric patients. The 42
students of the control group only participated in the theoretical part while the
42 students of the intervention group took part in the complete course. The
effects were assessed by an objective structured clinical examination (OSCE)
[2] consisting of five practical and five theoretical tasks testing for clinically
applied knowledge and communication skills. In addition, a questionnaire was
conducted asking for students’ opinions about course structure, relevance of
teaching content and overall satisfaction.
The intervention group achieved a significantly better overall result in the
OSCE assessment (46.4±9.5 vs. 28.2±9.0 of 90 points; p<0.001) with most
positive effect in assessed communication skills (27.4±5.4 vs. 16.3±6.0 of 40
points; p<0.001). The performance in the theoretical tasks was poor (12.1±4.1
vs. 8.1±3.2 of 30 points; p<0.001) indicating that knowledge-based applied
clinical pharmacy skills still need to be improved. This could be achieved by
more practical training in clinical pharmacy. The findings are supported by the
questionnaire: 93% of the students rated the course as practice-orientated,
90% felt better prepared for patient contact and 92% gave a positive answer
when asked for overall satisfaction. Many students suggested an extension of
the course in the free text field of the questionnaire.
In conclusion, the results of this quantitative teaching study demonstrate
significant learning benefits by the teacher practitioner course and the high
usefulness of bed-side teaching in pharmacy student education. Hence, it
should be implemented as a mandatory course in the pharmacy course
curriculum in Germany.
1 6 9 15 10 5 14 8 11 12 2 7 4 3 13
-3
CP.08
In vitro 03
microdialysis characterising vancomycin as precondition
lab. #
Dissolution
z-score
for an upcoming trial in neonates
8
1
7 15 13 9
6 14 2
5
4 12 3 11 10
-3
lab. #
Figure 1: Z-score charts: The z-scores of each participant are shown
for assay and dissolution. A z-score between -2 and 2 (white area) is
very good. Z-scores between -3 to -2 and 2 to 3 (black area) acceptable.
CP.07
The Erlangen
03 Teacher Practitioner Project: Advances in Clinical
Pharmacy Teaching in Germany
Dircks, M.G.1,2; Dörje, F.2; Friedland, K.1
1 Molecular
and Clinical Pharmacy, Friedrich-Alexander University Erlangen-Nürnberg,
Cauerstraße 4, 91058 Erlangen, Germany
2 Pharmacy Department, Erlangen University Hospital, Palmsanlage 3, 91054 Erlangen,
Germany
Kauzor, D.1; Schröpf, S.²; Fürtig, M.1; Ruhe, D.1; Kloft, C.1
1 Dept.
Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,
Kelchstr. 31, 12169 Berlin, Germany
2 Dept. of Pediatrics, Dr. von Haunersches Kinderspital, Lindwurmstraße 4, 80337 Munich,
Germany
Background: Infections with gram-positive bacteria are commonly treated with
vancomycin. This wide use leads to increasing rates of vancomycin-resistant
bacteria which are favoured by suboptimal dosing. To avoid non-therapeutic
vancomycin concentrations, the pharmacokinetics (PK) has to be well
characterised as has been done for adults, but not in neonates. Extrapolation
of PK parameters from adults to other patient populations may increase the
risk of adverse effects (overdosing) or therapy failure (underdosing). Especially
in neonates PK studies are difficult to perform with multiple blood sampling due
to the limited availability of blood volume. An alternative is the microdialysis
technique which benefits from sampling without removing body fluids: a
catheter with a semipermeable membrane is inserted in the particular
interstitial
fluid
(ISF)
allowing
measurements of drug concentrations continuously and directly at the target site. In an
upcoming trial, microdialysis will be performed in the ISF of subcutaneous
tissue of neonates treated with vancomycin (VAN). Prior to clinical trials, an in
vitro microdialysis characterisation of the particular drug is crucial in order to
149
set optimal settings for a consistent relative recovery (RR) in vivo; being the
‘catheter calibration factor’ as the ratio between measured concentrations in
microdialysate samples and ISF in neonates.
Methods: In vitro characterisation of vancomycin was performed with a
standardised in vitro microdialysis system [1] and CMA 63 microdialysis
catheters (membrane length 10 mm, molecular weight cut-off 20 kDa) which
are approved for neonates and will be used in the clinical trial. Three catheters
were used simultaneously for the characterisation of VAN to investigate
intercatheter variability. Drug concentration was set to 50 µg/mL of vancomycin for all investigations; the surrounding medium was heated to 37° C to
mimic body temperature. As perfusate, Ringer’s solution (RS) was used, and
the surrounding medium consisted of phosphate buffered saline (PBS) to
mimic ISF with a constant pH value. RR was determined for different flow rates
(2.0, 1.0 and 0.5 µL/min) and pH values (6.6, 7.0, 7.4 and 8.0) of the surrounding medium in both, delivery (VAN diluted in RS in the perfusate) and recovery
(VAN diluted in PBS in the surrounding medium) experiments, to assess
equality of RR in both diffusion directions. Five replicates were sampled in
each experiment. An HPLC assay for the quantification of VAN
from microdialysate samples was developed and validated according the EMA
guideline [2].
Results: RR was found to be dependent of flow rate and pH value. RR
increased with lower flow rates and was highest at a pH value of 7.0 in delivery
as well in recovery experiments. Highest RRs were obtained with a flow rate of
0.5 µL/min (pH: 7.0). Lowest RRs were observed at a pH value of 8.0 and a
flow rate of 2 µL/min. RRs calculated from delivery were on average approximately 20% lower than RR from recovery experiments.
Conclusion:
The results demonstrate a strong dependency of RR from flow rate, as well as
from pH value of the surrounding medium. Based on the RR findings, a flow
rate of 1 µL/min is to be recommended for the clinical trial due to a compromise in high RR, acceptable µD sampling intervals and VAN concentration
changes over time. As RR seems to be dependent on the pH value of the
surrounding medium, monitoring of pH values of the microdialysate samples as
surrogates for the pH values in interstitial fluid shall be performed in the trial.
Equality of RR in both directions was not observed in vitro and has to be
further investigated. As next step, the RR dependency of drug concentration
shall be examined to further establish conditions in the upcoming trial which
shall contirubute to improve vancomycin therapy and might support the
implementation of therapeutic drug monitoring.
We thank Prof. Markus Zeitlinger, UKH Wien for the support concerning microdialysis
issues.
References:
1. Kirbs et al.: EJPS 2014.
2. EMA guideline on bioanalytical method validation 2012.
CP.09
03and future directions for the implementation of a
Status quo
Medication Management Service – An online survey in German
Pharmacy Stores
Britz, H.*; Keßler, V.*; Schneider, I.*; Müller, C.*; Schäftlein, A.; Lehr, T.
1 Saarland
University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany
* Equally contributing first-authors
The implementation of medication management as a standard service for
German pharmacies is under discussions [1]. Despite the increasing interest,
there might be open issues for German pharmacist around medication
management, like a common definition of the process or reimbursement. Aim
of this project was to develop and conduct an online survey for German
pharmacists on the medication management service in German pharmacy
stores to depict the Status Quo of knowledge about the medication management and the expectations about the potential future service in a huge
population of officinal pharmacists.
Methods
An anonymized online survey for German pharmacies on medication management was developed [2]. Besides questions about demographic data and
information on the respective pharmacy store, pharmacists were asked
questions on status quo and future directions of medication management. The
150
survey is accessible online [2] and will be promoted in August and September
2014. The survey will be continued until at least 200 responses are available.
Results from the survey were summarized electronically; statistics and
graphics were generated using SAS 9.3.
Results
So far, 12 pharmacists responded to the online survey. Even so the number is
small so far, a few interim results are presented in the following, final results
will be presented at the meeting.
Overall, 70% of the pharmacists perform already medication management in
their pharmacy store. However, there is a huge diversity in what is actually
done and what is believed to be part of the medication management service.
Only 30% of the pharmacists are aware of the definitions about medication
management provided by the German pharmaceutical society (DPhG) [3].
After providing the definition to the pharmacists, 80% would consider the
definition of an “extended” medication management including information
about and through the patient as implementable. Pharmacists believe that total
costs for medication management should be shared as follows: 91% should be
paid by health care providers and 9% by patients. Pharmacists expected that
they would spend on average 50 minutes for a first face-to-face meeting, 60
minutes for the analysis of the medication and 25 minutes for a follow up
meeting. On average, a reimbursement of €50 per hour was considered as
acceptable for a medication management service by pharmacists.
Conclusion
Medication management is recognized by German pharmacists and considered as a potential new standard service in pharmacy stores. The results of
this survey may help to the guide the future implementation of a regular
medication management service in pharmacy stores.
References:
1. http://www.abda.de/leitbild.html
2. www.medikationsmanagement.info
3. http://www.dphg.de/news-folder/detailansicht/implementierung-desmedikationsmanagements-als-neue-pharmazeutischedienstleistung/f196c965646ee2fac82c21c165e1f939/
CP.10
Acceptance
03 of a Medication Management Service in German
Pharmacy Stores – A Saarland Patient Survey
Müller, C.*; Schneider, I.*; Keßler, V.*; Britz, H.*; Schäftlein, A.; Lehr, T.
Saarland University, Clinical Pharmacy, Campus C 2 2, Saarbrücken, 66123, Germany
* Equally contributing first-authors
Medication management is considered as an important tool to improve the
safety and efficacy of patient’s therapy. The implementation of medication
management as a standard service for German pharmacies is intensively
discussed these days [1]. However, little is known about the interest and
perception of patients on the potential future service. Aim of this project was to
develop and conduct a survey in patients about the acceptance of medication
management service in German pharmacy stores.
Methods
A patient dedicated anonymized survey on medication management was
developed. Besides age and sex, various questions were asked about patient’s
medication (number, self-reported knowledge, resource, contact person). Next,
medication management was introduced to the randomly selected patients by
a standardized paragraph which was recited by the interviewer. Afterwards,
questions about medication management (interest, willingness to pay, sharing
of costs) were asked. The survey was conducted in the pedestrian zone in
Saarbrücken by four undergraduate pharmacy students in April 2014. Results
from the survey were summarized manually; statistics and graphics were
generated using SAS 9.3.
Results
Overall, 212 patients (60% females) participated in the survey. Most patients
(48.9%) were between 51 and 70 years old. 74% of the patients had a regular
intake of medications; the average number of medications per patient was 3.
The self-reported knowledge of the patients about their medications was high
(average 82%). The majority of the patients had a fix pharmacy store (67%)
where they receive their medication from and which the contact for questions
about the medication.
In total, 70% of the patients are willing to use a medication management
service. Of this proportion, 71% were willing to pay on average €18 out of their
pocket for this service. Patients believe that total costs for medication
management should be shared as follows: 67% paid by health care providers,
22% by patients, 9% by pharmacists and the remaining 2% by doctors.
Conclusion
Medication management was perceived very well by the patients as a potential
new standard service in pharmacy stores. In addition to the acceptance of the
new service provided, patients were also willing to pay for medication
management. Even so the study presented may have a recall bias of the
patients and potentially a selection bias of the patients selected by the
interviewer, the results are promising. The outcomes of this survey may help to
the guide the future implementation of a regular medication management
service in pharmacy stores.
Reference:
1. http://www.dphg.de/news-folder/detailansicht/implementierung-desmedikationsmanagements-als-neue-pharmazeutischedienstleistung/f196c965646ee2fac82c21c165e1f939/
CP.11
Utilisation
03of electronically generated medication plans in
German community pharmacies: status quo
Botermann, L.1,2, Griese, N.2, Krueger, K.2, Eickhoff, C.2, Schulz, M.2, Kloft, C.1
1 Dept.
of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet
Berlin, Kelchstr. 31, 12169 Berlin, Germany
2 Dept. of Medicine, ABDA – Federal Union of German Associations of Pharmacists,
Jaegerstr. 49/50, 10117 Berlin, Germany
Objectives: A current and complete medication plan is of central importance for
medication safety. Therefore the German Ministry of Health (BMG) integrated
a standardised medication plan (MP) as a safety measures in their “Action
Plan to promote medication safety in Germany”. A specification1 defines the
requirements for the standardised MP in form and content. The aim of this
study was to evaluate the status quo of the technical capabilities and the
utilisation of electronically generated printable MPs for patients in German
community pharmacies.
Methods: An online survey was developed and sent to 3,380 community
pharmacists. The questionnaire contained items on the digital storage of
patient data, the technical capabilities to generate and print a MP for patients
and its utilisation in pharmacy practice. Pharmacists were also asked to mail
an example of a medication plan. These sent documents were then analysed
according to specific criteria: The first criterion was whether it was actually to
be considered as a plan for patients and not for health care professionals. In
the next step it was analysed to which extent the document followed the
specification for a standardised MP by the BMG.
Results: The response rate was 10% (n=326). 80% (n=261) of the respondents
saved patient-based medical data digitally. For 35% (n=91) of these pharmacists this also included dosages for selected patients. 44% (n=144) of all
respondents indicated that their pharmacy software offered the technical
capabilities to print a medication plan, whereas 36% (n=117) were uncertain
about this. 15% (n=42) of the questioned pharmacists stated to print MPs or
selected ambulant patients. Only two of the sent medication plans were for
patients and based on the BMG specification.
Conclusions: At present not all German community pharmacies have the
technical capabilities to generate a MP with their pharmacy software systems.
Furthermore, other medication lists are mistaken to be a MP for patients. For a
successful nationwide implementation of the standardised MP it is necessary
to improve (i) the technical capabilities of pharmacy software systems and (ii)
the knowledge of community pharmacist.
References:
1. Spezifikation für einen patientenbezogenen Medikationsplan, Version 2.0 - für
Modellvorhaben (2013). (Accessed July 10, 2014, at
http://www.akdae.de/AMTS/Medikationsplan/index.html)
CP.12
Systemic
03review of treatment options for functional
gastrointestinal diseases: STW 5 is equivalent to MCP in the
treatment of functional dyspepsia and has a superior safety
profile
Madisch, A.1; Vinson, B.R.* 2; Kelber, O.2; Kraft, K.3; Storr, M.4
1 Gastroenterology,
Medical Clinic I, KRH Clinic Siloah , Hannover,
Research / Medical Information, Scientific Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt,
3 Chair of Naturopathy, Center for Internal Medicine, University Medicine, Rostock,
4 Neurogastroenterology, Medical Clinic and Policlinic II, Clinic of Munich University
Munich-Großhadern, Munich, Germany
2 Clinical
INTRODUCTION: The use of metoclopramide (MCP) has recently been
restricted due to the risk of extrapyramidal side effects, based on provisions of
the European Regulatory Agency EMA and national regulatory authorities.
Thus, MCP is no longer available for the treatment of chronic conditions such
as dyspepsia and gastro-oesophageal reflux diseases. This necessitates
revisiting of the clinical data for alternative treatments with equivalent clinical
efficacy and a more benign safety profile, which were less visible in the past.
AIMS & METHODS: As herbal medicinal products (HMPs) are widely used for
functional gastrointestinal diseases including functional dyspepsia (FD) and
irritable bowel syndrome (IBS) and with a favorable safety profile, a systematic
review was conducted, in compliance with the PRISMA statement, to identify
HMPs with a therapeutic efficacy comparable to MCP, using a data base
search complemented by cross referencing and hand searching for assuring
completeness.
RESULTS: Six comparison studies using HMPs and MCP were identified, four
of them with Ginger in the treatment of emesis and therefore out of scope,
while two were conducted in FD and were therefore included into the evaluation. In these studies, STW 5, an herbal combination medicine was used. One
study is an RCT in 94 patients [1], showing despite its small size a clear
equivalence of both treatments, with a trend towards a faster onset of action
and a lower number of UEs in the STW 5 group. The second, a retrospective
epidemiologic analysis study was conducted in 960 patients treated with MCP
or STW 5 [2] and confirmed these data in routine clinical practice, with a
significantly higher proportion of symptom-free patients and a lower number of
days off work in the STW 5 group. A literature search assessing the safety of
STW 5 identified studies in more than 50.000 patients with FD and IBS [3], with
a high degree of safety also in children [4, 5] and without any severe side
effects and without an interaction potential with other medicines.
CONCLUSION: For the treatment of functional gastrointestinal diseases such
as FD, STW 5 was identified as a treatment with clinical efficacy equivalent to
MCP but a superior safety profile. This allows an excellent long term treatment
for patients with FD in the post MCP age.
References:
1. Nicolay, K. et al.: Gastro-Entero-Hepatologie 1984, 4: 24-28.
2. Raedsch, R. et al.: Z Gastroenterol 2007, 45: 1041-1048.
3. Ottillinger, et al.: Wien Med Wochenschr 2013, 163: 65-72.
4. Kelber, O.: Z Phytotherapie 2010, 31: 40-47.
5. Radke, M.: Gastroenterologe 2011, 6: 486-495.
CP.13
1 + 1 ≠ 03
2? Gene expression profiling as a novel approach for
developing fixed drug combination products
Kelber, O.1; Abdel-Aziz, H.1; Okpanyi, S.N.1; Ulrich-Merzenich, G.2
1
Scientific Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany;
for Internal Medicine, Medical Clinics III, University Clinics Bonn, Bonn, Germany
2 Center
Introduction: Combinations of drugs are common in many indications. But
despite their established positive effects with regard to compliance, the
number of available fixed combination products is comparatively small.
Aim: The regulatory rules of FDA and EMA for the justification of fixed
combination products are based up to now on the theses of Crout from 1974
[1].
Methods: Therefore an overview of the present state of scientific approaches
in this field is given.
Results: The equivalence or superiority of combinations, in comparison to the
combination partners, is shown in studies with several arms, as the Vishnu-
151
or the Diener study [2, 3]. In contrast, gene expression profiling allows a
description of the pharmacological profile of the combination in comparison to
the combination partners. According to this, the profile of the combination is
different to that of the partners and the risk profile can be more favourable
compared to that of the partners [4].
Discussion: A combination is, according to the equation 1 + 1 ≠ 2, a
completely new active substance with an own pharmacological profile [4].
Instead of the combination partners [1], therefore the standard therapy in the
indication should be used in comparison studies, as in other new active
substances.
Acknowledgments: This contribution is dedicated to Prof. Dr. Dr. h.c. mult. Hildebert
Wagner, who has described in a multitude of scientific contributions the multi-target effect
of multi-drug-combinations.
To Dr. Christiane Otto, Dr. Uwe Gessner, Dr. Carolin Stäbler and Dr. Christoph Theurer,
Bayer Vital, Leverkusen, Prof. Dr. Thomas Effert, Universität Mainz and Prof. Dr.
Alexander Panossian, Swedish Herbal Institute, Vallberga, best thanks for scientific
discussions.
accept a change to DOAC. Patients willing to accept a change in therapy were
slightly younger than those who refused. Gender differences were not
observed. 74.4 % of the currently VKA-treated patients reported stable INR
values. 52.1% of patients who had already switched to DOAC indicated
complications in maintaining adequate INR-values prior to the change. The
following factors were statistically significantly associated with the acceptance
to change therapy: lack of a need for coagulation monitoring, doctor’s
recommendation, personal interest in alternative drugs, prospect of an
improvement in health-related quality of life, as well as the personal characteristics extrovert/introvert.
Conclusion: About 50 % of the participants of our survey were willing to
change anticoagulant therapy from VKA to DOAC. High variation in INRvalues, opinion of the practitioner and several personality traits motivate
patients to accept a change. Further investigations are required to prove the
validity of these results.
References:
1. Crout, J.: J Clin Pharmacol 1974; 14:249.
2. Eckles, R. and Voelker M.: Clin Pharm Drug Dev 2014; 3:118-125.
3. Diener, H.C. et al.: Cephalgia 2005; 25:776-787.
4. Ulrich-Merzenich et al.: Phytomedicine 2012; 19:322-329.
References:
1. Moser, M., Bode, C.: Kardiologe 2012, 6:148–156.
2. Harenberg, J., Weiss, C.: Haemostaseologie 2013, 33:62-70.
3. Zolfaghari, S. et al.: Semin Thromb Hemost 2014, 40:121-128.
4. Fahrenberg, J., Hampel, R. and Selg, H. (2010): Freiburger Persönlichkeitsinventar, 8.
Auflage. Hogrefe Verlag Göttingen
CP.14
Investigations
03 on the acceptance of new direct oral anticoagu-
CP.15
Caco-2 03
cells – a suitable in vitro model to predict drug-drug
lants in long-term treatment of thrombo-embolic prophylaxis in
patients with atrial fibrillation
Hergenröther, A.1; Thuermann, P.A.2 ; Weiss, C.3; Harenberg, J.1
Instiute of Clinical Pharmacology, Medical Faculty Mannheim, Heidelberg of University,
Germany
2 Institute of Clinical Pharmacology, University of Witten/ Herdecke, Germany
3 Department of Medicial Statistics, Medical Faculty Mannheim, Heidelberg of University,
Germany
1
Background and aims: Oral anticoagulants are world-wide frequently
prescribed for prevention and treatment of thrombotic and embolic diseases
[1]. However, severe and life-threatening side effects, numerous drug
interactions as well as regular blood coagulation monitoring limit their use.
Direct oral anticoagulants (DOAC) such as dabigatran, rivaroxaban, apixaban
and edoxaban became recently available as therapeutic alternatives. DOAC
can be administered without regular coagulation monitoring with a similar
therapeutic efficacy and a positive risk/benefit profile compared to VKA.
Disadvantages include higher expenses than their conventional competitors
[2]. In the present study we aimed to analyse whether patient’s personal
characteristics may contribute to the decision-making process concerning the
prescription and application of the DOAC as opposed to the conventional VKA.
The superior aim of this study is to optimise treatment and therapeutic success
by integrating a patient’s personal characteristics and preferences into the
prescribing process.
Materials and methods: Data were acquired over a period of 15 months.
Patients treated (for a minimum of 2 months) with either VKA or DOAC were
recruited from registered physicians and public pharmacies and written
informed consent was obtained according to ethical approval of the study. The
anticoagulation therapy survey was developed based on the results of a
previous focus group interview [3]. In addition to a standardised questionnaire
to assess personality traits (Freiburger Persönlichkeitsinventar, FPI-R) [4], a
new questionnaire was developed and validated. After a pilot study (44
participants) the full-scale survey on anticoagulation therapy employing a
shortened version of the questionnaire was completed. Biographic data and
medical history, concomitant diseases and comedication were documented in
an EXCEL datasheet (EXCEL, Microsoft Office, 2007). Statistical analyses
using the chi-squared test (SAS version 9.2) were performed investigating the
association between personal characteristics and the patient’s disposition for a
change in anticoagulation therapy.
Results: In total 117 anticoagulation patients aged between 24 and 92 years
with 39.3 % female participants were included. 70.9 % of them specified atrial
fibrillation as indication for anticoagulant therapy. At the time of the interview
84 participants were on VKA, whereas 33 patients took one of the DOAC. 23 of
these patients had been previously treated with VKA. Although patients on
VKA were quite satisfied with their treatment, 39 (47 %) of them were willing to
152
interactions caused by induction of drug transporters?
Brück, S.; Strohmeier, J.; Busch, D.;Siegmund, W.; Oswald, S.
Clinical Pharmacology, University of Greifswald, Center of Drug Absorption and Transport
(C_DAT), Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany
Background: Intestinal absorption of many drugs is influenced by transport
proteins. Induction of these proteins by concomitant treatment with inducers
such as rifampicin or St. John’s wort (SJW) was shown to cause unwanted
drug-drug interactions (DDIs). Although it would be desirable to predict these
interactions using well established cellular transporter models, in vitro methods
for evaluation of transporter induction are not yet established. Therefore, we
investigated whether Caco-2 cells may be a suitable experimental model to
predict the aforementioned DDIs.
Methods: For induction studies, Caco-2 cells were incubated for 48 hours with
pharmacologically relevant but non-toxic concentrations of carbamazepine
(500 µM), efavirenz (10 µM), the SJW constituents hypericin (0.5 µM) and
hyperforin (1 µM), and rifampicin (100 µM), respectively. Afterwards, mRNA
expression and protein abundance were determined using TaqMan® low
density arrays and LC-MS/MS-based targeted proteomics. Functional studies
with respect to ABCB1 were performed using Transwell® assays with [3H]digoxin and [3H]-talinolol as substrates. In parallel, mRNA expression and
protein content of clinically relevant intestinal transporters in native Caco-2
cells and jejunal tissue specimens were compared.
Results: After incubation with prototypical inducers, effects on mRNA
expression and protein content could not be observed in Caco-2 cells.
Consequently, the efflux ratios (B to A/A to B) for ABCB1 probe drugs in Caco2
monolayers were not significantly affected by the tested inducers. In contrast to
this, ABCB1 function was nearly abolished by PSC833. With the exception of
ABCB1, the gene expression and protein content of ABCC2, ABCC3, PEPT1,
OATP2B1 and others were markedly different in Caco-2 cells compared to that
from human jejunum.
Conclusion: Established in vivo inducers of drug transport do not influence
expression and function of drug transporting proteins in Caco-2 cells.
Therefore, Caco-2 cells are no suitable in vitro model to predict transporter
induction in man.
DRUG DESIGN/MEDICINAL CHEMISTRY/ANALYTICS (MC01-MC62)
MC.01
Synthesis, Enantiomeric Separation and Biological Evaluation of
Fosmidomycin Thia Isosters as 1-deoxy-D-xylulose 5-phosphate
reductoisomerase Inhibitors
Lienau, C.1; Kunfermann, A.2; Illarionov, B.3; Held, J.4; Gräwert, T.3; Behrendt,
C.T.1; Werner, P.3; Hähn, S.1; Eisenreich, W.2; Riederer, U.3; Mordmüller, B.4;
Bacher, A.3; Fischer, M.3; Groll, M.2; Kurz, T.
1 Institut
für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität
2 Center for Integrated Protein Science Munich, Lehrstuhl für Biochemie, Technische
Universität München, Lichtenbergstr. 4, 85747 Garching, Germany
3 Hamburg School of Food Science, Universität Hamburg, Grindelallee 117, 20146
Hamburg, Germany
4 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074
Tübingen, Germany
Multidrug-resistant pathogens endanger human health worldwide. Antiinfective drugs addressing novel targets are therefore urgently needed. Being
absent in mammalians, the non-mevalonate pathway of isoprenoid biosynthesis (MEP pathway) is a promising target for the development of new antibiotics
[1,2]. In several pathogenic bacteria and apicomplexan protozoa - including
Plasmodium spp. - the MEP pathway is the only source of isopentenyl
diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). 1-Deoxy-Dxylulose 5-phosphate reductoisomerase (Dxr, IspC), a key enzyme within the
MEP pathway, can be inhibited by the natural product forsmidomycin. Reverse
α-aryl substituted carba- and oxa-analogs of fosmidomycin are highly active
Dxr inhibitors [3,4,5]. We report on the synthesis, enantiomeric separation and
biological evaluation of reverse α-aryl substituted β-thia isosters of fosmidomycin, which show potent antiplasmodial in vitro activity [6].
Magnesium supplementation by means of organic magnesium salts is a very
popular practice [1] e.g. with magnesium bis(L-hydrogenaspartate) dihydrate.
This substance is monographed in the European Pharmacopoeia. Here, we
examined the enantiomeric purity using a chiral capillary zone electrophoresis
applying (2-hydroxypropyl)-β-cyclodextrin as a chiral selector coupled to laser
induced fluorescence detection after CBQCA derivatization [2] and a HPLCfluorescence method with chiral derivatization using o-phthaldialdehyde and
N-acetyl-L-cysteine [3] as an orthogonal method. Two API batch samples and
three drug products of the salt were analyzed by means of both methods. The
concentration of the D-enantiomer of aspartic acid ranged from 0.03 to 0.12 per
cent.
The substance is prepared from different magnesium salts and L-aspartic acid
via neutralization and subsequent precipitation [4]. A closer look to the
synthesis revealed the elevated D-aspartic acid content to be caused by the
dissolution of L-aspartic acid at acidic pH values following the reaction pathway
shown below [5].
Acknowledgements: Thanks are due to the Federal Institute of Drugs and Medical Devices
(Bonn, Germany) for financial support and the European Directorate for the Quality of
Medicines & HealthCare for the sample and reference substance supply.
References:
1. Garrison, S.R. et al.: Magnesium for skeletal muscle cramps, Cochrane Db. Syst. Rev.,
2012.
2. Novatchev, N., Holzgrabe, U.: J. Pharm. Biomed. Anal., 2002, 28(3-4): 475-486.
3. Aswad, D.W.: Anal. Biochem., 1984, 137(2): 405-409.
4. Shaolin Huang, H.J., Feng Liu, Fei Wang, Yong Zhang: Method for preparing
magnesium aspartate, CN 101239925 (A), Beijing Jingwei Xinkang Pharma, China, 2008.
5. Erbe, T., Bruckner, H.: Eur. Food Res. Technol., 2000, 211(1): 6-12.
MC.03
Analytical technologies for the detection of counterfeit medicines: Are simple field assays still suitable for the routine quality
control of pharmaceuticals in developing countries?
Höllein, L.; Holzgrabe, U.
University of Würzburg, Institute of Pharmacy and Food Chemistry, Am Hubland, 97074
Würzburg
Fig. 1 S-Enantiomer of fosmidomycin thia isoster 5a in complex with PfIspC
References:
1. Jomaa, H. et al.: Science 1999, 285: 1573-1576.
2. Rohmer, M. et al.: Curr. Opin. Investig. Drugs 2004, 5: 154-162.
3. Behrendt, C.T. et al.: J. Med. Chem. 2011, 54: 6796-6802.
4. Behrendt, C.T. et al.: ChemMedChem 2010, 5(10):1673-1676.
5. Brücher, K. et al.: J. Med. Chem. 2012, 55: 6566-6575.
6. Kunfermann, A., Lienau, C. et al.: J. Med. Chem. 2013, 56: 8151-8162.
MC.02
Enantiomeric purity of Magnesium bis(L-hydrogenaspartate)
dehydrate
Wahl, O.; Holzgrabe, U.
University of Würzburg, Institute for Pharmacy and Food Chemistry, 97074 Würzburg,
Germany
The increasing prevalence of poor-quality pharmaceuticals circulating in lowand middle-income countries, especially in the field of antibiotic and antimalarial medicines, is alarming because side effects, resistances or complete
therapy failures may occur after the ingestion of such products [1]. Regulatory
authorities in the developing world are scarcely able to control the situation
within the national markets. The lack of human resources, limited technical
equipment or narrow financial means at the respective testing institutions are
only few reasons why counterfeit and substandard medicines can be easily
distributed in these countries.
Laboratories in resource-restraint environments are not always capable of
adhering to compendial methods which are described in the major pharmacopoeias, e.g. the European Pharmacopoeia. The application of sophisticated
High-Performance Liquid Chromatography (HPLC) found in almost every
monograph is still considered the gold standard, but demands for delicate
consumables (chemicals, solvents, columns), expensive apparatus or skilled
analysts for operation and maintenance [2]. These are critical bottlenecks
during laboratory work in developing countries making a routine application
almost impossible.
The provision of adapted technologies for the analysis of essential medicines
has been anticipated since many years and one of the major achievements in
this area was the introduction of the Minilab® through Merck and the Global
Pharma Health Fund in the late 1990s using colour reactions for identification
153
and thin-layer chromatography (TLC) for a semiquantitative determination of
the content for meanwhile 70 essential active pharmaceutical ingredients
(APIs) [3]. Beside the simplicity of its protocols it also holds certain drawbacks
being a failure rate of approximately 60 % during the quantitative assays [4].
Therefore we suggest the application of simplified liquid chromatographic
methods instead which we developed for common antimalarial agents [5]. Only
very simple chemicals and columns (e.g. no ion-pairing reagents) are
necessary and they are able to quantitatively determine the respective APIs,
resolve distinct impurities and can detect replacements or mix-ups. Our
methods can be run on basic HPLC instruments at simple laboratories (e.g. no
gradient pumps are required) and can be used for intermediate testing
between the Minilab screenings and confirmatory assays using established
HPLC protocols. Combining these technologies to three analytical ‘levels’
could be a promising solution to reliably detect poor-quality medicines
circulating in the distribution chains.
References:
1. Tremblay, M.: Curr. Drug Saf. 2013, 8: 43-55.
2. Deconinck, E. et al.: J. Chromatogr. Sci., 2013, 51: 791-806.
3. Jahnke, R.W.O. et al.: Drug Inf. J. 2001, 35: 941-945.
4. World Health Organization: Survey of the Quality of Selected Antimalaria Medicines
Circulating in Six Countries of Sub-Saharan Africa. 2011.
5. Hoellein, L. and Holzgrabe, U.: J. Pharm. Biomed. Anal, 2014, article in press.
MC.04
Development of MIP-Inhibitors
Seufert, F.1; Juli, C.1; Hein, M.1; Norville, I.H.2; Jenner, D.2; Sarkar-Tyson, M.2;
Harmer, N.J.3; Weiwad, M.4; Schweimer, K.5; Rösch, P.5; Stacy, R.6; Myler, P.6;
Begley, D.7; Fox, D.7; Lorimer, D.7; Sotriffer, C.A.1; Holzgrabe, U.1
1 Institute
of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074
Würzburg, Germany
2 Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK
3 Biocatalysis Centre, University of Exeter, Devon, EX4 4QD, UK
4 Institute of Biochemistry and Biotechnology, MLU Halle-Wittenberg, Weinbergweg 22,
06120 Halle, Germany
5 Department of Biopolymers, University of Bayreuth, Universitätsstrasse 30, 95447
Bayreuth, Germany
6 Seattle Structural Genomics Center for Infectious Disease, Seattle, WA
7 Emerald BioStructures, Bainbridge Island, WA
Macrophage infectivity potentiator proteins (MIP proteins) are virulence factors
that facilitate the infection of cells with different pathogenic bacteria like
Legionella pneumophila (Lp) or Burkholderia pseudomallei (Bp). Both
pathogens manifest in different diseases, Lp is the causative agent of
Legionnaires‘ disease, whereas Bp induces melioidosis. MIPs of both gramnegative bacteria show peptidyl prolyl cis/trans isomerase (PPIase) activity.
These surface proteins belonging to the family of the FKBP binding protein
form a stable complex with the immunosuppressive drugs FK506 or rapamycin
which inhibit the enzymatic function of the mentioned MIPs (1, 2). Due to the
immunosuppressive effects Legionnaires‘ disease and melioidosis can’t be
treated with these drugs; thus novel inhibitors have to be developed. With the
aid of crystal structures and nuclear magnetic resonance analyses of Bp and
Lp MIP a key chemical scaffold was identified. Biophysical characterisation of
this scaffold showed that binding site specificity in solution is possible. This
series purveyed a low-micromolar affinity for Bp and Lp MIP without displaying
toxicity in human macrophages or having immunosuppressive effects. In an in
vitro assay, the compounds substantially reduce the Bp MIP activity, leading to
a decreased macrophage infectivity and intracellular growth of Bp (3). This, the
skeleton (1) can be regarded as a lead structure. The derived compound
library was tested towards several MIP proteins. SARs were established.
MC.05
Nonyl-linked bis-tacrine as a promising candidate for inhibition
of Trypanothione reductase
Schmidt, I.1; Schurigt, U.2; Krauth-Siegel, R.L.3; Jiménez-Ruiz, A.4; Holzgrabe,
U.1
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074
Würzburg, Germany
2 Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str. 2,
97080 Würzburg, Germany
3 Center of Biochemistry, University of Heidelberg, Im Neuenheimer Feld 328, 69120
Heidelberg, Germany
4 Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Carretera
Madrid-Barcelona km 33,600, E-28871 Alcalá de Henares, Madrid,Spain
1
Parasites belonging to the order of Trypanosomatida are the causative agents
for a variety of important infectious tropical diseases like African sleeping
sickness, Chagas` disease and Leishmaniasis. Unfortunately, therapy is
limited to the low number of currently available drugs and, due to spreading
drug resistance, to their effectiveness. Therefore the development of new and
cheap agents is urgently needed. A promising target for anti-trypanosomal
drug development is the trypanothione reductase (TR) system required for
DNA synthesis and defense against oxidative stress. Since it is unique for the
parasites and absent in mammalian hosts, no selectivity problems arise. The
search for inhibitors of TR is difficult because of its large binding pocket and,
besides, the TR activity would need to be reduced by >85 % to prevent
parasite proliferation [1].
The antiprotozoal activities of dimeric tacrine derivatives against Trypanosoma
brucei and Leishmania major in a full cell assay were in high nanomolar range
of concentration. In search of the mode of action, we found out that the TR of
Trypanosoma cruzi, Trypanosoma brucei and Leishmania infantum is inhibited
by our compounds. So further kinetic experiments were conducted and
resulted in a competitive mode of inhibition. To give an example, Compound 1
possesses a very low Ki-value (< 1 µM) for Trypanosoma cruzi and Trypanosoma brucei TR and doesn´t inhibit human glutathione reductase, which is a
cognate enzyme to TR in humans.
Compound 1
Acknowledgments
We acknowledge the Deutsche Forschungsgemeinschaft (SFB 630) for support.
References:
1 Krauth-Siegel R.L., Leroux A.E.: Antioxid Redox Signal 2012, 17(4):583-607.
MC.06
Development and validation of an HPLC method for determination of vancomycin in human plasma and its comparison with
PETINIA (Siemens)
Usman, M.; Hempel, G.
Institute of Pharmaceutical and Medicinal Chemistry - Clinical Pharmacy, Westfälische
Wilhelms Universität Münster, Corrensstr. 48, 48149 Münster, Germany
Acknowledgments: Financial support DFG (SFB 630)
References:
1. Norville I.H. et al.: Infection and Immunity 2011, 79(11): 4299–4307.
2. Juli, C. et al.: J. Med Chem. 2011, 54: 277–283.
3. Begley D. et al.: Antimicrob Agents Ch. 2014, 58(3): 1458-1467.
154
Vancomycin (VAN), a glycopeptide antibiotic is used against infections caused
by gram positive bacteria particularly methicillin-resistant staphylococcus
aureus (MRSA) infections [1-4]. Under dosing of VAN leads to drug resistance
in bacteria and over dosing is associated with toxicity [5] therefore therapeutic
drug monitoring (TDM) is recommended for optimizing VAN therapy [6]. For
TDM plasma/serum-level determination of VAN is inevitable.
In this investigation, a selective and sensitive method of high performance
liquid chromatography (HPLC) was developed and validated for the quantification of vancomycin (VAN) in human plasma. The separation was carried out by
using a mobile phase NH4H2PO4(50 mM)-acetonitrile (88:12, v/v) (pH 2.2) at
isocratic flow rate 0.360 mL/min on a nucleodur C18 column (125 mm × 4.6
mm, particle size 5 µm) with UV detection at 205 nm. Sample preparation was
done by deproteination of plasma with 70 % perchloric acid and a liquid/liquid
extraction to remove lipophilic compounds. Validation was performed
according to the European Medicines Agency (EMA) guideline. The method
showed linearity over the range of 0.25-60 mg/L with a coefficient of determination r2 ≥ 0.998 and a lower limit of quantification of 0.25 mg/L. No interference was observed in blank plasma samples at the retention time of VAN (Fig.
1). The percentage recovery and coefficient of variation (C.V %) values for
accuracy and precision were within the acceptable limits with a mean
percentage recovery (n = 5) of VAN between 91.46 % and 115.03 %. The latter
value is for LLOQ where the acceptable limit is ±20 %. For precision, the C.V
is ≤ 13.48 % except for LLOQ (17.82 %) where acceptable limit is 20 %.
Stability was proved at room temperature for 24 hours, after repeated freezethaw cycles and storage at -20ºC for three months. A good correlation was
observed (r2 = 0.898) by comparing with the results of particle enhanced
turbidimetric inhibition immunoassay (PETINIA, Siemens) technique in 216
serum samples (Fig. 2).
ATP binding site of EGFR. Some of these inhibitors including WZ-4002[6] and
CO1686[7] have shown great potential in overcoming drug resistance in
EGFR-T790M and are currently in further clinical development.
Within the spectrum of kinase inhibitors, covalent-reversible inhibitors (CRI)
represent another class of interesting inhibitors that can be optimized for
extended drug-target residence times. For CRI’s it was shown that the fast
addition of thiols to electron-deficient olefins leads to a covalent bond that can
break reversibly under proteolytic conditions (Fig. 1).[8] Although CRI’s are only
tool compounds at this point, they might very well develop into innovative
therapeutics to overcome some of the problems associated with conventional
covalent inhibitors, such as toxicity or off-target activities.
Figure 1 Schematic representation of the mechanism of covalent reversible
inhibition.
Here, we aim towards a better understanding of the kinetic mechanisms of
CRI’s and to investigate their potential in targeting EGFR-T790M in cellular
and in vivo systems. In a proof of concept study, we set out to design,
synthesize and test compounds that can address C797 in drug resistant
EGFR-T790M with an electron-deficient olefin.
Figure 1: Chromatograms of blank plasma (a) and plasma spiked with
vancomycin 60 mg/L (b)
References:
1. Wong, K.K.: Lung cancer 2008, 60: Suppl 2, S10.
2. Zandi, R. et al.: Cellular signalling 2007, 19: 2013.
3. Heuckmann, J.M. et al.: Journal of clinical oncology 2012, 30: 3417.
4. Kubo, K. et al.: Journal of medicinal chemistry 2005, 48: 1359.
5. Rabiller, M. et al.: Archiv der Pharmazie 2010, 343: 193.
6. Zhou, W. et al.: Nature 2009, 462: 1070.
7. Walter, A.O. et al.: Cancer discovery 2013, 3: 1404.
8. Serafimova, I.M. et al.: Nature chemical biology 2012, 8: 471.
MC.08
Figure 2: Correlation of VAN concentrations in 216 serum samples analyzed
by PETINIA (Siemens) and HPLC.
Acknowledgments: We acknowledge the help of Dr. Manfred Fobker, Universitätsklinikum
Münster (UKM) for this project.
References:
1. Van Bambeke, F.: Curr. Opin. Pharmacol. 2004, 4(5): 471-478.
2. Lundstrom, T.S.; Sobel, J.D.: Infect. Dis. Clin. North. Am. 2004, 18(3): 651-668, x.
3. Wilhelm, M.P.; Estes, L.: Mayo Clin. Proc. 1999, 74(9): 928-935.
4. Kullar, R. et al.: Clin. Infect. Dis. 2011, 52(8): 975-981.
5. Ingram, P.R. et al.: J. Antimicrob. Chemother. 2008, 62(1): 168-171.
6. Martin, J.H. et al.: Clin. Biochem. Rev. 2010, 31(1): 21-24.
MC.07
Design and synthesis of covalent-reversible Inhibitors to overcame the drug-resistant gatekeeper Mutation in EGFR.
Smith, S.; Basu, D.; Engel, J..; Rauh, D.
Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, D-44227,
Dortmund, Germany
Acquired drug resistance is a major bottleneck in targeted cancer therapies.
Cancer patients that suffer from non-small cell lung cancer (NSCLC)[1] caused
by a point mutation (L858R) in EGFR (epidermal growth factor receptor),
exhibit a dramatic relapse which is caused by secondary drug resistance
mutations. The first-line treatment with erlotinib and gefitinib[2] proofs
ineffective because of the newly formed gatekeeper mutation (T790M)[3],
which increases the off-rate if the conventional reversible inhibitors[4].
Therefore new drug scaffolds with a minimal off-rate and a maximal drug-target
residence time had to be developed.[5] Covalent inhibitors are equipped with
Michael-acceptors that target a unique cysteine (C797) located at the lip of the
Structure-Based Design, Synthesis and Biochemical Evaluation
of Ligands Addressing a Lipophilic Binding Site in the
MAP-Kinase p38α
Bührmann, M.; Wiedemann, B.M.; Hardick, J.; Ecke, M.; Rauh, D.
Fakultät für Chemie und Chemische Biologie, Technische Universität, D-44227 Dortmund,
Germany
Protein kinases catalyze the phosphotransfer from ATP to protein substrates
and are involved in a number of signaling pathways. Hence, dysfunction of the
fine-tuned regulation of kinase activity may result in diseases such as
inflammation and cancer. Targeting kinase function with small organic
molecules represents promising therapeutic strategies. However, due to the
conserved nature of the kinase active site, ATP competitive inhibitors are often
plagued by limited selectivity [1]. Allosteric ligands bind to distal binding sites
and often stabilize catalytically inactive conformations. Such ligands are
valuable starting points towards the development of more selective kinase
inhibitors [2,3].
The mitogen-activated protein kinase (MAPK) p38α is a key player in
inflammation and contains a unique hydrophobic pocket at its C-terminus
about 30 Å away from the ATP-pocket. This allosteric site has so far no known
biological function [4]. In a chemical biology approach, we aim to generate
potent binders that may serve as molecular probes and help to dissect the
functional role of this pocket in p38α. Recently, we have developed a
fluorescence-based high throughput screening (HTS) direct binding assay for
the allosteric pocket of p38α and identified scaffolds which bind exclusively to
this site [5]. Using protein X-ray crystallography, we validated the screening
results (Figure 1). We now present the structure-based design, organic
synthesis and biochemical evaluation of follow-up compounds.
155
References:
1. Schiffmann, R. et al.: Proc Natl Acad Sci USA 1998, 95: 1207-1212.
2. Altarescu, G. et al: Neurology 2002, 59: 306-313.
3. Grimm, C. et al: Chem. Biol. 2010, 17: 135-148.
MC.10
Synthesis and pharmacological evaluation of Dithiocarbamates
as novel anthelmintic inhibitors against Schistosomiasis
Mäder, P.1; Blohm, A.2; Grevelding, C.G.2; Schlitzer, M.1
1 Institut
für Pharmazeutische Chemie, Philipps-University Marburg, Marbacher Weg 6-10,
35032 Marburg
2 Institut für Parasitologie, Justus-Liebig-Universität Gießen, Schubertstraße 81, 35392
Gießen
Figure 1. Crystal structure of p38α in complex with 1 bound to the lipid binding
site at the C-terminus (boxed)[5].
References:
1. Rabiller, M. et al.: Arch. Pharm. 2010, 343: 193–206.
2. Fang, Z.; Grütter, C.; Rauh, D.: ACS Chem. Biol. 2013, 8(1): 58–70.
3. Schneider, R. et al.: J. Am. Chem. Soc. 2012, 134(22): 9138–9141.
4. Diskin, R.; Engelberg, D.; Livnah, O.: J. Mol. Biol. 2008, 375(1): 70–79.
5. Getlik, M. et al.: PLoS ONE 2012, 7(7): e39713.
MC.09
Synthesis of small molecules restoring the function of TRPML1
mutant isoforms causing mucolipidosis type IV
Keller, M.1; Cheng-Chang, C.2; Kortum, F.1; Hawarden, A.1; Prothiwa, M.1;
Hüwel, D.1; Schiffmann, R.3; Urban, N.4; Schaefer, M.4; Wahl-Schott, C.2; Biel,
M.2; Grimm, C.2; Bracher, F.1
für Pharmazie – Zentrum für Pharmaforschung, Ludwig-MaximiliansUniversität München, Butenandtstraße 5-13, 81377 München, Germany
2 Department für Pharmazie – Center for Integrated Protein Science Munich (CIPSM),
Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München,
Germany
3 Institute of Metabolic Disease, Baylor Research Institute, Dallas, TX, USA
4 Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Germany
Schistosomiasis, also known as bilharzia, is a chronic parasitic disease
infecting more than 250 million people, and at least 200.000 deaths are
associated with the disease.[1] After Malaria, it is the second most important
parasitic disease worldwide occurring in over 70 countries in tropical and
subtropical regions. [2] The schistosome species are all transmitted through the
contact with water containing free-living larval forms of the parasite (cercariae)
originating from intermediate host snails. [2] Due the lack of a vaccine, the
therapy is currently limited to a single drug, praziquantel, which is the only drug
effective against all schistosome species, and it has been used for more than
40 years. Every year, millions of people are treated with praziquantel, so that
upcoming fear of drug resistance encourages the search for novel anthelmintic
drugs against schistosomiasis. [2] [3]
Dithiocarbamates as anthelmintic compounds were identified from a screening
with disulfiram. Disulfiram was used for the treatment of chronic alcoholism by
inhibiting the acetaldehyde dehydrogenase that converts acetaldehyde to
acetic acid. [4] It has been observed that disulfiram also disintegrates the
surface structure of schistosomes, the tegument, leading to the death of the
parasites. We synthesized 22 dithiocarbamates instead of dithiocarbamate
disulfides. The basic structure is shown in figure 1.
1 Department
Mucolipidosis type IV (ML IV) is a very rare autosomal recessive lysosomal
storage disorder (LSD) characterized by severe neurological problems,
progressive neurodegeneration, and ophthalmologic abnormalities [1].
Mutations in the TRPML1 gene are causative for ML IV. Some of these
mutations lead to a loss or partial loss of function but do not cause severe
mislocalization of the protein [2]. Starting from SF-22 [3], a TRPML1 activator
discovered recently in a random screening, we generated several series of
chemically modified SF-22 analogues with the aim to further improve the
efficacy and potency profile of SF-22. Therefore we performed systematic
modifications in any structural motif of the lead structure. These compounds
were tested for their potential to restore TRPML1 mutant channel function.
Using the whole-lysosome planar patch-clamp technique, we found out that
some of the synthetic small molecules are acting as agonists and strongly
increase the activity of the TRPML1 receptor. Trafficking defects as well as
accumulation of heavy metals (zinc) in lysosomes of ML IV patient-derived cell
lines could be rescued by the treatment with these small molecules. These
findings demonstrate that small molecules can be used to restore TRPML1
channel function and to rescue disease associated symptoms in patient cells
expressing certain TRPML1 point mutations.
156
Figure 1: basic structure.
All compounds with a methyl-, amide-, carboxylic acid or methylester as
substituent for R3 are inactive, but the compounds with a lipophilic substituent
for R3 such as benzyl or cyclic acetal showed a much better activity than
disulfiram.
The significant effects of dithiocarbamate compounds on adult schistosomes in
vitro provides convincing evidence that these compounds have promise with
respect to a new direction for chemotherapy of human schistosomiasis
References:
1. Maha-Hamadien, A. et al.: PLoS Medicine. 2007, 4(1): 130-138.
2. Thétiot-Laurent, S. et al.: Angew. Chem. Int. Ed. 2013, 52: 7936-7956.
3. Sayed, A. et al.: Nature Medicine. 2008, 14(4): 407-412.
4. Wickström, M. et al.: Biochem. Pharm. 2007, 73: 25-33.
MC.11
Fatty acid composition analysis in polysorbate 80 with high
performance liquid chromatography coupled to charged aerosol
detection
will need to bear additional functional groups, requiring protective groups,
exocyclic amidation is the last step of the synthesis.
We extend this by discussing a stereoselective synthesis for 2,3diaminobutyric acid by a Mitsunobu reaction starting from allo-threonine
needed as starting material for 12-membered cyclic peptides, replacing both
ester bonds by amide bonds.
Pyridomycin
Ilko, D.1; Braun, A.1; Germershaus, O.2; Meinel, L.1; Holzgrabe, U.1
Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE97074 Wuerzburg, Germany
2 Institute for Pharma Technology, University of Applied Sciences Northwestern
Switzerland, Gruendenstrasse 40, 4132 Muttenz, Switzerland
1
Polysorbate 80 (PS 80), a sorbitan oleic acid ester copolymerized with about
20 mole of ethylene oxide per mole sorbitan, is a frequently-used non-ionic
surfactant and stabilizing agent in protein formulations. Its fatty acid (FA)
composition is characterized by gas chromatography after derivatization in the
European (EP) [1] and United States Pharmacopoeia (USP) [2]. We developed
an alternative method using HPLC in combination with a Corona ® charged
aerosol detector (CAD). After saponification with potassium hydroxide the FA
fraction was collected with liquid-liquid extraction using methyl-tert-butyl ether.
Method validation in terms of specificity, repeatability, limits of quantification,
linearity, range, accuracy and robustness was performed. In addition, the
method was expanded to the evaluation of the free FAs. Having determined
the entire FA composition, the acid value according to EP and USP can be
calculated.
The characterization of four different PS 80 batches revealed considerable
variability regarding their FA composition. Two FAs currently not mentioned in
pharmacopoeias were found additionally. Petroselinic acid, a double-bond
positional isomer to oleic acid was present in every batch. In addition, 11hydroxy-9-octadecenoic acid, an oxidation product of oleic acid was found by
means of HPLC-MS/MS.
References:
1. Polysorbate 80 Monograph 01/2011:0428 in European Pharmacopoeia, 8th Edition,
European Directorate for the Quality of Medicines & HealthCare (EDQM), 2014,
Strasbourg, France.
2. Polysorbate 80 Monograph in United States Pharmacopoeia, USP 37 NF 32, The
United States Pharmacopeial Convention, 2014, Rockville, MD, USA.
References:
1. Maeda, K. et al.: J Antibiot (Tokyo) 1953, 6(3): 140.
2. Koyama, G. et al.: Tetrahedron Lett. 1967, 37: 3587–3590.
3. Hartkoorn, R.C., et al.: EMBO Mol Med 2012, 4: 1–11.
4. Hartkoorn, R.C., et al.: Nature Chemical Biology 2014, 10: 96–98.
5. Laqua, K. et al.: paper in preparation.
6. Horlacher et al.: ACS Med. Chem. Lett. 2013, 4(2): 264–268.
MC.13
Molecular details of the reaction of nitro, nitroso, hydroxylamino
and amino benzothiazinones with Mycobacterium tuberculosis
DprE1
Richter, A.1; Rudolph, I.1; Chung, C.2; Singh, O.2; Argyrou, A.2; Ballell, L.3;
Voigt, K.4; Möllmann, U.4; Imming, P.1
Institut fuer Pharmazie, Martin-Luther-Universitaet, Wolfgang-Langenbeck-Str. 4, Halle
(Saale), 06120, Germany
2 GSK Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, United
Kingdom
3 Diseases of the Developing World, GSK, Severo Ochoa 2, Tres Cantos, Madrid, 28760,
Spain
4 Leibniz Institute for Natural Product Research and Infection Biology Hans-Knöll- Institute,
Beutenbergstr. 11a, Jena, 07745, Germany
1
MC.12
Synthesis of simplified amide analogues of the antimycobacterial
cyclodepsipeptide pyridomycin
Klemm, M.; Imming, P.
Martin-Luther-Universitaet Halle-Wittenberg, Institut fuer Pharmazie, WolfgangLangenbeck-Str. 4, 06120 Halle (Saale), Germany
Maeda et al. isolated pyridomycin from cultures of Streptomyces pyridomyceticus in 1953 [1]. Pyridomycin is a twelve-membered cyclic depsipeptide
containing five stereocentres, substituted with a semicyclic (Z)-s-butylidene
group, 3-pyridylmethyl side chain and an exocyclic hydroxypicolinoyl ring [2]. It
shows activity against Mycobacterium tuberculosis at 0.6 –1.2 µg/ml by
inhibiting NADH-dependent enoyl acyl carrier protein reductase InhA directly
[3] and has the unique feature to block both substrate and co-factor binding
sides of InhA [4]. As little is known about pyridomycin SAR, analogues are
promising candidates for further investigation.
In our group, we synthesized simplified analogues of pyridomycin. We
established a sequence with formation of the amide bond first to reduce the
risk of inter- or intramolecular transacylations followed by the formation of the
two ester bonds. [5] The semicyclic (Z)-s-butylidene group was replaced by
different aliphatic side chains as it was recognized to be unessential for
biological activity [6]. The ring closure poses the crucial step of the whole
synthetic procedure because of possible di/polymerization, resulting in difficult
purification.
We decided to synthesize cyclic depsipeptides in which both or either of the
ester bonds is replaced by amide bonds combining the advantages of less
likelihood for transacylations with better yields for the ring closure step
implementing macrolactamization instead of macrolactonization [5, 6].
We present synthetic approaches to a pyridomycin-analogous backbone
replacing one of the ester bonds by an amide bond. The cyclic depsipeptides
are synthesized via a building block strategy along the amide and ester bonds
implying the need for orthogonal protecting groups. As the exocyclic side chain
Benzothiazinones (BTZs) display very strong antimycobacterial activity in vitro
[1]. Some derivatives of this compound class, for example BTZ043 [2] and
PBTZ169 [3], are very active against Mycobacterium tuberculosis in vitro with
MICs < 1nM. The outstanding in vitro activity is not yet fully translated to in vivo
effectivity, with relatively high doses of 50-200 mg/kg daily required for
considerable reduction of CFUs in mice [1,4].
BTZs inhibit the mycobacterial enzyme decaprenylphosphoryl-β-D-ribose-2epimerase (DprE1), interfering with the construction of a functioning cell wall.
Important for the activity is a nitro group in the molecule, which is reduced by
FADH2/DprE1 to a nitroso BTZ that binds covalently to the thiol group of Cys387 near the catalytic centre [5]. In Fig. 1 we fully depict the complex process
of this two-step mechanism-based inhibition:
Figure 1. Steps of DprE1 inhibition by BTZs
BTZs with nitro, nitroso, hydroxylamino and amino groups were directly
incubated with DprE1 of Mycobacterium tuberculosis. The nitroso BTZs were
synthesized and purified for the first time [6].
The covalent binding to the target was corroborated by mass spectral data,
consistent with previous findings [7, 8]. BTZ-DprE1 X-ray structures with
157
different BTZs were solved and indicated a very similar binding mode of all
BTZs investigated, notwithstanding their different in vitro or in vivo activities.
For a deeper insight into the molecular activity of BTZs, long-term enzyme
kinetic data were collected and evaluated. This information improves the
understanding of the complex BTZ-DprE1 interaction better, trying to find an
answer to the question what are the key determinants for highly active DprE1
inhibitors.
References:
1 Makarov, V. et al.: Science 2009, 324: 801 – 804.
2 Möllmann, U.; Makarov, V.; Cole, S.T.: WO2009010163A1 2009.
3 Makarov, V.; Cole, S.T.: WO2012/066518A1 2012.
4 Makarov, V. et al.: EMBO Mol. Med. 2014, 6 (3): 372-383.
5 Trefzer, C. et al.: J. Am. Chem. Soc. 2012, 134 (2): 912 – 915.
6 Davey, M.H. et al.: J. Org. Chem. 1999, 64 (13): 4976-4979.
7 Trefzer, C. et al.: J. Am. Chem. Soc. 2010, 132 (39): 13663 – 13665.
8 Neres, J. et al.: Sci. Transl. Med. 2012, 4 (150): 150ra-121.
MC.14
Synthesis of peptide and depsipetide analogues of Hirsutellide A
using a solid phase peptide synthesis approach for antimycobacterial activity testing
Asfaw, H.; Imming, P.
Martin-Luther-Universitaet Halle-Wittenberg, Institut fuer Pharmazie, WolfgangLangenbeck-Str. 4, 06120 Halle (Saale), Germany
Hirsutellide A is an 18-membered symmetrical cyclic depsipeptide that was
isolated from a cell extract of the entomopathogenic fungus Hirsutella
kobayasii BCC 1660 and reported to exhibit antimycobacterial activity with a
minimum inhibitory concentration (MIC) of 6–12 µg/mL towards M. tuberculosis
H37Ra [1]. The structure of Hirsutellide A was elucidated by analyses of
spectroscopic data and shown to contain L-allo-isoleucine, (R)-2-hydroxy-3phenylpropanoic acid and sarcosine.
Though a synthetic strategy for one of stereoisomers of Hirstullide A was
reported using a standard solution phase approach [2], it was found to be time
consuming and purification of the intermediate products was troublesome.
Hence, to synthesize a variety of derivatives for SAR study, a more rapid and
efficient synthetic route is needed.
We report a new solid phase peptide synthetic approach for preparation of
some peptide and depsipeptide analogues of Hirsutellide A to define its SAR
with a hope of generating potent antituberculotic agents.
The synthesis involved a stepwise solid phase synthesis of the linear
hexapeptide and hexadepsipeptide precursors based on an Fmoc protecting
group strategy on chlorotrityl resin followed by macrocyclization in solution
phase under conditions of highly dilution. All amide bond formations on the
solid support were done by using 1-[bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) with diisopropylethylamine (DIPEA) as the coupling agent. The ester bond of the linear
hexadepsipetide was formed using N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC.HCl) mediated coupling. While ring
closure for the peptide analogue was achieved via macrolactamization using
HATU/DIPEA, the cyclic depsipeptide analogue was formed via macrolactonization using 2-methyl-6-nitrobenzoic anhydride (Shiina’s reagent) [3].
References:
1. Vongvanich, N. et al.: J. Nat. Prod. 2002, 65: 1346-1348.
2. Xu, Y. et al.: Tetrahedron Lett. 2005, 46: 437- 4379.
3. Shiina, I. et al.; Tetrahedron Lett. 2002, 43, 7535-7539.
Because of a global increase of infections by microorganisms and viruses,
there is a continuous demand for rapid detection methods. In the here
presented approach, the surface of polyethylene microparticles was modified
in a manner that bacteria and fungi will be bound reversibly. The polyethylene
particles were filled in a chromatographic tube and retention was investigated
for artificial microparticles as surrogates for cells, for bacteria, and fungi. A light
scattering detector was used for analysis. The goal of this investigation is the
rapid separation and detection of microorganisms in a chromatography-like
manner.
Introduction: Bacterial and viral infections have increased rapidly during last
years, especially in developing countries. There are many reasons for this as
incorrect application of antibiotics, the increasing globalization, contamination
of large amounts of foods, etc. Conventional methods for the detection of
bacteria are culture techniques (cultivation on selective agar plates and
examining the cultures by metabolic markers) as also various immunoassays.
The major drawback is that these methods are labour-intensive and it takes
several days to obtain results and confirmation (between 2 and 3 days for
immunoassays and up to 7-10 days for culture methods). There are also some
biosensoric approaches for rapid detection of bacteria [1]. But nevertheless,
there is an increasing demand for inexpensive, rapid and sensitive diagnostic
methods for the analysis of microorganisms. The here presented approach is
based on polymer surfaces with various chemical compositions [2].
Polyethylene seems to be a suitable material [3].
Results and Discussion: Preliminary investigations showed that surface
modified polyethylene beads can be used for separation of functionalized
microparticles. As shown in Figure 1, amino-functionalized polyethylene beads
were filled in a column and microparticles [carboxy-functionalized
polymethylmethacrylate (PMMA-COOH)] could be eluted under various pH
conditions. PMMA-COOH particles were firstly dispersed in water and than
applied to the column. During the elution with water, no particles could be
detected (Fig 1, violet). Only when using sodium hydroxide solution (pH 11) as
an eluent, the detector showed a significant signal (Fig 1 green). PMMACOOH particles without H2O washing led to rapid elution (Fig 1, red).
Additionally, experiments with the bacteria Escherichia coli and the yeast
Saccharomyces cerevisiae were carried out also showing different reversible
retentions of these cells. The ongoing research is focussed on improved
polyethylene beads as well as forming monolithic filling materials for the
column.
Figure 1: Elution of 1 µm PMMA-COOH particles from amino–polyethylene
beads under various conditions.
Conclusions: Surface functionalized polyethylene beads seem to be a
promising material for the separation of microparticles, especially
microorganisms.
Acknowledgments: The Authors kindly want to thank Mr. Van Elsäcker for his support.
MC.15
Detection and separation of microparticles using surface
modified polymer particles
Shopova, T.1; Holländer, A.2; Keusgen, M.1
1 Institute
for Pharmaceutical Chemistry, Philipps-Universität Marburg, Marbacher Weg 6,
D-35032 Marburg, Germany
2 Fraunhofer Institut for Applied Polymer Research, Gieselbergstraße 69, D-14476
Potsdam, Germany]
158
References:
1. Mazumdar, S. et al.: Biosensors and Bioelectronics 2007, 22: 2040-2046.
2. Kumar, A. et al.: Nature protocols 2010, 5: 1737-1747.
3. Holländer, A.: Patent WO002006077020A2 2006.
Substitution layout of the diaryl
(thio)ethers:
MC.16
Inhibitors of “New Permeability Pathways”
Steiner,
I.S.1;
Baumeister,
S.2;
Lingelbach,
K.2;
Schlitzer,
X = NO2, CF3, NH2, H, COOH,
aromatic or aliphatic residues
M.1
Y = S, C, O
Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6,
35037 Marburg
2 Philipps-Universität Marburg, Entwicklungsbiologie und Parasitologie, Karl-von-FrischStraße 8, 35043 Marburg
1
Upon Malaria-infection intensive remodeling of the host red blood cell takes
place. Novel pathways, commonly known as “New Permeability Pathways”
(NPP), are established to take up solutes.
These novel structures are not fully identified yet, but at least some may have
several characteristics of anion channels.[1] Examples of known NPP inhibitors
are furosemide and NPPB.[2]
We have prepared several new compounds which inhibit solute uptake of
cultured malaria parasites. As an additional design feature these compounds
are fluorescent. These fluorescent compounds are intended to be used as
tools for further identification and characterization of proteins which constituted
the NPP:
Inhibitor-Design
NPPB
On long term some of these structures may ultimately be used as basis for a
development of drug candidates.
References:
1. Baumeister, S. et al.: Protoplasma 2010, 240: 3-12.
2. Kirk, K.; Horner, H.A.: Biochem. J. 1995, 311: 761-768.
MC.17
Synthesis, characterization and screening of Dengue Virus S2BNS3 protease inhibitors
Wu, H.1; Holloway, S.1; von Hammerstein, F.1; Berger, T.1; Weidner, T.1, Kiefer,
W.1, Bodem, J.2; Bock, S.2; Snitko, M.2; Kanitz, M.3 Steuber, H.3; Diederich,
W.3; Schirmeister, T.1
Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099
Mainz, Germany
2 Institute of Virology and Immunology, University of Würzburg, Versbacher Strasse 7, D97078 Würzburg, Germany
3 Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6-10, D35032 Marburg, Germany
1
Dengue fever is a mosquito-borne tropical disease caused by the dengue virus
(DENV). It is transmitted to humans by the Aedes aegypti and Aedes
albopictus mosquitoes. [1] This viral infection is becoming continually a global
threat, as there are nearly 3.6 billion people living in the areas, tropical and
subtropical regions of the world (predominantly in Southeast Asia, Africa and
the Americas), where the infection is common. [2,3] The DENV infection can
result in classic dengue fever, dengue hemorrhagic fever (DHF) or dengue
shock syndrome (DSS). [4] Worldwide there are over 50 million infections
reported annually and the infection causes over 20,000 deaths each year. [5]
DENV is a single positive-stranded RNA virus of the family Flaviviridae with
four distinct serotypes. [6,7] The dengue virus genome encodes a serine
protease with a classical catalytic triad (His51, Asp75 and Ser135) [8,9] which
is responsible for the post-translational proteolytic processing of the polyprotein precursor and essential for the viral replication [10,11], making it an
important and attractive therapeutic target. [12]
Our projects include synthesis, characterization and testing of DENV2/3 NS2BNS3pro inhibitors based on the structure of variously substituted diaryl
(thio)ethers. The possible binding modes are analyzed by docking studies. The
synthesized compounds are screened both in vitro in fluorometric enzyme
assays using different fluorogenic AMC-derived substrates and in cell culture.
Additionally, Microscale Thermophoresis (MST) is used to investigate the
binding of selected diaryl (thio)ethers to DENV NS2B-NS3pro.
Z = variously substituted
aromatic, heterocyclic or
aliphatic residues
[University of Mainz for financial support]
References:
1. Yildiz, M. et al.: ACS Chem. Biol. 2013, 8: 2744-2752.
2. Murray, N.E.A. et al.: Clinical Epidemiology 2013, 5: 299-309.
3. Guzman, M.G. et al.: Nat. Rev. Microbiol. 2010, 8: S7-S16.
4. Martina, B.E. et al.: Clin. Microbiol. Rev. 2009, 22: 564-581.
5. Wilder-Smith, A. et al.: Arch. Med. Res. 2002, 33(4): 330-342.
6. Li, H. et al.: J. Virol. 1998, 73(4): 3108-3116.
7. Chambers, T.J. et al.: Annu. Rev. Microbiol. 1990, 44: 649-688.
8. Bazan, J.F.; Fletterick, R. J.: Virilogy 1989, 171: 637-639.
9. Melino, S., Paci, M.: FEBS Journal 2007, 274: 2986-3002.
10. Falgout, B. et al.: J. Virol. 1991, 65: 2467-2475.
11. Zhang, L.; Mohan, P.M.; Padmanabhan, R.: J. Virol. 1992, 66: 7549-7554.
12. Zheng, Y. et al.: Bioorg. Med. Chem. Lett. 2006, 16: 36-39.
MC.18
The Apicoplast: An Organelle of particular Interest in antimalarial
Drug Development
Schäfer, E.-M.1; Ortmann, R.1; Degenhardt, I.; Boomgaren, M.; Dahse, H.-M.,
Hillebrecht, A.; Seeber, F.2; Baumeister, S.3; Lingelbach, K.3; Klebe, G.1;
Schlitzer, M.1
1 Institut
für pharmazeutische Chemie, Fachbereich Pharmazie, Philipps-Universität
Marburg, Marbacher Weg 6, D-35032 Marburg, Deutschland.
2 Robert-Koch-Institut, FG 16: Erreger von Pilz- und Parasiteninfektionen und Mykobakteriosen. D-13353 Berlin, Deutschland
3 Institut für Entwicklungsbiologie und Parasitologie, Fachbereich Biologie, PhilippsUniversität Marburg, Karl-von-Frisch-Str.8, D-35043 Marburg, Deutschland.
Among infectious diseases Malaria still is one of the biggest problems for
public health especially in developing countries.[1] Due to rising resistance the
clock’s been ticking on the development on highly active antimalarial drugs.
The apicoplast represents a crucial target with its various singular and
essential pathways and functions. Fosmidomycine is a drug in clinical
development that inhibits DOXP-reductase, an enzyme of the non-mevalonate
pathway of isoprenoid synthesis that is located in the plasmodial apicoplast
and thus is active against erythrocytic phases of the parasite. We were able to
increase the in vitro activity of FR900098.[2]
An important house keeping function of the apicoplast is the modification of
proteins. At the end of protein biosynthesis a formyl residue needs to be split
off the N-terminal of the protein for its activation. This is done by the enzyme
peptiddeformylase. Actinonine is a known natural product that not only inhibits
this enzyme but also the proliferation of malaria parasites and thus is not
applicable. We were able to find a synthetic ten fold more active inhibitor of the
peptiddeformylase.
159
inhibitor segment
O
H
N
S
O2
O
O
NH
H
N
NH2
N
palmitoyl chain
MI-908
MI-902
HN
O
O
O
O
O
N
H
O
N
H
O
References:
1. World Malaria Report 2013, World Health Organisation, Genf, Schweiz.
2. Bormann, S. et al.: JID, 2004, 189: 901-908.
3. Seeber, F.; Aliverti, A.; Zanetti, G.: Curr. Pharm. Des. 2005, 11: 3159-3172.
O
N
H
S
H
NH
H
HN
O
linker
A promising target in the apicoplast is the redox-system ferredoxin/ferredoxinNADPH-reductase. This system is essential for the synthesis of iron-sulphur
clusters and its distribution to enzymes of various essential pathways like the
lipoic-acid synthesis or the non-mevalonat isoprenoid synthesis. Therefore an
inhibition of this system meant an inhibition of these essential pathways and
lead to the parasites death. Some active compounds have been found and
represent a basis for further development.[3]
O
linker
Reference:
1. Steinmetzer, T. et al. : ChemMedChem. 2012, 7: 1965-1973.
MC.20
Regulation of cNMP concentrations in RFL-6 fibroblasts by
activators and stimulators of soluble guanylate cyclase
Böttcher, M.M.1; Kaever, V.1, 2, Stasch, J.-P.3 Seifert, R.1
1 Institute
50
**
Basal
40
5 min
***
10 min
30
20 min
***
20
*
10
M
cU
M
cG
P
P
0
P
Thrombin is a serine protease and the final enzyme of the blood coagulation
cascade. Meanwhile, various direct thrombin inhibitors such as oral dabigatran
etexilate and parenteral r-hirudin, bivalirudin or argatroban have been
approved and can be used as anticoagulants for various prophylactic and
acute applications. Moreover, thrombin is easily available and there are
several methods to determine its activity in vitro. Therefore, it is an excellent
tool for studying receptor/ligand interactions. Many peptidic protease inhibitors
suffer from poor bioavailability, metabolic instability or short half-life due to
rapid elimination. In case of parenteral inhibitors it is possible to overcome
some of these problems by modification with larger polymers, such as
polyethylenglycols.
Here we describe a different strategy by developing potent and selective
thrombin inhibitors suitable for presenting them on the surface of liposomes.
For this purpose our previously described thrombin inhibitors containing Lamino acids in P3 position such as Lys or Asp were used. X-ray structure
analysis of the complex with thrombin revealed that the side chain of their P3
residue is directed into the solvent and might be used for further modification
without losing potency [1]. Therefore, we have prepared several new thrombin
inhibitors, whereby the P3 side chain was modified with short and defined
amino- or carboxyl-functionalized ethylene glycol linkers. These derivatives
can be either used for direct anticoagulant modification of artificial surfaces
and are suitable for further coupling with fatty acids and incorporation into
liposomes.
Several of the palmitoylated derivatives inhibit thrombin in the subnanomolar
range, e.g., an inhibition constant of 0.65 nM was determined for analogue MI902. This inhibitor was used for the preparation of liposomes. Analysis of the
supernatant after liposome preparation by HPLC, MS and thrombin activity
tests revealed that the inhibitor was completely incorporated. These liposomes
possess potent thrombin inhibitory activity in enzyme kinetic studies and strong
anticoagulant activity in plasma clotting assays. Replacement of the thrombin
inhibitor by biotin provided a related analog MI-908. This compound together
with the thrombin inhibitor MI-902 was used for the development of bifunctionalized liposomes, as shown below. The additional biotinylation can be used for
the further characterization of the liposomes.
Our studies show that it is possible to develop liposomes suitable for the
protease inhibition. This concept should be applicable for other enzyme or
protease inhibitors as well.
M
2
cC
Institute of Pharmaceutical Chemistry, Philipps University, 35032 Marburg, Germany
Institut of Pharmaceutical Technology and Biopharmacy, Philipps University, 35032
Marburg, Germany
1
P
Endreas, W.1; Brüßler, J.2; Bakowsky, U.2; Steinmetzer, T.1
Soluble guanylate cyclase (sGC) plays an important role in the physiology and
pathophysiology of numerous cardiovascular diseases [1]. sGC is activated by
binding of nitric oxide (NO) to the heme group of the enzyme. Recently, two
classes of compounds have been discovered that amplify the function of sGC,
the so-called sGC stimulators and sGC activators. The sGC stimulators, such
as BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)1H-pyrazolo[3,4-b]pyridine) directly stimulate sGC, both independently of NO
and in synergy with NO. In contrast, sGC activators, such as BAY 58-2667 (4[[4-carboxybutyl-[2-[2-[[4-(2phenylethyl)phenyl]methoxy]phenyl]etyl]amino]methyl]benzoic acid) preferentially activate sGC, when it is in an oxidized or heme-free state [2].
Purified sGC does not only form cGMP, but also synthesizes other cyclic
nucleotides such as cAMP, cCMP and cUMP [3]. Therefore, the objective of
this project is to examine whether sGC stimulators and activators induce
production of other cyclic nucleotides in intact cells. As model system we use
RFL-6 lung fibroblasts that express sGC endogenously [4]. RFL-6 fibroblasts
were treated with BAY 41-2272 and BAY 58-2667. Their effects on cNMP
concentrations were determined by high-performance liquid chromatographytandem mass spectrometry [5]. BAY 41-2272 increased cGMP in a timedependent manner. Moreover, at later time points, BAY 41-2272 also
increased cAMP. Currently, we examine the effects of a combination of the
sGC stimulator and NO as well as with a phosphodiesterase (PDE)5 inhibitor
on cNMP concentrations. In future studies, longer time course experiments will
be conducted in order to detect a potential increase in cCMP or cUMP
concentration. The time course studies are particularly important in view of the
fact that in sGC-transfected HEK293 cells, NO increased cCMP and cUMP
only after 60- 120 minutes [5].
cA
M
Synthesis and characterization of liposome-bound thrombin
inhibitors
cNMP [ pmol/ mg protein]
MC.19
of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625
Hannover, Germany
2 Research Core Unit Metabolomics, Hannover Medical School, Carl-Neuberg-Str. 1,
30625 Hannover, Germany
3 Cardiology Research, Bayer HealthCare AG, Apratherweg 18 a, 42096 Wuppertal,
Germany
Fig. 1: Effect of BAY 41-2272 [10 µM] on cNMP concentrations in RFL-6 cells.
(Two-way ANOVA with Bonferroni post-test. *: p < 0.05; **: p < 0.01; ***: p <
0.001)
References:
1. Stasch, J.P. et al.: Circulation 2011, 123: 2263-2273.
160
2. Follmann et al.: Angew Chem Int Ed Engl. 2013, 52: 9442-9462.
3. Beste, K.Y. et al.: Biochemistry 2012, 51: 194-204.
4. Förstermann, U. et al.: Proc. Natl. Acad. Sci. USA 1991, 88: 1788–1792.
5. Bähre, H. et al.: Biochem. Biophys. Res. Commun. 2014, 443: 1195-1199.
MC.21
In silico library design for BID inhibitors
Wegscheid-Gerlach, C.1; Barho, M.T.1; Kraus, A.L.1; Oppermann, S.2;
Culmsee, C.2; Schlitzer, M.1
1 Institute
for Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps-Universität
Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
2 Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, PhilippsUniversität Marburg, Karl-von-Frisch-Str. 1, D-35032 Marburg, Germany
Bid has been discovered as a potential target for progressive neuronal death in
age-related neurological diseases. By inhibition of the BH3-only protein BID, a
prevention of mitochondrial damage and a delayed neuronal death after
oxygen-glucose-deprivation can be achieved. Recently, two new structural
classes of lead structures have been identified [1, 2], which prevent tBIDinduced toxicity.
To obtain more insights in the structure-activity relationships of these
compounds a virtual library design approach was chosen. Building blocks were
handpicked by application of basic medicinal chemistry rules to introduce
structural variations. Furthermore, a structure-based approach called KNOBLE
[3] was used to select building blocks with direct information of the binding
pocket of BID. Bound fragments are extracted using structural information
derived from similar subpockets which can be identified within the Cavbase [4],
a database developed from the database RELIBASE. Subsequently, a
substructure search taking feasible synthetic strategies into account was
performed giving rise to a subset of putative building blocks with suitable
functional groups for the later synthesis. Afterwards, an enumeration of all
compounds is done. A fast filtering is achieved by evaluation of the entirely
assembled molecules‘2D and 3D descriptors. Finally, all compounds with
desired properties are evaluated by a docking run using FlexX as implemented
in LeadIT [5].
References:
1. Oppermann, S. et al. J Pharmacol Exp Ther. 2014, 350(2):273-289.
2. Barho, M. et al. ChemMedChem 2014, accepted.
3. Gerlach, C. et al. Angew Chem Int Ed Engl. 2007, 46(47):9105-9109.
4. S. Schmitt, D. Kuhn, G. Klebe, J. Mol. Biol. 2002, 323: 387.
5. a) Rarey, M., et al. J. Mol. Biol. 1996, 261(3):470–489. b) BioSolveIT, St. Augustin,
Vers. 2.1.7
MC.22
Validation of an analytical method for simultaneous quantification of Midazolam and 1’-Hydroxymidazolam in human plasma,
using QTOF liquid chromatography/mass spectrometry.
Radke, C.1; Fabian, J.2; Hempel, G.1
MA, USA) was used for separation of the samples which was performed on a
Luna C18(2) (2 x 100 mm, particle size 3 µm, Phenomenex, Torrance, CA,
USA) using a gradient method at a flow rate of 0,3 ml/min and an injection
volume of 3 µl. For sample acquisition and quantification, a micrOTOF-Q II
(Bruker Daltonik, Bremen, Germany) mass spectrometer, operating at a mass
range from m/z 290 to m/z 360, was used. Validation was assessed according
to the EMA guideline on bioanalytical method validation.
Results:
The method showed valid results which meet the specifications of the EMA
guideline. Using a 1/x weighted quadratic regression, the calibration curve
showed an excellent fit over a concentration range of 3.5 – 1500 ng/ml for
both, MDZ and its main metabolite 1’-OHM. Within-run accuracy for MDZ
ranged between 97.7 – 99.7% with a precision of 2.0 – 6.2%, whereas the
results for 1’-OHM ranged between 99.5 – 107.2% and 3.9 – 6.4%. Betweenrun accuracy for MDZ and 1’-OHM ranged over 98.7 – 100.7% and 96.5 –
101.7%, with a corresponding precision of 6.2 – 9.2% and 6.9 – 10.7%,
respectively. Absolute recoveries for MDZ, 1’-OHM and Clobazam were
78.5±4.2%, 76.0±5.3% and 68.0±2.1% (mean ± SD), respectively.
Selectivity was examined using six individual sources of blank plasma.
Furthermore three possibly co-administered drugs (Bisoprolol, Piperacillin/Tazobactam, Dobutamin) as well as the constitutional isomer
4-Hydroxymidazolam were examined for interference. No interfering chromatographic and mass spectrometric peaks could be detected at the respective
retention times neither in blank plasma samples nor in the drug-spiked
samples. In addition, no matrix effects could be detected for the three
substances.
A dilution factor of five and ten was tested and accuracy and precision were
within the specified limit of ± 15%.
Stability of stock solutions and sample probes were not studied, because
former investigations determined a long-term stability of up to ten months at 20°C, a short-term stability of up to three days at room temperature and no
affection of stability due to three freeze-thaw cycles [1].
Conclusion:
Although triple quadrupole (QqQ) mass spectrometer are nowadays state of
the art for drug quantification, this investigation is the first method to determine
the concentration of MDZ and its main metabolite 1’-OHM in human plasma,
using a QTOF mass spectrometer. The wide concentration range of the
calibration curve offers the possibility to monitor increasing plasma concentrations, due to constant drug infusion, which might be useful for pharmacokinetic
studies in special subpopulations, e.g. critically ill patients.
Acknowledgments: This investigation was supported by Bayer Technology Services
GmbH (Leverkusen, Germany).
Reference:
1. Shimizu, M. et al.: J Chromatogr B Analyt Technol Biomed Life Sci. 2007, 847(2): 275 –
281.
MC.23
Active Site Inhibitors Disturbing the Dimerization of tRNAGuanine Transglycosylase (TGT)
Ehrmann, F.R.1; Barandun, L.J.2; Debaene, F.3; Betz, M.1; Heine, A.1; SanglierCianférani, S.3; Diederich, F.2; Klebe, G.1
of Pharmaceutical and Medicinal Chemistry – Clinical Pharmacy, University
of Münster, Corrensstrasse 48, 48149 Münster, Germany
2 Department of Pharmaceutical and Medicinal Chemistry, University of Münster,
Corrensstrasse 48, 48149 Münster, Germany
of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6,
2 Laboratory of Organic Chemistry, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093
Zurich, Switzerland
3 Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC-DSA, Université
de Strasbourg, CNRS UMR7178; 25 rue Becquerel, 67087 Strasbourg, France
Introduction:
Midazolam (MDZ) is a widely used sedative in intensive care units, especially
for mechanically ventilated critically ill patients (e.g. septic patients). It is often
administered as a continuous infusion and therefore increasing concentrations
of MDZ and its main metabolite 1’-Hydroxymidazolam (1’-OHM) can be
expected. The aim of our investigation was to develop and validate a quadrupole time-of-flight (QTOF) based mass spectrometry method for the simultaneous quantification of both, MDZ and 1’-OHM, in human plasma, suitable to
assess increasing concentrations in septic intensive care patients.
Method:
Blank plasma samples were spiked with the analyte and the internal standard
(IS) Clobazam and were afterwards extracted by a liquid-liquid extraction (LLE)
procedure. A Dionex Ultimate 3000 LC system (ThermoScientific, Waltham,
tRNA-Guanine Transglycosylase (TGT) is a putative target enzyme to fight
pathogenicity of Shigella flexneri, the causative agent of Shigellosis. This
bacterial dysentery occurs predominantly in developing countries and is
responsible for several million cases of death per year.
The bacterium is usually transmitted via fecal-oral ingestion through contaminated food and water or through person-to-person contact.
Shigellosis is highly infectious, as only 10–100 bacteria are sufficient to
cause the disease in an adult person. After oral uptake, the bacteria
invade the intestinal mucosa, which causes the typical symptoms such
as bloody diarrhea, abdominal cramps, and dehydration.
The primary goal in the treatment is the replacement of fluid and salt loss
caused by diarrhea. In severe cases, antibiotics (such as Ampicilin, Cotrimox-
1 Department
1 Institut
161
azole, Nalidixic acid, and Ciproflaxin) are administered, but they are often not
accessible in developing countries.
Recent studies show a
dramatic growth of Antimicrobial resistance pattern for the different
resistant
strains,
Shigella species
making the develop100%
ment of new drugs an
90%
80%
urgent need.
70%
60%
50%
TGT catalyses only as
40%
30%
a functional homodi20%
10%
mer a base exchange
0%
reaction in the wobble
position of tRNAHis, Tyr,
Asp, Asn.
Here, guanine is
S. flexneri (n=1976)
S. boydii (n=189)
replaced by a modified
base which leads to an
effective translation of
several
virulence
Break apart of the TGT homodimer
factors of Shigella.
Thus, inhibition of TGT
reduces the pathogenicity of Shigella
significantly.
We have developed a small series of lin-benzoguanine type inhibitors
substituted with different modified ribose sidechains. All inhibitors exhibit
binding affinities in nanomolar range and confirm the assumption as promising
parental compounds which fit perfectly into the active site.
Interestingly, a superposition of crystal structures with spiking ligands,
designed to destabilize and break the interface formation, show that one ribose
ligand imposes similar effects onto a flexible loop (β1α1-loop), which shields
the interface from water access.
Native mass spectrometry (nanoESI-MS) confirmed that this ligand has a
similar effect onto dimer stability as the spiking ligands and represents an
active site inhibitor with a dual mode-of-action.
We acknowledge the beamline support of Bessy II in Berlin for practical help and the HZB
for travel grants
References:
1. Immekus, F. et al: ACS Chem Biol 2013, 8(6): 1163-1178.
2. Barandun, L.J.: Dissertation 2013, ETH Zurich, Zurich (Switzerland).
3. von Seidlein, L. et al: PLoS Med 2006, 3(9): e353.
MC.24
Fast Estimation for Cellular Metal Ion Uptake Using Affinity
Capillary Electrophoresis (ACE)
Alhazmi, H.A.1; Nachbar, M.1; Mozafari, M.1; El Deeb, S.1,2; Albishri, H.M.3; ElHady, D.A.3,4; Wätzig, H.1
1 Institute
Saudi Arabia.
Developing organometallic complexes is still growing for the treatment of
different disorders such as cancer and infection. In fact, these complexes are
pro-drugs, and have the role to transfer metal ions to target binding sites [1].
Therefore, investigations of metal ions behaviors and their interactions with the
plasma proteins such as human serum albumin and transferrin could give a
preliminary information about the cellular metal ion uptake prior to the
development of a new pro-drug. Hence, techniques to reliably investigate
these interactions are urgently needed. Recently, Affinity Capillary Electrophoresis (ACE) has been provided as an important tool for this purpose [2,3]. In a
recent study, the performance of this technique has been improved by applying
an appropriate rinsing protocol. Using 0.1 M EDTA in the rinsing protocol
became mandatory to desorb the metal ion from the capillary wall and prevent
the influence of EOF changes on the precision and accuracy of results. ACE
can now be performed in approximately 10 min including rinsing procedures.
An excellent precision, corresponding to RSD% of < 1.0% was achieved. The
162
influence of the noble metal ions (Pd2+, Ir3+, Ru3+, Rh3+, Pt4+, Os3+, Au3+, Au+,
Ag+, Cu2+) on plasma proteins were investigated by ACE, giving deep insight
into the strength of the interactions between these species and plasma
proteins. Accordingly, the cellular uptake of these species could be estimated.
Furthermore, the interaction of the recently developed Rh1+ N-heterocyclic
carbene complex (prospective anticancer) with plasma proteins were
investigated [4]. The results showed that the cellular uptake of Rh 1+ could be
decreased in the presence of BSA while could be increased in the presence of
HSA or transferrin. Hence, ACE technique could become first choice for in-vitro
studies of the cellular metal ion uptake and protein metal ion interactions.
Acknowledgment: We gratefully acknowledge the financial support by the funding of King
AbdulAziz University, Jeddah, Saudi Arabia, provided by Vice President Prof. Dr. A. O.
AlYoubi. We also thank Jazan University and Saudi Arabian cultural office in Berlin for
supporting our work by a grant to H. AlHazmi. Furthermore, we thank Polymicro
Technologies for providing the capillaries.
References:
1. Romero-Canelón, I., Sadler, P.J.: Inorg. Chem. 2013, 52: 12276 - 12291.
2. AlHazmi, H. A. et al.: submitted to Electrophoresis.
3. El Deeb, S. et al..: Trends Anal. Chem. 2013, 48: 112-131.
4. Oehninger, L. et al.: Chem. Eur. J. 2013, 19: 17871-17880
MC.25
Synthesis, Biological Evaluation and Binding Modes of Reverse
Fosmidomycin Analogs against the Antimalarial Drug Target
IspC
Konzuch, S.1; Umeda, T.2; Held, J.3; Brücher, K.1; Lienau, C.1; Behrendt, C.T.1;
Gräwert, T.4; Bacher, A.4; Illarionov, B.4; Fischer, M.4; Mordmüller, B.3,5;
Tanaka, N.2; Kurz, T.1
1 Institut
für Pharmazeutische und Medizinische Chemie, Heinrich-Heine Universität
2 School of Pharmacy, Showa University, Tokyo 142-8555, Japan
3 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074
Tübingen, Germany
4 Hamburg School of Food Science, Universität Hamburg, Grindelallee 117, 20146
Hamburg, Germany
5 Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon
Inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase of Plasmodium
falciparum (PfIspC) of the non-mavalonate isoprenoid pathway (MEP pathway)
is a promising strategy for the development of novel antiplasmodial drugs.[1,2]
The enzyme is inhibited by the antibiotic fosmidomycin, which has been used
successfully to treat malaria patients in clinical studies.[1,2] Reverse α-aryl
substituted carba-, oxa- and thia- analogs of fosmidomycin are well-known as
highly potent inhibitors against PfIspC.[3,4,5] In this work a series of new
reverse fosmidomycin derivatives was synthesized and biologically evaluated
in order to provide new insights into their structure activity relationships. The
most potent α-aryl substituted analog 2c inhibits PfIspC as well as Plasmodium
falciparum growth and exceeds the inhibitory potential of the natural product
fosmidomycin by more than one order of magnitude. The binding mode of
three derivatives in complex with PfIspC, NADPH and Mg2+ was clarified by Xray analysis.[6]
Figure 1: Interactions of the S-enantiomer of inhibitor 2c in the active site of
PfIspC.
References:
1. Jomaa, H. et al.: Science 1999, 285: 1573-1576.
2. Rohmer, M. et al.: Curr. Opin. Investig. Drugs 2004, 5: 154-162.
3. Behrendt, C.T. et al.: J. Med. Chem. 2011, 54: 6796-6802.
4. Brücher, K. et al.: J. Med. Chem. 2012, 55: 6566-6575.
5. Kunfermann, A. et al.: J. Med. Chem. 2013, 56: 8151-8162.
6. Umeda, T. et al.: Sci. Rep., 2011, 1: 1-8.
MC.27
Molecular Docking Studies to Explain SARs of Tertiary Amine
Substituted Acetylcholinesterase Inhibitors
MC.26
Wehle, S.1; Darras, F.H.2; Sotriffer, C.A.1; Decker, M.1,2
Triarylmethyl radicals: Synthesis, EPR characterization and
pharmaceutical applications
Elewa, M.; Frank, J.; Mäder, K.; Drescher, S.; Imming, P.
Institut fuer Pharmazie, Martin-Luther-Universitaet, Wolfgang-Langenbeck-Str. 4, D06120, Halle (Saale), Germany
Triarylmethyl (trityl) radicals are
paramagnetic spin probes which are
used in electron paramagnetic
resonance (EPR) spectroscopy and
imaging. Tris(tetrachloro, tetrathia,
and tetraoxatriaryl)methyl radicals
show intense, single and narrow EPR
lines at concentrations in the µM
range. Furthermore, they show good water solubility when present as
tricarboxylates, and a good stability in biological systems.
Trityl radicals (Figure 1) were synthesized and characterized. The influence of
pH, viscosity, oxygen concentration and solvent parameters on the EPR signal
of tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl radical (PTMTC) are shown
in Figures 2, 3 and 4.
PTMTC contains three carboxylic acid groups leading to pH sensitivity. To
investigate the effect of viscosity on line width, different glycerol-water mixtures
were tested (Figure 2). Between 10 and 40 % (m/V) glycerol content, a slight
decrease in the EPR signal line width was detected. This could be due to the
decrease of oxygen solubility with increasing percentage of glycerol [1]. The
increase above 60 % reflects the effect of increased viscosity by increasing
glycerol content. Apparently, viscosity by itself up to a value of about 40 % of
glycerol has no effect on the EPR signal line width. This fact is important for invivo applications as blood has a viscosity of 3–4 (mPa.s) at 37 °C [2], which is
only reached at 50 % of glycerol in water at 40 °C [3].
EPR signal line width increase is directly proportional to oxygen concentration
[4, 5]. A 1 mM solution of PTMTC in phosphate buffer (pH 7.4) shows a linear
relationship between line width and oxygen concentration (Figure 3). The small
slope of the line is due to the fact that oxygen concentration hardly increases
with increasing oxygen partial pressure, a physicochemical fact that should be
kept in mind for any oxygen determination in water.
A 1 mM solution of the radical in methanol-water mixtures (10 % – 100 %
methanol) were tested (Figure 4). On increasing methanol concentration, the
EPR signal line width increased because oxygen solubility increases considerably with increasing methanol concentration [1], leading to line broadening [4].
In conclusion, PTMTC is an interesting paramagnetic spin probe with
favourable EPR spectroscopy and imaging characteristics (single EPR signal
with narrow line width). Line width is affected by different parameters (viscosity, oxygen concentration and pH [not shown]). Each of these parameters can
be measured accurately when others are kept constant. In aqueous solutions,
viscosity and oxygen partial pressure do not have a pronounced effect on line
width.
0.24
0.06
0.22
0.20
0.16
0.14
0.12
0.10
0.08
Line width (mT)
0.20
0.18
Line width (mT)
Line width (mT)
Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative
brain disorder and the most common form of dementia with 24 million people
affected worldwide [1]. No truly disease-modifying therapies are known so far,
and cognitive deficits that characterize AD can only be addressed by symptomatic pharmacotherapy. Currently, the most common clinical treatment for AD
is the use of acetylcholinesterase (AChE) inhibitors which increase acetylcholine levels in the brain and symptomatically improve cognition and memory.
Quinazolinones are moderate micromolar AChE inhibitors both at the electric
eel and human isoforms of the enzyme [2]. Quinazolines (i.e. reduced form of
quinazolinones) connected with tertiary amines of variable size via a threecarbon-linker have been identified as highly potent dual-acting hAChE
inhibitors and hH3 antagonists [3]. Analogous substitution of quinazolinone
scaffolds leads to a loss in hH3 affinity, but to a remarkable increase in AChE
inhibitory affinity up to the nanomolar range [4]. A correlation of basicity with
IC50 values is observed. Computational docking methods were applied to
investigate the putative binding mode of this group of inhibitors in the human
AChE gorge and, concomitantly, an inhibitor library was synthesized for these
studies and for further optimization.
The 36 compounds of the compound library were docked to the binding site
of the human AChE crystal structure (PDB-ID: 4EY7) using GoldSuitev5.2 and
v5.1 by incorporation of seven structural water molecules in the docking
process [4, 5]. These water molecules were found to be essential for inhibitorprotein stabilization by at least one hydrogen bond of the quinazolinone
carbonyl to a water molecule. An “inverted binding mode” was found in 88% of
all top-5 poses. These poses show the basic amine placed at the active center
near the choline-attracting residue Trp86 (human AChE numbering), potentially
stabilized by a cation-π interaction, whereas the quinazolinone is placed at the
entrance of the binding gorge showing aromatic interactions with Trp286 and
hydrogen bonds to the water molecules. The “classical” orientation with the
scaffold placed near the catalytic active site (CAS) was found in just 12% of
the top-5 poses. Therefore, the high affinity and the surprisingly high AChE
selectivity over butyrylcholinesterase is only partly due to the heterocyclic part
of the molecules; SARs are mainly dependent on the tertiary amine structure. It
seems essential for the optimization of novel heterocyclic AChE inhibitors to
investigate the putative binding mode by SARs in connection with molecular
docking studies.
0.24
0.22
0.04
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.06
0.04
0.04
0.02
Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg,
Am Hubland, D-97074 Würzburg, Germany
2 Institut für Pharmazie, Universität Regensburg, Universitätsstraße 31, D-93053
Regensburg, Germany
1
0
10
20
30
40
50
60
70
Glycerol conc. (%)
80
90 100
0.02
0
10
Oxygen conc. (%)
20
0.02
0
10
20
30
40
50
60
70
Methanol conc. (%)
80
90 100
Figure 2: Change in line width of the EPR signal at different glycerol conc. Figure 3: Change in line width of the EPR signal at different oxygen conc. Figure 4: Change in line width of the EPR signal at different methanol conc.
References:
1. Ballard, C. et al.: Lancet 2011, 377(9770): 1019-1031.
2. Decker, M. et al.: Bioorg. Med. Chem. 2006, 14(6): 1966-1977.
3. Darras, F.H. et al.: ACS Chem. Neurosci. 2014, 5(3): 225-242.
4. Darras, F.H., Wehle, S. et al.: Bioorg. Med. Chem. 2014, in press.
5. GOLDSUITE 5.1, GOLDSUITE 5.2, CCDC Software, www.ccdc.cam.ac.uk
Acknowledgments: We thank the Egyptian Ministry of Higher Education and Scientific
Research for financial support to M.E.
References:
1. Kutsche, I. et al.: J. Chem. Eng. Data. 1984, 29(3): 286–287.
2. Joubert-Huebner, E. et al.: Perfusion 2000, 15: 69–76.
3. Physical properties of glycerine and its solutions (Glycerine Producers Association)
1963.
4. Driesschaert, B. et al.: Bioorg. Med. Chem. Lett. 2008, 18: 4291–4293.
5. Kuppusamy, P. Zweier, J.L.: NMR Biomed. 2004, 17: 226–239.
163
MC.28
Synthesis of L- S-(2-pyrrolyl)-cysteine sulphoxide
Feizabad, M.S.; Keusgen, M.
Institute of Pharmaceutical Chemistry, Philipps-Universität Marburg D-35037, Germany
The genus Allium L. has a large diversity, and more than 800 species
worldwide are described until now. Nearly all of them occur in the semiarid
regions of Europe, North America, North Africa and Asia. The most common
Allium species are garlic, (Allium sativum) and Onion (A. cepa) [1, 2]. In Middle
Asia, Allium species of the subgenus Melanocrommyum have a large range of
usage. In several cases, the amount of cysteine sulphoxides and their
metabolites are rather high, so that the plants are used as spicy vegetables or
are even not edible [3]. A recently discovered, new cysteine sulphoxide
containing a pyrrolyl residue seems to typical for many bulbs of species
belonging to the subgenus Melanocrommyum. Typical examples are A.
giganteum and A. rosenorum, found in Central Asia and West Asia, especially
in Iran, Tajikistan, Uzbekistan and Afghanistan. This new cysteine sulphoxide
has been identified as L-S-(2-pyrolyl)-cysteine sulphoxide [5]. The members of
the above mentioned subgenus have antibiotic antifungal, antidiabetic, or
wound healing effectivity that shows the medical importance of synthesizing
this cysteine sulphoxide found in them. The aim of this study was to synthesis
this pyrrole-containing cysteine sulphoxide.
5. Chemical structure of L-S-(2-pyrrolyl)-cysteine sulfoxide
Synthesis has been done in three steps. At first, N-benzyl-pyrrole and 3-chloroL-alanine hydrochloride were used as starting blocks, with the addition of
thiourea, iodine and potassium iodide, in extreme alkaline medium (sodium
hydroxide and hydrazine), soved in aqueos ethanol (50%-50%). The intended
product was 3-(1-benzyl-1H-pyrrol-2-yulsulphanyl) propionic amino acid [4]. In
the next step, this product was oxidized using hydrogen peroxide in acetic acid
as solvent, in order to obtain S-(1H-benzyl-2-pyrrole)-cysteine sulphoxide. In
the last step, the benzyl group should be reductively removed with palladium/C
20% and hydrogen in methanol as solvent.. With the exception of the last step,
all reaction pruducts could be confirmed by TLC, HPLC-MS and NMR. For
most important products, preparative HPLC was performed.
Acknowledgments: We are grateful to Matthias Brauschke for technical support.
Refrences:
1. Yoshida, H. et.al.: Biosci., Biotechnol. Biochem. 1999, 63: 588-590.
2. Kumari, K.; Augusti, K.T.: Planta Medica.1995, 61: 72-74
3. Keusgen, M. et.al.: J. Ethnobiol. Ethnomed. 2006, 2: 18.
4. Rudyakova, E.V. et.al.: Russian Journal of Organic Chemistry. 2008, 44(10): 1539-1543
MC.29
New tripeptide inhibitors of the West Nile virus NS2B-NS3
protease
Kouretova, J.1, Hammamy, M.Z.1, Haase, C.2, Hilgenfeld, R.2, Steinmetzer, T.1
1 Institute
of Pharmaceutical Chemistry, Philipps University, Marbacher Weg 6, D-35032
Marburg, Germany
2 Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of
Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
West Nile virus (WNV) is a mosquito-borne flavivirus, which was first identified
in the West Nile part of Uganda and has spread later to Asia, America and
Europe. The majority of the infected humans shows no symptoms but may
develop a mild flu-like illness. A small number of infected people, mainly
children and the elderly, develop fatal meningitis or encephalitis leading to a
mortality rate of around 10 %. Despite increasing demand, there is no specific
treatment of WNV infections available, so far. A potential target for the
treatment of WNV infections could be the viral NS2B-NS3 protease, which is
essential for cleaving the WNV-polyprotein into various mature viral proteins.
The NS3 protein contains a serine protease domain, which cleaves their
substrates preferentially at the C-terminus of two basic amino acids.
164
Based on the preferred P3-P1 amino acids containing an N-terminal phenylacetyl protection [1] we have prepared a series of peptide derivatives as WNV
NS2B-NS3 protease inhibitors by modifying the prime site segment. Surprisingly, the strongest inhibitory potency was found for simple protected tripeptides containing a C-terminal arginylamide moiety, all elongated derivatives
exhibit reduced activity. Incubation experiments and subsequent HPLC
analysis revealed that these compounds are nearly stable against cleavage by
the WNV-protease, only a minor amount of the cleavage product was detected
after 4 hours. Therefore, these compounds seem to be very poor substrates of
the WNV NS2B-NS3 protease, which can be considered as competitive
inhibitors. The best Ki-values < 0.15 µM were obtained for peptides with the
general structure phenylacetyl-Lys-Lys-Arg-NH2 containing an N-terminal
guanidinomethyl substitution at the P4-residue.
NH2
H
N
3
HN
H 2N
4
O
O
NH
N
H
H
N
O
NH2
O
K i = 0.13 µM for C3 modification
K i = 0.12 µM for C4 modification
NH
NH2
HN
NH2
References:
1. Hammamy, M.Z. et al.: ChemMedChem 2013, 8: 231-241.
MC.30
Improving binding free energy estimates for InhA inhibitors by a
hybrid scoring function/molecular dynamics approach
Narkhede, Y.; Sotriffer, C.A.
Institute for Pharmacy and Food Chemistry, Am Hubland, D– 97074, Wuerzburg,
Germany
Due to the high infection rates and the recent emergence of extremely drug
resistant forms, infection by Mycobacterium tuberculosis still represents a
significant challenge for global health [1]. The NADH-dependent enoyl-ACPreductase InhA of the type II mycobacterial fatty acid biosynthesis pathway is a
well-validated target for inhibiting mycobacterial growth [2]. Numerous
inhibitors of InhA have already been synthesized and tested for in-vitro activity.
Despite the progress in this area, very few drug candidates have been
reported or are in development [3]. Of all inhibitors reported in literature, the
diaryl ethers, pyrrolidine carboxamides and arylamides are the most prominent
chemical classes for further development [3].
The present work illustrates the use of hybrid scoring approaches consisting of
classical scoring functions and molecular dynamics based methods to improve
the prediction of binding free energies of pyrrolidine carboxamides as InhA
inhibitors. The pyrrolidine carboxamides with a narrow pIC 50 range coupled
with diverse binding modes presented a challenge for molecular docking as
well as the ensuing binding free energy calculations using the Linear Interaction Energy approach (LIE) [4]. Starting from representative crystal structures,
the binding modes for 44 compounds were predicted using molecular docking.
The Glide XP [5] and SFC scoring functions [6] together with the LIE method
were used in multiple logistic regression models to classify the compounds into
low-, medium- and high-affinity groups, respectively. Taking into consideration
the experimental uncertainty of binding activity data, this approach appears
more reasonable and practically useful than classical regression methods and
pure correlation-based metrics.
References:
1. WHO Global TB report, 2013, 1-3.
2. Molle, V. et al: Mol. Microbiol., 2010, 78: 1591–1605.
3. Pan, P. et al.: Curr. Top. Med. Chem. 2012, 12(7):672-693.
4. Gutiérrez-de-Terán, H. et al.: Computational Drug Discovery and Design, (Springer
New York) 2012, 890: 305-323.
5. Glide, version 5.8, Schrödinger, LLC, New York, NY, 2012
6. Sotriffer, C.A. et al.: Proteins, 2008, 73: 395–419.
MC.31
Catechol-based substrates of Chalcone Synthase as a scaffold
for novel inhibitors of PqsD
Allegretta, G.; Weidel, E.; Empting, M.; Hartmann, R.W.
Helmholtz Institute for Pharmaceutical Research Saarland – Department of Drug Design
and Optimization, Saarland University, Campus C 2.3, 66123 Saarbrücken, Germany
A new strategy for treating Pseudomonas aeruginosa infections could be
disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS)
system. The goal is to impair the communication among the cells and, hence,
reduce the expression of virulence factors and the formation of biofilms. PqsD
is an essential enzyme for the synthesis of PQS [1] and shares some features
with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa.
Both proteins are quite similar concerning the size of the active site, the
catalytic residues and the electrostatic surface potential at the entrance of the
substrate tunnel. [2,3,4] Hence, we evaluated selected substrates of the
vegetable enzyme as potential inhibitors of the bacterial protein. This similarityguided approach leads to the identification of a new class of PqsD inhibitors
having a catechol structure as an essential feature for activity, a saturated
linker with two or more carbons and an ester moiety bearing bulky substituents. The developed compounds showed PqsD inhibition with IC 50 values in
the single-digit micromolar range. The binding mode of these compounds was
investigated by SPR experiments revealing that their interaction with the
protein is not influenced by the presence of the anthranilic acid bound to active
site cysteine. Importantly, some compounds reduced the signal molecule
production in cellulo.
Acknowledgments: “Helmholtz Institute for Pharmaceutical Research Saarland –
Department of Drug Design and Optimization”, Maurer, C., Kirsch, B.
References:
1. Dulcey, C.E. et al.: Chem. & Biol. 2013, 20(12): 1481–1491.
2. Bera, A.K. et al.: Biochem. 2009, 48(36): 8644–8655.
3. Ferrer, J.–L. et al.: Nat. Struct. Biol. 1999, 6: 755–784.
4. Dao, T.T.H.; Linthorst, H.J.M.; Verpoorte, R.: Phytochem. Rev. 2011, 10(3): 397–412.
MC.32
Johannes Franz Wilhelm Valentin (1884–1959) - a pioneering
pharmacist in the field of chromatography
Michler, V.1; Friedrich, C.1
Institut für Geschichte der Pharmazie, Universität Marburg, Roter Graben 10, 35037
Marburg, Germany
The chromatographic technique was invented by an an italian-born russian
biologist named Mikhail Semjonovich Tswett (1872–1919).[1] He separated
plant pigments in 1903 by allowing them to percolate down columns of calcium
carbonate or inulin. This new procedure attracted little attention until it was
rediscovered by a group of german chemists at the University of Heidelberg in
1931.[2] After that chromatography entered a period of intensive development
and is nowadays the most versatile technique in analytical chemistry.
However, thus far little is known how this technique entered the pharmaceutical analytics.
Based on our research, Johannes Franz Wilhelm Valentin (1884–1959), a
pharmacist from the University of Königsberg, was the first german scientist
who used the chromatography to analyze pharmaceutical products.[3] The
initial publication of Valentin was released in Mai 1935.[4] He made the
technique of chromatography available for analyzing pharmaceutical preparations. The first pharmaceutical substances he investigated were Peru Balsam
and Tinctura Digitalis.[4] He demonstrated how easy, fast and more feasible
these methods are compared to the existing monographs in the German
Pharmacopoeia 6 in 1926. This poster will summarize the life and work of
Hannes Valentin, a pioneering pharmacist from the University of Königsberg
and later on from the University of Greifswald.
References:
1. Michler, V.: CVET, Michail Semënovič. In: Personendatenbank zum Vorhaben
"Wissenschaftsbeziehungen im 19. Jahrhundert zwischen Deutschland und Russland auf
den Gebieten Chemie, Pharmazie und Medizin" bei der Sächsischen Akademie der
Wissenschaften zu Leipzig. Online-Ausgabe: http://drw.saw-leipzig.de/10283.html (23. Mai
2014)
2. Kuhn, R.; Winterstein, A.; Lederer, E.: Hoppe Seyler‘s Zeitschrift für physiologische
Chemie, 1931, 197: 141–161.
3. Friedrich, C.; Seidlein, H.- J.: Pharmazie, 1984, 39 : 262–269.
4. Valentin, J.: Pharmazeutische Zeitung, 1935, 80 : 469-471.
MC.33
Targeting drug resistance in EGFR with covalent inhibitors – a
structure-based design approach
Engel, J.1; Getlik, M.1; Richters, A.1; Heuckmann, J.M.2; Grütter, C.1; Thomas,
R.K.2; Rauh, D.1
Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, D44227, Dortmund, Germany
2 Universität zu Köln, Abteilung Translationale Genomik, Weyertal 115b, D-50931
Cologne, Germany
1
Aberrant activity of the epidermal growth factor receptor kinase (EGFR) plays a
pivotal role in the development and growth of tumor cells and is associated
with the onset and progression of non small cell lung cancer (NSCLC).[1,2]
Patients harboring activating mutations in the catalytic domain of EGFR show
a significant clinical response to reversible Type-I inhibitors gefitinib and
erlotinib.[3] However, patients responding to these drugs develop secondary
drug resistance mutations and suffer from a dramatic relapse. In 50% of these
cases, Thr790 at the gatekeeper position of the kinase domain, was mutated to
a sterically more demanding methionine.[4] In order to overcome this
resistance, covalent inhibitors represent the most promising strategy by
covalently targeting the unique Cys797 in the active site of EGFR.[5,6,7]
Strong efforts were directed to the development of irreversible inhibitors and
led to compound CO-1686 which remarkably takes advantage of increased
residence time to EGFR by alkylating Cys797 simultaneously preventing toxic
effects. [6]
Here, we present the structure-based approach to design novel and covalent
EGFR inhibitors based on a screening hit that was identified in a phenotype
screen of 80 NSCLC cell lines against about 1500 compounds.
Using protein X-ray crystallography we determined the binding mode of this
inhibitor in the related tyrosine kinase cSrc. The complex crystal structure did
not only provide valuable insights into the mode of action but also highlighted
strategies for chemical optimization that led to a series of derivatives which
displayed strong inhibitory activities against EGFR and its mutant variants.
References:
1. Zandi, R. et al.: Cell Signaling 2007, 19(10): 2013-2023.
2. Heuckmann, J.M. et al.: J. Clin. Oncol. 2012, 30(27): 3417-3420.
3. Pao, W. et al.: Proc. Natl. Acad. Sci. USA 2004, 101(36): 13306-13311.
4. Kobayashi, S. et al.:N. Engl. J. Med. 2005, 352(8): 786-792.
5. Zhou, W. et al.: Nature 2009, 462(7276): 1070-1074.
6. Walter, A.O. et al.: Cancer Discov. 2013, 3(12): 1404-1015.
7. Sos, M.L. et al.: Cancer Res. 2010, 70(3): 868-874.
MC.34
Buffer conditions regulate the binding pose of dualsteric ligands
at the muscarinic M2 receptor
Krebs, F.; Chirinda, B.; Mohr, K.
Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, 53121
Bonn, Germany
G-protein-coupled receptors, the largest superfamily of membrane bound
receptors, control and regulate several processes in the human body and
165
therefore serve as targets for a variety of drugs. Muscarinic acetylcholine
receptors, with their subtypes M1-M5, belong to this superfamily of receptors.
Throughout the five subtypes, the orthosteric binding site is highly conserved
[1]. Consequently, synthetic drugs that target this binding site generally lack
subtype selectivity. In contrast, the allosteric vestibule is not as highly
conserved among the subtypes and therefore could function as a possible
binding epitope for allosteric modulators yielding certain subtype selectivity.
Recently the muscarinic M2 receptor was crystallized in its active state bound
simultaneously with both an orthosteric agonist and a specifically designed
allosteric modulator [2].
Dualsteric ligands are small molecules, which consist of covalently linked
moieties that address the orthosteric and the allosteric binding site, respectively. As a consequence, the binding pose of such a bitopic ligand depends on
the individual affinities of these moieties for their respective binding site [3]. A
previous study showed that the binding affinity of positively charged allosteric
ligands is highly sensitive to the cation concentration of the incubation buffer
[4]. Here the aim of the study was to investigate the influence of different salt
compositions in the buffer solution on the binding pose. The affinities of the
bitopic ligands and their orthosteric and allosteric fragments were investigated
in either equilibrium binding or dissociation kinetics experiments with the
radioactive tracer [³H]NMS. All experiments were conducted with membrane
homogenates from CHO cells stably expressing the human M2 receptor.
A change of buffer conditions from HEPES-buffer (10 mM HEPES, 10 mM
MgCl2, 100 mM NaCl, pH = 7.4) to Na,K,Pi-buffer (1 mM KH2PO4, 4 mM
Na2HPO4, pH = 7.4) led to a prominent increase in affinity. In contrast, the
affinity for [³H]NMS remained unchanged. The increased affinity for the
allosteric fragment in Na,K,Pi-buffer conditions caused the bitopic compound to
bind in a higher fraction in the purely allosteric pose, which was reflected by a
promoted [³H]NMS binding to the receptor. In contrast, under HEPES-buffer
conditions the affinity of the orthosteric fragment dominated the binding pose of
the dualsteric compound, as reflected by displacement of [³H]NMS from the
orthosteric pocket.
These findings show that modification of buffer conditions is a new means to
gain insight into the binding pose of orthosteric/allosteric class A GPCR
ligands.
Test compounds were kindly provided by Prof. Dr. U. Holzgrabe, Würzburg, Germany,
and Prof. M. De Amici, Milan, Italy and co-workers.
F.K. is a member of the research training group GRK 1873 - funded by the German
Research Foundation (DFG).
B.C. is a member of the research training group BIOTECH PHARMA.
References:
1. Wess, J. et al.: Crit Rev Neurobiol 1996, 10: 69-99.
2. Kruse, A. et al.: Nature 2013, 504: 101-106.
3. Bock, A. et al.: Nat Chem Biol. 2014, 10: 18-20.
4. Schröter, A. et al.: Naunyn Schmiedebergs Arch Pharmacol. 2000, 362: 512-519.
MC.35
Aldose Reductase as a target to prevent diabetic complications:
Investigations of the structural and thermodynamic background
of the opening of the specificity pocket
Rechlin,C.1; Scheer,F.2; Toth, P.2; Heine, A.1; Diederich, W.2; Klebe, G.1
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6,
2 Zentrum für Tumor- und Immunbiologie, Philipps-Universität Marburg, Hans- MeerweinStr. 3 , 35032 Marburg, Germany
1
Diabetes is a global burden of which 347 million people suffered in 2008 alone.
Estimations indicate that this number will increase so that by 2030 4.4 % of all
age groups will suffer from this disease. [1] Diabetes does not only have
immediate symptoms but also leads to some long-term complications.
Blindness, end-stage renal disease and different neuropathies are microangiopathies which count to these complications. [2]
The human aldose reductase (ALR2) is involved in the first reaction step in the
so-called polyol pathway which is one of the major mechanisms for the
development of diabetic complications. Glucose is reduced to sorbitol with the
help of the cofactor NADPH. Higher oxidative stress is the result of the
reduced NADPH level. Based on this knowledge inhibitors of the human
aldose reductase (ARIs) have been evaluated in several clinical studies
concerning the effectiveness and safety.[3] They seem to have an effect
166
especially on motor nerve function in patients with mild to moderate diabetic
sensorimotor polyneuropathy. [4]
Despite these promising findings the ALR2 is a challenging target for drug
design, as it exhibits an highly conserved anion binding pocket and the very
flexible specificity pocket forming the binding site. The specificity pocket only
opens if the inhibitors provide favorable interactions. In the past this pocket has
been addressed to obtain selective inhibitors for ALR2. Taking into account
that ALR2 belongs to a large protein family, selectivity is a requirement for
successful drug design. [5]
We would like to understand how the opening of the specificity pocket works in
detail. The structure of the crystallization buffer ingredient citrate complexed to
the binding site of ALR2 (pdb: 2j8t) has been determined. As citrate is only
partially occupied this structure provides some insights into the putative apostructure and suggests the specificity pocket is closed. This indicates that the
enzyme with an opened pocket does not correspond to the energetically most
favorable situation. An inhibitor that protrudes into this area of the binding site
has to open this pocket first. By how much does the inhibitor pay for the
opening and is the contribution compensated by suitable interactions in the
newly formed pocket?
We are investigating this process using derivatives of the so called “IDDligands”, a series of 2-benzylcarbamoyl-phenoxy-acetic acids. Members of this
class could be found which leave the pocket closed while the most potent ones
open the pocket. Our aim is to determine the smallest sidechain which is able
to open the pocket. X-ray crystallography and Isothermal titration calorimetry
(ITC) were used to follow the opening of the pocket from the structural and the
energetic point of view.
References:
1. Danaei et al.: LANCET. 2011, 378: 31-40.
2. Brownlee: NATURE. 2001, 414: 813-820.
3. Hu et al.: PLOS ONE. 2014, 9: 2.
4. Bril et al.: DIABETES CARE. 2009, 32;7: 1256-1260.
5. Sotriffer et al.: PROTEINS. 2004, 56: 52-66.
MC.36
Chiral separation of amino acids by CE and HPLC using online
derivatization with ortho-phthalaldehyde and a chiral mercaptane.
Kühnreich, R.; Holzgrabe, U.
Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074
Würzburg, Germany
The aim of this study was to develop a method for the separation of chiral
racemic amino acids, which is fast and allows for automatization. Orthophthalaldehyde (OPA) is a commonly used derivatization reagent for amino
acids to improve sensitivity and retention on reversed phase HPLC columns.
By using a chiral mercaptan as coreagent for the derivatization, enantiomeric
compounds can be converted to diastereomers, allowing a separation without
the use of an expensive chiral column. Amino acids were derivatized with
ortho-phthalaldehyde, using N-acetyl-L-cysteine (NAC) or N-isobutyryl-Lcysteine (NIBLC) as mercaptan reagent in a 20 mM borate buffer (pH 10.0) to
form diastereomeric isoindole derivatives, which can be separated by HPLC
using reversed phase columns or capillary electrophoresis (CE) using
selectors like cyclodextrines (CD). Besides enantioseparation, CDs can be
used for the separation of closely related compounds, which have similar
electrophoretic mobilities. For the online derivatization on CE, a modified
method by Kaale et al.1 was used. The derivatized amino acids were separated
in a 50 mM borate buffer (pH 9.2) and different neutral CDs (di-o-methyl-βcyclodextrine, tri-o-methyl-β-cyclodextrine, hydroxypropyl-β-cyclodextrine, βcyclodextrine, γ-cyclodextrine, hydroxypropyl-γ-cyclodextrine) and charged
CDs (carboxymethyl-β-cyclodextrine, sulphated β-cyclodextrine) were tested.
The derivatization on HPLC was performed with a custom injection program of
the autosampler, where the derivatization takes place in the needle directly
before injection. For the separation, a Kinetex PFP column (150x4.6 mm; 2.6
µm) and a Phenylhexyl column (150x4.6 mm; 3.0 µm) was used. The mobile
phase consists of various buffers with pH in the range from 2.5 to 5.5 and
acetonitrile. For both, HPLC and CE, separation conditions for the separation
for 17 amino acids were found. The resolution was found to be at least 1.5.
Acknowledgments: We like to thank BfArM for their financial support.
References:
1. Kaale, E. et al.: Electrophoresis 2001, 22(13): 2746–2754.
MC.37
Decoration of living cell surfaces by copper catalyzed azidealkyne cycloaddition
Gutmann, M.1; Wurzel, J.1; Memmel, E.2; Seibel, J.2; Meinel, L.1; Lühmann, T.1
1 Institute
for Pharmacy and Food Chemistry, University of Würzburg, Germany
² Institute for Organic Chemistry,University of Würzburg,Germany
The cell surface is responsible for the interaction among cells and plays a key
role in anchorage of the cell in macromolecular systems such as the extracellular matrix (ECM). This study aims at the modification of cell surfaces by
deploying the bioorthogonal copper catalyzed azide-alkyne cycloaddition
(CuAAC) and by studying the associated copper toxicity in the cellular system.
We aimed to address two essential points, (i) the modification of the cell
surface after CuAAC in respect to cell viability and (ii) the characterization of
the cell surface with fluorescent dyes and a model protein plk-eGFP after click
chemistry over time.
The cell surface of adherent NIH3T3 fibroblasts and suspended HEK 293 F
cells was modified to introduce azide-functionalities by a glycoengineering step
using tetraacylated-N-azidoacetylglucosamine as previously described [1]. To
proof the presentation of the azide-modified glycoproteins on the cell surface,
we applied the azide-alkyne click reaction (50 µM CuSO4 / 250 µM THPTA /
2,5 mM sodium ascorbate for 5 minutes at room temperature), followed by
surface staining with the fluorescent dyes sulfo-Cy5-alkine or acetylene-fluor
488, respectively [2, 3]. Moreover, an enhanced green fluorescent protein (plkeGFP) modified with propargyl-L-lysine in position 5 was recombinantly
expressed in BL21-DE3 cells and purified and used in a similar manner for cell
surface modification. Site-directed immobilisation was investigated by confocal
laser scanning microscopy (CLSM) at different time points. Copper cell toxicity
after CuAAC was monitored by qRT-PCR analysis of apoptotic genes and
FACS measurements thereafter [4].
High viability of the cells after the glycoengineering step and optimized CuAAC
was shown. Here, we investigated both the apoptotic downstreaming process
by use of qRT-PCR and the metabolic and membranous status using a
fluoresceindiacetate (FDA) and propidiumiodide (PI) staining monitoredby
FACS analysis. An overall good survivability ~80-100% was observed up to 20
minutes under click conditions regarding the expression of the examined
apoptotic genes and the ratio of FDA and PI staining analyzed by FACS. In a
subsequent set of experiments, the cell surface was decorated by means of
metabolic glycoengineering and further modified by CuAAC between azidofunctionalized NIH3T3 fibroblasts and HEK 293 F cell surfaces and alkyne
groups carrying fluorescent dyes. Strong fluorescence was observed on the
cell membrane with some background scattering within the cytosol directly
after the click reaction by CLSM. Later time points (12 hours) demonstrated
reduced membrane fluorescence. To introduce a more relevant biomacromolecule onto the cell surface, we chose as a model protein, plk-eGFP, for sitedirected immobilisation. In line with the optimized protocols, we confirmed the
site-directed immobilisation of plk-eGFP onto the cell surface. After click
reaction between azido-functionalized NIH3T3 fibroblasts and plk-eGFP,
specific fluorescence of eGFP was observed on the cell membrane with fast
reduction (1 hour) in fluorescence over time.
References:
1. Homann, A. et al.: Beilstein journal of organic chemistry 2010, 6: 24.
2. Memmel, E. et al.: Chemical communications (Cambridge, England) 2013, 49 (66):
7301-7303.
3. Uttamapinat, C. et al.: Nature protocols 2013, 8 (8): 1620–1634.
4. Hong, V. et al.: Bioconjugate Chemistry 2010, 21: 1912-1916.
Introduction
Myostatin is a potent negative regulator of myogenesis and inhibits myoblast
differentiation in compromised skeletal muscles. As myostatin is upregulated
during aging and in muscle-wasting diseases including sarcopenia, we
targeted this protein by 20 – 32 mer peptide antagonists in order to provide an
effective therapy. By introducing a propargyl-derivative of glycine into the
molecule, thus rendering it amenable for click chemistry, we featured the
therapeutic for functionalized surface decoration.
Materials and Methods
The myostatin inhibitors were manufactured by solid phase peptide synthesis
following the protocol of [1] with a propargyl-modified glycine analogue
introduced into the sequence for the click chemistry option. Following
purification using reversed phase chromatography on an ÄKTA purifier system,
the synthesized peptides were analyzed by RP-HPLC and MALDI-MS for the
verification of successful synthesis and characterization of stability upon freeze
drying. Potency was profiled by a luciferase-based reporter gene assay in HEK
293 cells stably expressing the SBE-luciferase reporter gene [2] and bioactivity
of the alkyne-functionalized peptides collated with the unmodified sequences.
A C2C12 myoblast differentiation assay was deployed for the evaluation of the
inhibitory effect on myostatin as determined with fluorescence microscopy and
Western Blot. The accessibility of the alkyne group for click chemistry was
demonstrated by performing click reaction with the fluorescent dye Cy3 azide.
Successful synthesis and purification of the functionalized peptides was
demonstrated by mass spectrometry and HPLC. The luciferase assay
confirmed the inhibiting activity on myostatin signalling and comparison of the
potency of functionalized and original peptides resulted in similar outcome. The
restoration of C2C12 differentiation ability after incubation of myostatin treated
cells with the peptide inhibitor indicated bioactivity as demonstrated by the
formation of multinucleated myotubes and positive MyHC expression of
differentiating myoblasts. Performance of the click reaction with Cy3-azide [3]
proved the functionality of the ‘clickable’ group. The successful coupling of the
myostatin antagonists to azido-silk fibroin coated polystyrene particles
demonstrated the ability for surface decoration. In conclusion, a potent
‘clickable’ myostatin inhibitor was developed for advanced controlled release
approaches. This system is further profiled for boosting muscle function and
regeneration in ongoing studies.
Acknowledgements: The financial support from the Bavarian Research Foundation
(FORMOsA grant) is gratefully acknowledged.
References:
1. US 2004/0181033 A1
2. Cash, J.N. et al.: J. Biomol. Screen. 2013, 18: 837-844.
3. Lutz, J.F. et al.: Adv. Drug Deliv. Rev. 2008, 60: 958–970.
Part of this research has been presented at the CRS Local Chapter meeting in Kiel
(27./28. February 2014)
MC.39
Screening, synthesis and characterization of novel ligands for
Farnesoid X Receptor (FXR)
Flesch, D.; Achenbach, J.; Gabler, M.; Merk, D.; Steri, R.; Proschak, E.;
Schubert-Zsilavecz, M.
Goethe University Frankfurt, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9,
60438 Frankfurt
MC.38
Site-directed modification of myostatin-inhibitors for muscle
regeneration
Braun, A.1; Gutmann, M.1; Li, L.1; Ebert, R.2; Jakob, F.2; Lühmann, T.1; Meinel,
L.1
1 Institute
for Pharmacy and Food Chemistry, University Würzburg, Germany
Center for Musculoskeletal Research, Würzburg, Germany
2 Orthopedic
The Farnesoid X Receptor (FXR) is not only a key regulator of bile acid
metabolism, furthermore this nuclear receptor acts in various pathways of the
fatty acid- and carbohydrate metabolism as well as liver protection and regeneration [1,2]. Therefore it is an attractive target to treat several metabolic
disorders. A Phase III study with Obeticholic acid (OCA, 6α-ethylchenodeoxycholic acid, 6-EDCA, INT-747) in primary biliary cirrhosis and
phase II studies in non-alcoholic hepatosteatosis and alcoholic hepatitis are
underway to verify the value of an FXR agonist in disease models of hepatic
injury [3].
To generate starting points for novel synthetic FXR modulators we initiated an
in silico search with a combined ligand- and structure-based virtual screening.
167
The most promising structures to be identified were an anthranilic acid
derivative and an imidazo[1,2-a]pyridine, which showed moderate activation of
the receptor [4]. Subsequently these scaffolds were chemically modified to
generate structure-activity-relationships (SAR) revealing an insight into
pharmacophore properties of the specific residues. All structures were
characterized biologically in a luciferase-based, full-length FXR transactivation
assay [5].
4. Elgaher, W.A.M. et al.: RSC Adv. 2014, 4: 2177−2194.
MC.41
Mobility Shift Affinity Capillary Electrophoresis: A Fast and
Suitable Method for Early Stage Protein Metal Ion Interaction
Screenings
Nachbar, M.1; Mozafari, M.1; Alhazmi, H.A.1; Albishri, H.M.2; El-Hady, D.A.2,3; El
Deeb, S.1,4; Redweik, S.1; Wätzig, H.1
1 Institute
Saudi Arabia.
In detail the imidazo[1,2-a]pyridine scaffold was chemically altered considering
the thiophene- and methylenedioxyphenyl partial structures as well as the
substitution pattern of the imidazo[1,2-a]pyridine core. The published "miniSAR" was enlarged to systematically and gradually evaluate modifications to
the structure to activity and maximal activation towards FXR. Therefore several
derivatives were synthesized using the groebke-blackburn three-component
synthesis of imidazo[1,2-a]pyridines using the respective aminopyridines,
aldehydes and isocyanides. The results of biological evaluation promote
ligands that activate FXR in a nanomolar activity range and that can be
optimized regarding physicochemical properties for further in vitro and in vivo
characterization.
The work has been supported by the Else Kröner-Fresenius Foundation (EKFS),
Research Training Group Translational Research Innovation - Pharma (TRIP)
References:
1. Wang Y.D. et al.: Cell Res. 2008, 18(11):1087-1095.
2. Fan M. et al.: Biochim Biophys Acta. 2014 May 27. pii: S1874-9399(14)00135-7. [Epub
ahead of print]
3. https://clinicaltrials.gov/ct2/results?term=obeticholic+acid&Search=Search
4. Achenbach J. et al.: Med. Chem. Commun.,2013, 4: 920-924
5. Merk D. et al.: Bioorg Med Chem. 2014, 22(8): 2447-2460.
MC.40
Design, synthesis, SAR exploration and optimization of novel
bacterial RNA polymerase inhibitors targeting the switch region
Elgaher, W.A.M.; Sahner, J.H.; Groh, M.; Haupenthal, J.; Hartmann, R.W.
Helmholtz Institute for Pharmaceutical Research Saarland, Department of Drug Design
and Optimization, Campus C2.3, Saarland University, 66123 Saarbrücken, Germany
The evolution of antibiotic resistant bacteria represents a true threat to public
health that demands the development of new antibiotics with alternative mode
of action. [1] The “switch region” of RNA polymerase (RNAP), targeted by the
α-pyrone antibiotics e.g. myxopyronins, was proved to be a promising target
for antibacterial drug discovery. [2] Based on a hit candidate discovered by
virtual screening, a small library of 5-aryl-3-ureidothiophene-2-carboxylic acids
(class I) was synthesized resulting in compounds with increased RNAP
inhibition. Hansch analysis revealed π (lipophilicity constant) and σ (Hammett
constant) of the substituents at the 5-aryl moiety to be crucial for activity. [3]
In a further step, six new classes were synthesized via two analog design
approaches: First, design of regioisomers to explore the optimum configuration
of the aryl-ureidothiophene-carboxylic acids (class II‒IV). Second, bioisosteric
replacement of the thiophene core by different heterocyclic rings to improve
the physicochemical properties (class V‒VII). Structure activity relationship
(SAR) studies, supported by molecular modeling, revealed the structural
requirements necessary for interaction with the binding site. The new
compounds displayed good antibacterial activities against Gram positive
bacteria and Gram negative E. coli TolC, accompanied by a reduced resistance rate compared to the known antibiotic rifampicin together with a low
mammalian cytotoxicity. [4]
References:
1. Coates, A.R.M., Halls, G., Hu, Y.: Br. J. Pharmacol. 2011, 163: 184−194.
2. Srivastava, A. et al.: Curr. Opin. Microbiol. 2011, 14: 532−543.
3. Sahner, J.H. et al.: Eur. J. Med. Chem. 2013, 65: 223−231.
168
The importance of protein drugs for inflammatory and antitumor therapy is
growing steadily. Hence, for reaching an optimal therapeutic success the right
conformation and therefore the overall charge is of major interest. Protein
conformations are not only highly affected by the pH, temperature and the ionic
strength of the surrounding solution; they are also influenced by the containing
ions. These ions can change the conformation by binding to specific motifs at
the protein e.g. the EF hand motif or by unspecific bonds like electrostatic
attraction between a cation and negatively charged amino acids. Changes of a
protein's charge can easily be examined using affinity capillary electrophoresis
(ACE). This work reveals how protein drugs can be affected by metal ions
using a proline-rich digest obtained from galectin-3 (CBPep) as a model for
these interactions. The mode of operation for ACE is based on the shift of
electrophoretic mobility after ions bind to a protein. Thus, the time until the
analyte is detected is changed according to the change of the overall charge of
the protein. So if the protein is getting more negative the time is increased or if
the protein is getting more positive the time is decreased.
The influence of various ions was determined by using mobility ratios of the
EOF marker and the protein to prevent effects of migration time shifts which
are not related to interactions. The difference of the mobility ratio of the protein
with the ligand (Ri) and without the ligand (Rf) was normalised to Rf (ΔR/Rf)1.
The result demonstrates the change in the overall charge of the protein-ioncomplex and also the strength of the interaction.
During the experiments various metal ions e.g. Mn2+, Cu2+ and Ba2+ as well as
complexes of metal ions which have a low solubility at physiological pH (e.g.
Fe3+) were tested to determine their ability to interact with CBPep.
The analysis of CBPep did not show the strong, sequence-specific interactions
with calcium ions that were reported for mass spectrometry binding experiments in the vacuum state2. Only very weak interactions with other metal ions
e.g. (Mg2+, Mn2+, Ba2+ , Sr2+, SeO32-, Fe3+, Ni2+, Cu2+, Zn2+, Au3+) were found.
These findings suggest that the binding mode in aqueous solutions is different,
which is also encouraged by preliminary molecular modelling results for the
CBPep-Ca2+-complex in vacuum state.
References:
1. Redweik S., Xu Y., Wätzig H.:ELECTROPHORESIS 2012, 33(22):3316-3322
2. Lehmann WD. et al.: RAPID COMMUN MASS SP 2006, 20(16):2404-2410
MC.42
C2- and O-Linked Melatonin Dimers as Bivalent Ligands Targeting Dimeric Melatonin Receptors
Zlotos, D.P.1; Sadek, M.S.1; Tadros, S.A.A.1; Gerbier, R.2,3,4; Jockers, R.2,3,4
1 The
German University in Cairo, Dept. of Pharmaceutical Chemistry, New Cairo City,
11835 Cairo, Egypt
2 Inserm, U1016, Institut Cochin, Paris, France
3 CNRS UMR 8104, Paris, France
4 Univ. Paris Descartes, Sorbonne Paris Cite, Paris, France
Melatonin MT1 and MT2 receptors are among the first G-protein coupled
receptors whose homo- and hetero-dimerization have been demonstrated
using bioluminescence resonance energy transfer (BRET) [1,2]. Recently, we
have reported the synthesis and pharmacological evaluation of a series of
dimeric melatonin analogues obtained by connecting two melatonin molecules
through N1 with spacers of 15-24 atoms [3]. The BRET studies provided
evidence for the binding of these compounds to MT1 and MT2 homo- and
heterodimeric receptors. Here, we describe the synthesis and pharmacological
evaluation of two novel series of dimeric melatonin analogues obtained by
linking two melatonin pharmacophores through C2 and O with spacers of 1824 atoms. The findings are important for the design of novel bivalent melatonergic ligands selectively targeting MT1/MT1-homodimers, MT2/MT2homodimers, and MT1/MT2-heterodimers.
AcHN
H
H
N (CH )m N
2
O
N
H
N
H
m = 4,6,8,10,12
N
H
AcHN
NHAc
O
MeO
NHAc
O
O
O
O
O
H
N (CH 2)n
NH
NH
OMe
n = 6,8,10,12
N
H
NH
O
AcHN
O
NHAc
O
MeO
N
H
OMe
O
H
N (CH 2) 12
NH
NH
NH
N
H
O
Acknowledgments: Prof. Dr. Ulrike Holzgrabe, Würzburg University, Deutscher Akademischer Austauschdienst (DAAD)
References:
1. Ayoub, M.A. et al.: J. Biol. Chem. 2002, 277: 21522.
2. Zlotos, D.P. et al.: J. Med. Chem. 2014, 57: 3161.
3. Journé, A-S. et al.: Med. Chem. Commun. 2014, 5: 792.
References:
1. Saur, O. et al.: Arch. Pharm. Chem. Life Sci. 2007, 340(4): 178−184.
2. Höfling, S.B. et al.: Angew. Chem. 2010, 122(50): 9963−9966.
3. Jasch, H. et al.: J. Org. Chem. 2012, 77(3):1520−1532.
4. Fehler, S.K. et al.: Chem. Eur. J. 2014, 20(2): 370−375.
MC.44
Performance Qualification in Surface Plasmon Resonance
Analysis
MC.43
Phenylazocarboxamides as structural analogues for cinnamoyl
amides in D3 receptor ligands
Bartuschat, A.L.1; Fehler, S.K.1; Hübner, H.1; Prante, O.2; Gmeiner, P.1;
Heinrich, M.R.1
1 Department
of Chemistry and Pharmacy, Medicinal Chemistry, FAU Erlangen-Nürnberg,
Schuhstr. 19, 91052 Erlangen, Germany
2 Department of Nuclear Medicine, Molecular Imaging and Radiochemistry, FAU ErlangenNürnberg, Schwabachanlage 6, 91052 Erlangen, Germany
Cinnamoyl amide derivatives such as 1 represent a potent class of dopamine
D3 receptor ligands, as shown by Saur et al..[1] By replacing the C-C double
bond with the structurally comparable azo unit we were able to find a new
class of dopamine D3 receptor ligands 2. These ligands are readily accessible
from phenylazocarboxylic esters which are valuable building blocks for
combinatorial synthesis.[2,3] Due to the electron-withdrawing properties of the
azocarbonyl moiety, the aromatic core of phenylazocarboxylic esters is highly
activated towards nucleophilic substitution. Besides the introduction of
aromatic amines, aliphatic amines and phenols, substitutions with [18F]fluoride
can be conducted at short reaction times, under mild conditions and with high
yields, which in turn allows a very efficient access to [18F]-labeled D3 ligands
such as 3.[4] Moreover, new results on the effects resulting from structural
variations of the lead compound 2 will be presented.
Steinicke, F.; Oltmann-Norden, I.; Wätzig, H.
TU Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany
Surface Plasmon Resonance (SPR) is a dominant tool for characterization of
biomolecular interaction. This technique facilitates label-free binding analysis
studies of biomolecules such as affinity, kinetic, thermodynamics and specifity
analysis in real-time. Therefore SPR is an important application in drug
discovery and proteomics.
In this study a concept was investigated for performance qualification, longterm precision and accuracy for kinetic measurements of biomolecular
interactions using SPR on a Biacore X100 instrument. So far there is no
research done which offers reliable data for the parameters of our interest. The
method used was an already established single cycle kinetic assay by GE
Healthcare to monitor human beta-2-microglobulin (b2m) with a covalently
bound anti-beta-2-microglobulin antibody from mouse. The first step of the
assay was the direct immobilization of anti-b2m antibody to a CM5 sensor
chip. The antibody was covalently bound to the carboxymethylated dextran
layer of the sensor chip surface using the amine coupling method. The sensor
chip was reusable for several runs. In order to reduce bulk effects all assay
solutions have been prepared with the running buffer, a HBS-EP buffer
(HEPES Buffer Saline-EDTA Polysorbate 20) pH 7,4 containing 0.01 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.15 M NaCl, 3 mM
EDTA and 0.005% (v/v) Polysorbate 20. Each day a freshly prepared serial
dilution of b2m (2 nM, 4 nM, 8 nM, 16 nM and 32 nM) was employed in singlecycle kinetic measurements. In this assay all five analyte dilutions were
sequently measured followed by one regeneration step with glycine hydrochloride 10 mM pH 2.5. All binding experiments were carried out at 25°C and with
a flow rate of 30 µL/minute. The sensorgrams for each single-cycle kinetic
assay were analyzed by curve fitting based on a 1:1 binding model using the
Biacore X100 evaluation software (version 2.0). The parameters that have
been analyzed include the maximal theoretical Response Units (Rmax), the
dissociation constant (KD), the residuals from the optimal fitted curve and the
peak absorption value for every concentration. These parameters were
evaluated and then plotted into control-charts. In addition to these data we
watched for external influences such as mechanical resilience of the chip and
data deviations for example caused by pipeting-errors or material impact. The
first series contains 27 cycles and shows a percental relative standard
deviation of 4.9% for the KD.
169
MC.45
Synthesis and characterization of ‘clickable’ alkyne-PEGylated
FGF2 and its site-directed immobilization to azide modified
microspheres via click chemistry
Heusler, E.; Jones, G.; Zhao, H.; Lühmann, T.; Meinel, L.
Growth factors such as vascular endothelial growth factor (VEGF) [1] and bone
morphogenetic protein 2 (BMP2) [2] had been covalently immobilized to
biomaterials by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/Nhydroxysuccinimide (NHS) chemistry with the aim to (re-)engineer lost tissues.
A remarkable drawback of this EDC/NHS chemistry is that most biologic
molecules carry both carboxyl and amino groups yielding crosslinking (intraand/or intermolecular) and loss of function. Therefore, we used Cu(I) catalyzed
azide-alkyne cycloaddition chemistry (click chemistry) to overcome this
disadvantage [3]. The suitability of this decoration method for biologics was
studied with fibroblast growth factor 2 (FGF2), a heparin-binding growth factor
affecting the proliferation, differentiation and migration of many cell types of
mesodermal and neuroectodermal origin, stimulating tissue regeneration and
having therapeutic potential in wound healing [4].
Firstly, we PEGylated the FGF2 in a site-directed pattern by deploying free
thiol groups (four cysteine residues: two pointing outwards, two pointing into
the protein core) with thiol reactive 10 kDa PEGs carrying ethinyl groups as
described before with modification [3]. Alkyne-PEGylated FGF2 was purified
using heparin based affinity chromatography on an ÄKTA purifier system and
finally characterized by reducing SDS-PAGE, RP-HPLC and MALDI MS. The
bioactivity of alkyne-PEGylated FGF2 was determined by analysis of the
proliferation of NIH-3T3 cells. Alkyne-PEGylated FGF2 was coupled to azide
modified microspheres by click chemistry and analyzed by flow cytometry
(FACS) after incubation with FGF2 primary and FITC labelled secondary
antibody.
MALDI MS spectra of alkyne-PEGylated FGF2 indicated that either one or two
FGF2 cysteine residues were PEGylated, and the potency of alkynePEGylated FGF2 was demonstrated by NIH-3T3 cell proliferation assay. The
coupling of alkyne-PEGylated FGF2 to the azide modified microspheres was
confirmed by FACS analysis. Azide modified microspheres exposed to alkynePEGylated FGF2 in presence of copper showed stronger fluorescence than
controls in the absence of copper.
The functionalization of azide modified microspheres with FGF2 has been
successfully demonstrated using click chemistry. This chemical strategy for
covalent immobilization avoids the formation of intra- and/or intermolecular
covalent aggregates among biologics as well as among biomaterials and
consequently may improve the safety profile of functionalized biomaterials
compared to EDC/NHS chemical strategies [3].
Acknowledgments:
The financial support from the IZKF (Wuerzburg, Germany) with grant number D-218 is
gratefully acknowledged.
References:
1. Chiu, L.L.Y. and Radisic, M.: Biomaterials 2010, 31(2): 226-241.
2. Karageorgiou, V. et al.: Journal of biomedical materials research 2004, 71(3): 528–37.
3. Zhao, H., Heusler, E. et al.: Journal of structural biology 2014, 186(3): 420-430.
4. Gospodarowicz, D., Neufeld, G., Schweigerer, L.: Cell differentiation 1986, 19(1): 1–17.
MC.46
septic arthritis caused by gram-positive bacteria resistant to other first-line
antibiotics occurs, linezolid is given additionally. A prerequisite for antibiotic
efficacy are sufficiently high concentrations at the site of infection, i.e. the
synovial fluid. Due to potentially inadequate penetration of the antibiotics into
the synovial fluid, insufficient concentrations at the infection site may occur,
also accounting for the high morbidity and mortality rate of septic arthritis.
Therefore, it is important to measure drug concentrations at the site of
infection. A pharmacokinetic study of cefuroxime and linezolid in 10 patients
undergoing elective knee arthroscopy for meniscal repair was performed in
collaboration with the Department of Clinical Pharmacology, Medical University
of Vienna. Concentrations of linezolid and cefuroxime were measured in
microdialysis samples obtained in synovial fluid and interstitial, muscular tissue
fluid as well as in plasma samples to characterise the pharmacokinetics of both
antibiotic agents in combination. For the analysis of study samples an
appropriate HPLC assay for the determination of both antibiotics had to be
developed. In total, 25 concomitant drugs were given during the trial and
needed to be separated from the analytes.
Methods: For drug quantification in the biological fluids a Thermo Scientific
Dionex Ultimate 3000 HPLC with DAD 3000 detector was used. Detection
wavelengths for linezolid and cefuroxime were set based on previous
investigations. Separation was achieved by a Thermo Fisher Hypersil GOLD
Phenyl column (100 x 4.6 mm, 3 µm) with a Thermo Fisher Phenyl guard
column. To isolate cefuroxime and linezolid from the concomitant drugs
different modes and mobile phases were assessed. For the isocratic mode, a
mobile phase consisting of Milli-Q water and acetonitrile with and without 0.1%
trifluoroacetic acid and for the gradient mode, Milli-Q water with 0.1%
trifluoroacetic acid as mobile phase A and Milli-Q water and acetonitrile with
0.1% trifluoroacetic acid as mobile phase B were investigated in different
ratios. In addition, different injection volumes, flow rates, and column oven
temperatures were evaluated.
Results: Separation of linezolid and cefuroxime was achieved. Detection
wavelengths were set to 251 nm for linezolid and 271 nm for cefuroxime,
respectively. A gradient method was performed using Milli-Q water and 0.1%
trifluoroacetic acid as mobile phase A and Milli-Q water and acetonitrile 30:70
(v/v) and 0.1% trifluoroacetic acid as mobile phase B were applied at a flow
rate of 2.0 mL/min. The injection volume was amounted to 10 µL. The
temperature of autosampler and column oven was set to 4° C and 35° C,
respectively. The total run time was 20 minutes with retention times of 3.95
and 4.10 minutes for linezolid and cefuroxime, respectively. Except urapidil, all
concomitant drugs were successfully separated from the analytes.
Conclusion: A successful separation of linezolid and cefuroxime and isolation
from 24 of the concomitant drugs was achieved. As next step, an additional
HPLC assay has to be developed for the patient receiving urapidil. Both
assays shall be extended to plasma as matrix. Thereafter, the methods have to
be validated according to the criteria of the EMA Guideline on bioanalytical
method validation [1]. In total, concentrations of linezolid and cefuroxime in
synovial fluid and interstitial fluid of muscle tissue measured by microdialysis
and plasma will be determined and related to each other in order to investigate, if current antibiotic regimens will provide effective concentrations at the
particular site of infection.
Acknowledgement: We thank Dorothea Frenzel, Martin-Luther-Universitaet HalleWittenberg for her analytical support.
Reference:
1. European Medicines Agency (EMA): Guideline for bioanalytical method validation 2012
MC.47
HPLC method development for the determination of linezolid and
cefuroxime in synovial fluidsite microdialysis samples of arthritis
patients
Substituted vinyl sulfones as covalent and reversible cysteine
protease inhibitors
Appelt, A.K.1; Kauzor, D.1; Wicha S.G.1; Brosig, H.1; Zeitlinger, M.2; Kloft, C.1
Kesselring, J.1; Schneider, T.1; Grathwol, C.1; Weickert, A.2; Lee, W.2; Willmes,
T.3; Engels, B.2; Sotriffer, C.A.3; Schirmeister, T.1
Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet
2 Dept. of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20,
1090 Vienna, Austria
1
Objectives: Septic arthritis, a joint destructive infectious disease with the knee
as the most frequently involved joint, displays a high morbidity and mortality
rate. Cefuroxime is commonly used for perioperative antibiotic prophylaxis. If
170
1 Institute
of Pharmacy and Biochemistry, University of Mainz, GERMANY
of Physical and Theoretical Chemistry, Universiy of Würzburg, GERMANY
3 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY
2 Institute
Cysteine proteases play vital roles for the life cycles, nutrition and pathogenesis of a variety of parasites causing infectious tropical diseases [1,2]. Therefore
a promising strategy for the treatment of these diseases is the inhibition of
these proteases.
At present a large number of potent and irreversible cysteine protease
inhibitors of different classes, including peptidyl vinyl sulfones, have been
reported [3]. The inhibition mechanism of such vinyl sulfones results from the
addition of the enzyme’s active site cysteine residue in a Michael type reaction
[4].
QM and QM/MM calculations have proposed substituted vinylsulfones which
should be able to form a covalent, but reversible bond with the cysteine sulfur
of the protease’s active site. Such a reversible reaction should be possible by
position of the vinyl sulfone moiety, e.g. nitrile groups, halogens and thiolates,
respectively. This leads to a thermoneutral or slightly endergonic vinylic
substitution or addition reaction. To confirm these calculations a series of
highly functionalized vinyl sulfones with varying peptidic residues were
synthesized and reacted with phenylethanethiol as an analogue of the active
site’s cysteine residue. The progression of the reaction was monitored by
NMR, which allows the determination of rate constants, equilibrium constants,
and reaction energies. Additionally the reversibility of the reaction with the
enzyme could be proved by dilution and dialysis experiments. Furthermore the
inhibitory potencies and binding modes of the synthesized compounds were
evaluated by enzyme assays with various cysteine proteases.
References:
1. Hanzlik, R.P.: J. Med. Chem. 1984, 27(6): 711.
2. Rosenthal, P.J. and coworkers: Antimicrob. Ag. Chemother. 2003, 47(1): 154.
3. Powers, J.C. et al.: Chem. Rev. 2002, 12: 4639.
4. Palmer, J.T. et al.: J. Med. Chem. 1995, 38 (17): 3193 .
Acknowledgments: Deutscher Akademischer Austauschdienst, DAAD
References:
1. Dean, M.; Rzhetsky, A.; Alliknets, R.: Genome Res. 2001, 11(7): 1156-66.
2. Leslie, E.M.; Deeley, R.G.; Cole S.P.: Toxicol. Appl. Pharmacol. 2005, 204(3): 216-237.
3. Egger, M. et al.: Eur. J. Org. Chem. 2007, (16): 2643-2649
4. Kühnle, M. et al.: J. Med. Chem. 2009, 52(4): 1190-1197
5. Ochoa-Puentes, C. et al.: ACS Med. Chem. Lett. 2013, 4(4):, 393−396
6. Bauer, S.; et al.: ChemMedChem 2013, 8(11): 1773-1778
7. Leslie, E.M. et al.: Mol. Pharmacol. 2001, 59(5): 1171-1180.
8. Burkhart, C.A., et al.: Cancer Res., 2009, 69(16): 6573-6580
MC.49
MC.50
Virtual Screening and Biological Evaluation of new Inhibitors of
the ATPase-Domain in GyrB
Münsterberg, M.; Fransson, I.; Lemcke, T.
Institute of Pharmacy, University of Hamburg, Bundesstr. 45, 20146 Hamburg, Germany
MC.48
Flavonoid-based compounds as novel selective ABCC1 modulators
Obreque-Balboa, J.1; Sun, Q.2; Bernhardt, G.1; König, B.2; Buschauer, A.1
1 Institute
2 Institute
of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany
of Organic Chemistry, University of Regensburg, D-93040 Regensburg,
Germany
The superfamily of human ATP-binding cassette (ABC) proteins comprises 48
members divided into 7 subfamilies (ABCA - ABCG) [1]. In addition to
physiological functions, a number of these membrane proteins are known as
efflux pumps limiting the bioavailability and the access of a wide variety of
xenobiotics, including drugs, to the brain. Moreover, the ABCB1 (pglycoprotein), ABCG2 (BCRP, breast cancer resistance protein) and ABCC1
(MRP-1, multidrug resistance related protein 1) are the most prominent efflux
transporters contributing to multidrug resistance. For instance, the expression
of these transporters is related with high extrusion levels of chemotherapeutic
agent, such as anthracyclines, vinca alkaloids or epipodophyllotoxins, and poor
outcome of the treatment [2]. Subtype-selective and potent modulators of
these ABC transporters are required as pharmacological tools to investigate
their role in health and disease, to improve brain access or to overcome
chemoresistance.
Previously, potent and selective ABCB1 and ABCG2 modulators have been
developed in our laboratory [3-6]. Here we report on the synthesis and
characterization of a new class of ABCC1 modulators. The core structure of
flavonoids known both, as ABC substrates and inhibitors [7], served as
template. The synthesized compounds were investigated in vitro for transporter
inhibition in fluorescence based assays using ABCC1- (MDCKII-MRP1 cells),
ABCB1- (Kb-V1 cells) and ABCG2- (MCF-7/topo cells) overexpressing cells.
The potential to revert drug resistance was explored for selected inhibitors in a
kinetic chemosensitivity assay.
Among the synthesized compounds, the most promising modulators were
comparable to reversan [8] in potency, and by far superior to the reference
substance regarding selectivity. The most potent compound revealed an IC 50
value of 10.5 µM (ABCC1) and a maximal inhibitory effect (Imax) of 120%
compared to reversan (IC50=4.5 and Imax 100%). In contrast to the latter, which
is a combined ABCB1 and ABCC1 modulator, the title compounds were highly
selective for ABCC1.]
The development of new antibiotics is essential for the future of the treatment
of infection diseases. Although, this fact is well-known [1,2], it has already
become of political interest [3]. Gyrase is an important target for antibiotic
therapy. It is inhibited by the commonly-used fluoroquinolone family, which
interacts with the gyrase-DNA-complex. In contrast, aminocoumarines like
novobiocin inhibit the ATPase domain of the gyrase subunit B (gyrB).
Currently, no drug acting as gyrB ATPase inhibitor is used in therapy [4].
We performed a high-throughput virtual screening with the “Drug-Like” subset
of the ZINC database [5] targeting the ATPase domain of the gyrB-subunit of
E. coli. Starting with 11 million compounds we applied several filter criteria (like
MW, number of rotatable bonds) for the first substantial reduction of compound
numbers. The remaining 3.6 million compounds were docked with the newly
developed high-throughput screening tool TrixX [6] which allowed a multi target
approach because of its fast algorithm. In this way we were able to dock the
compounds in different conformations of the ATPase domain to consider the
flexibility of the enzyme. Together with a validated post-processing protocol the
amount of compounds could be reduced to several thousands. Subsequently,
these compounds were redocked with the glide docking tool of Schrödinger [7].
A crucial visual inspection resulted in 20 compounds which were selected and
purchased.
Finally, we tested these compounds in a fluorescence quenching and
supercoiling assay for their inhibitory activity of the E. coli gyrB. For the
fluorescence quenching we adopted the newly developed energy transfer strategy [8,9] in order to obtain a fast, simple and reliable screening
method.
In this poster we will present a successful, hit search protocol (see scheme
below) including the virtual screening and biological evaluation. This work will
be continued to further optimize our findings to a possible lead structure for the
inhibition of bacterial gyrB ATPase.
Scheme of the Hit Search Protocol
171
References:
1. Livermore D.M.: J. Antimicrob. Chemother. 2009, 64(suppl 1): i29.
2. Boucher H.W. et al.: Clin. Infect. Dis. 2009, 48(1): 1–12.
3. Theuretzbacher U.: Int. J. Antimicrob. Agents 2012, 39(4): 295–299.
4. Collin F.; Karkare S.; Maxwell A.: Appl. Microbiol. Biotechnol. 2011, 92(3): 479–497.
5. Irwin J.J. et al.: J. Chem. Inf. Model. 2012, 52(7): 1757–1768.
6. Schlosser J.; Rarey M.: J. Chem. Inf. Model. 2009, 49(4): 800–809.
7. Schrödinger, LLC 2014.
8. Zuck P. et al.: Anal. Biochem. 2005, 342(2): 254–259.
9. Miyata Y. et al.: J. Biomol. Screening 2010, 15(10): 1211–1219.
MC.51
Bifunctional phosphonates for the functionalization of metal
surfaces
Klitsche, F.; Maison, W.
Pharmaceutical and Medicinal Chemistry, University of Hamburg, Bundesstraße 45,
20146 Hamburg, Germany
The coating of metal surfaces is a valuable method to generate tailor-made
materials for a variety of applications. Due to the fact that the formation of
biofilms, also known as biofouling, occurs on almost all material surfaces in
biological systems it is a crucial issue in medicine, clinical hygiene, food
industry and marine technology [1,2]. The process of biofilm formation can be
described stepwise. In a first step organic molecules like proteins, polysaccharides or glycoproteins attach to the surface reversibly to form a molecular
monolayer. This conditioning film forms the soil for the colonization of
microorganisms. The next and final step of the so-called microfouling is the
formation of an inert bacterial biofilm that is protected by a surrounding
mucopolysaccharide layer [3]. At this stage, bacterial biofilms can no longer be
removed by antibiotics and cause therefore serious problems in a clinical
context and in the food industry. In implant medicine, for example, biofilms can
lead to inflammation, impair integration and final rejection of the implant. To
prevent biofouling several strategies have been applied which interfere with
different steps of the process. Examples include bactericide surfaces
generated with metals such as copper or silver. A second approach is the use
of repellant nanolayers such as polyethylene glycol (PEG) and other hydrophilic polymers preventing non-specific protein attachment in the first step of
biofouling [2,4,5]. A key aspect of this approach is the stable immobilization of
hydrophilic polymers on the material surface.
We have developed modular tripodal anchor molecules for the stable
immobilization of hydrophilic polymers on metal surfaces [6,7]. The synthesis
of tripodal phosphonates and their conjugation to PEG is described on the
poster. Moreover the immobilization of these conjugates on metal oxides of
clinical relevance and the analysis of the resulting antifouling surfaces is
reported.
MC.52
Synthesis of oxazinones and oxazinethiones with selective
inhibition of NAD+-dependent histone deacetylases (Sirtuins)
Beese, K.1; Swyter, S.2; Jung, M.2; Link, A.1
1 Institute
of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-JahnStr. 17, 17489 Greifswald, Germany
2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr. 25,
79104 Freiburg, Germany
The seven human sirtuins are NAD+-dependent histone deacetylases, which
are involved in epigenetic gene expression. The balance of acetylation and
deacetylation of histones and non-histone proteins is regarded to be relevant in
age-related diseases, e.g. cancer, neurodegenerative diseases like Parkinson's, Alzheimer's and Huntington's disease.
To obtain non-hydrolyzable analogs of the lactone splitomicin, we advanced βphenylsplitomicin analogs with lactam structure1 and synthesized novel
naphtho[1,3]oxazin-3-one and naphtho[1,3]oxazine-3-thione derivatives
performing a solvent-free one pot synthesis.
The substances were tested in a trypsin-coupled homogeneous assay, which
revealed a selective inhibition of Sirt2.
Acknowledgements: Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald,
Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg. Technical
assistance of Dr. A. Bodtke, K. Böheim and J. Technau is gratefully acknowledged.
References:
1. Neugebauer, R.C.; Sippl, W.; Jung, M.: J. Med. Chem. 2008, 51(5): 1203-13
MC.53
Design, Synthesis and Pharmacological Profiling of Dual Modulators of Soluble Epoxide Hydrolase and Peroxisome Proliferator
Activated Receptors
Blöcher, R.; Wittmann, S.K.; Lamers, C.; Weber, J.; Steinhilber, D.; SchubertZsilavecz, M.; Proschak, E.
Goethe University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438
Frankfurt am Main
Modular design principle of antifouling coatings [8]
References:
1. Banerjee, I.; Pangule, R.C.; Kane, R.S.: Adv. Mater. 2011, 23(6): 690-718.
2. Lejars, M.; Margaillan, A.; Bressy, C.: Chem. Rev. 2012, 112(8): 4347-4390.
3. Costerton, J.W.; Stewart, P.S.; Greenberg, E.P.: Science 1999, 284(5418): 1318-1322.
4. Fenton, J.W.; Fasco, M.J.: Thromb. Res. 1974, 4(6): 809-817.
5. Sisson, A.L.; Haag, R.: Soft Matter 2010, 6(20): 4968-4975.
6. Pannier, N.; Maison, W.: Eur. J. Org. Chem. 2008, 2008(7): 1278-1284.
7. Maison, W.; Frangioni, J.V.; Pannier N.: Org. Lett. 2004, 6(24): 4567-4569.
8. Franzmann, E. et al.: Chem.-Eur. J. 2011, 17(31): 8596-8603.
The basic idea of this study comprehends the development of polypharmacological agents for the treatment of the metabolic syndrome [1, 2, 3]. Several
dual modulators of the peroxisome proliferatior-activated receptors (PPARs) [4]
and the soluble epoxide hydrolase (sEH) [5] have been rationally designed,
synthesized and evaluated in vitro. It was possible to synthesis a number of
active compounds containing the common pharmacophores of both targets
(sEH/PPAR) [4, 5, 6]. The potency of sEH inhibition was determined in an in
vitro assay with recombinant enzyme [6]. The ability of the PPAR activation
was evaluated in a cell-based reporter-gene assay [7]. The sEH inhibtion was
achieved at a sub micromolar concentration of the compound. PPAR agonism
was reached at micromolar concentration. Relative activations could be
demonstrated, from full agonists to partial PPAR modulators. The compounds
targeted PPAR in a subtype selective as well as in a nonselective way. Future
in vivo studies will show the value of this approach for the therapy of metabolic
syndrome related diseases [8].
Acknowledgments:
Goethe University, Institute of Pharmaceutical Chemistry, Steinhilber, D.; SchubertZsilavecz, M. and Proschak, E.
Else Kröner-Fresenius-Foundation
172
References:
1. Eckel, R.H.; Grundy, S.M.; Zimmet, P.Z.: Lancet 2005, 365: 1415–1428.
2. Grundy, S.M. et al.: Nat. Rev. Drug Dis. 2006, 5: 295–309.
3. Page Acnp-Bc, J. et al.: J. Am. Acad. Nurse Prac. 2012, 24: 345–351.
4. Pirat, C.et al.: J. Med. Chem. 2012, 55: 4027–4061.
5. Hammock, B. et al.: J. Med. Chem. 2012, 55: 1789−1808.
6. Blöcher, R. et al.: J. Med. Chem. 2012, 55: 10771-10775.
7. Lamers, C.; Schubert-Zsilavecz, M.; Merk, D.: Exp. Opin. Ther. Pat. 2012, 22: 803–841.
8. Imig, J.D. et al.: Experimental Biology and Medicine 2012, 237: 1402-1412.
MC.54
Biological screening of newly synthesized thiocarbonic/carbonic
acid derivatives on isolated guinea pig tissue preparations
Hintersteininger, M.1; Erker, T.1; Studenik, C.R.2
1 Department
2 Department
of Pharmaceutical Chemistry, Althanstraße 14, 1090 Vienna, Austria
of Pharmacology and Toxicology, Althanstraße 14, 1090 Vienna, Austria
The aim of this project was to study the efficacy and the pharmacodynamics of
four compounds focusing on their inotropic, chronotropic, vasodilating and
spasmolytic action.
1
3
2
4
Compound 1 and Compound 3 are thiocarbonic acid derivatives, compound 2
and compound 4 are carbonic acid derivatives. Force of contraction (fc) on
electrically driven papillary muscles (1Hz), spontaneously beating right atria,
aortic-rings, arteria pulmonalis-rings (precontracted with 90 mM KCl) and
terminal ilea (precontracted with 60mM KCl) of guinea pigs was measured
using the method described by Reiter [1].
Compound 1 significantly reduced fc in papillary muscles with an EC50-value of
10 µmol/l. This derivative also caused a negative chronotropic effect (EC 50 =
48 µmol/l, right atria) as well as a vasodilating (EC50 = 12.5 µmol/l in aortic
rings and 13.7 µmol/l in pulmonalis rings) and spasmolytic effect (EC 50 = 9.3
µmol/l in terminal ilea). Compound 2 only showed a significant action on heart
muscle preparations with an EC50–value of 12.5 µmol (papillary muscles) and
44 µmol/l (right atria) and on the terminal ilea with an EC 50-value of 33.5
µmol/l. Compound 3 significantly decreased force of contraction in heart
muscle preparations with an EC50 – value of 5.45 µmol/l (papillary muscle) and
43,5 µmol/l (right atria) as well as in smooth muscle preparations with an EC50
of 91.5 µmol/l in aortic rings, 50 µmol/l in pulmonalis rings and 20.5 µmol/l in
terminal ilea. Compound 4 did not show significant effects on any preparations
with exception of a negative inotropic one on papillary muscles (EC50 = 30
µmol/l). We demonstrated that compound 1 and 3 had the most potent effects
on all tissue preparations possibly caused by H2S/COS release followed by
opening of KATP-channels. Compound 2 showed a negative inotropic, negative
chronotropic and spasmolytic effect, while compound 4 only excerted a
negative inotropic effect. Our results suggest that derivatives with a thiocarbonyl moiety possess a strong efficacy in all isolated tissues, while compounds
with a carbonyl moiety are less potent but more tissue selective.
References:
1. Reiter, M.: Drug. Res. 1967, 17:1249-1253.
MC.55
The mitochondrial Amidoxime Reducing Component (mARC) is
involved in energy metabolism
Jakobs, H.1; Mikula, M.2; Havemeyer, A.1; Ostrowki, J.2; Clement, B.1
1 Department
of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University,
Gutenbergstraße 76, 24118 Kiel, Germany
2 Department
of Oncological Genetcs, Maria Sklodowska-Curie Memorial Cancer Center
and Institute of Oncology, Warsaw, Poland
mARC is the fourth mammalian molybdenum enzyme and was recently
discovered in our lab [1]. It reduces N-oxygenated structures and is a
metabolic counterpart of P450s and FMOs [2]. However, mARC is not solely
active but requires cytochrome b5 and NADH-cytochrome b5 reductase for
electron transport [3]. The enzyme system is involved in N-reductive drug
metabolism and in the activation of amidoxime prodrugs [4,5].
Yet, little is known about the endogenous functions of the enzyme system.
Besides other functions [6,7], links to energy metabolism were demonstrated
like an association with diabetes in animal models [8]. Moreover, a mARC1
SNP is associated with altered blood lipids [9,10] and involvement in lipid
synthesis in 3T3 adipocytes was proven [11].
Based on the latter findings, we tested whether the enzyme system undergoes
changes due to glucose in hepatoma cell lines and due to diet in mice. We
tested mRNA and protein levels as well as N-reductive activity. Indeed the
studied proteins alter due to glucose in cell culture and both fasting and high
fat diet in mice. Taken together with other recent studies [9,10,11,12], it is
evident that the mARC protein is involved in energy and lipid metabolism. In
continuation, we tested the influence of fasting on N-reductive metabolite
concentrations in vivo and thus the potential impact on prodrug activation.
Albeit altered in vitro activity, no changes in the metabolite concentration in
vivo were detectable.
References:
1. Havemeyer, A.; Bittner, F.; Wollers, S.: J. Biol. Chem 2006, 281(46): 34796-34802.
2. Grünewald, S.; Wahl, B.; Bittner F.:J. Med. Chem. 2008, 51(24): 8173-8177.
3. Havemeyer, A.; Lang, J.; Clement, B.: Drug Metab. Rev. 2011, 43(4): 524–539.
4. Havemeyer, A. et al.: Drug Metab. Dispos. 2010, 38(11): 1917–1921.
5. Jakobs, H. et al.: ChemMedChem. 2014, DOI: 10.1002/cmdc.201402127.
6. Krompholz, N. et al.: Chem. Res. Toxicol. 2012, 25(11): 2443–2450.
7. Kotthaus, J. et al.: Biochem. J. 2011, 433(2): 383–391.
8. Malik, A.N. et al.: Biochem. Biophys. Res. Commun. 2007, 357(1): 237–244.
9. Teslovich, T.M. et al.: Nature 2010, 466(7307): 707–713.
10. Aslibekyan, S. et al.: PLoS ONE 2012, 7(10): e48663.
11. Neve, E.P.A. et al.: J. Biol. Chem. 2012, 287(9): 6307–6317.
12. Brown, S.D.M.; Moore, M.W.: Genome 2012, 23(9-10): 632–640.
MC.56
Development and validation of a new analytical method to
characterize PEG-asparaginase by flow field-flow fractionation
John, C.1; Herz, T.2; Hempel, G.2; Langer, K.1
Department of Pharmaceutical Technology and Biopharmacy - University of Münster,
Corrensstr. 48, 48149 Münster, Germany
2 Department of Pharmaceutical and Medicinal Chemistry - Clinical Pharmacy, University
of Münster, Corrensstr. 48, 48149 Münster, Germany
1
Background: PEG-asparaginase (PEG-ASNASE) is widely used in combination therapy for the treatment of acute lymphoblastic leukemia (ALL). The
PEGylated form is designed to reduce immunogenicity due to formation of
antibodies against the foreign protein, reduce its degradation process and to
decrease the clearance of the drug. Besides the physiological advances
PEGylated peptides also show better solubility thus making PEGylation
attractive to pharmaceutical companies and therefore increase the need for
suitable analytical methods.
Method: An AF 2000 MT-system (Postnova Analytics, Landsberg/Lech,
Germany) was used to perform an asymmetrical flow field-flow fractionation
(AF4) of PEG-ASNASE (Oncaspar®). Detection was performed by a dual
wavelength UV-VIS detector set to 220/280 nm, a refractive index detector (RI)
and a multi angle light scattering (MALS) detector. Additionally, a fraction
collector was connected to the AF 2000 MT-system gathering analyte of 5 min
periods for subsequent activity estimation using the well-established microplate
reader-based assay by Lanvers et al.[1]. Moreover, a protein determination
was carried out using a Pierce® BCA Protein Assay Kit (ThermoScientific,
Waltham, Massachusetts).
Validation was assessed by determining within and between run accuracy and
precision, stability tests including room temperature, fridge and freeze/thaw
cycles according to FDA guidelines (Guidance for Industry, Bioanalytical
Method Validation, May 2001).
Results: The method showed valid results for all measured parameters.
Linearity was achieved over the range of 15-750 IU/mL. Accuracy was 86.7%
to 102.2% for within run series with a precision of 1.2 to 10.2%. Between run
series showed an accuracy of 85.3% to 109.9% with a precision of 2.5 to 6.3%.
173
The results were verified by activity measurements. Here, accuracy for within
run and between run series was 88.3% to 137.0% and 97.4% to 114.3%,
respectively. The according precision was 2.4 to 9.3% and 0.1 to 12.3%,
respectively.
The samples stored at room temperature were measured after 24h, 7d, and
14d resulting in a recovery of 86.7±0.9%, 82.1±1.4%, and 77.3±4.8% (mean ±
SD), respectively. In an analogous manner analyzed samples stored at fridge
temperature presented a recovery of 101.2±5.2%, 92.6±7.9%, and 90.5±0.3%
(mean ± SD), respectively.
Analyzing the collected AF4 fractions for activity and protein content made it
possible to correlate them. The correlation showed that protein fragments
lacking enzymatic activity were formed in stressed samples whereas residual
unimpaired molecules (> 350 kDa) were characterized by high activity.
Conclusion: AF4 represents a useful tool for the analysis of PEG-ASNASE.
The successful validation according to FDA guidelines and also verification by
an established, clinically and routinely used activity assay could make it an
attractive method for product evaluation by pharmaceutical companies.
References:
1. Lanvers, C. et al.: Analytical Biochemistry 2002, 309: 117-126.
MC.57
MC.58
Dynamic virtual screening: reducing the search space within a
ligand library
2. Small-Molecule Drug Discovery Suite. 2014-1: QikProp, version 4.0: Schrödinger, LLC.
2014, NY.
MC.59
Development of partial farnesoid X receptor (FXR) agonists
Merk, D.1; Carrasco-Gomez, R.1; Flesch, D.1; Gabler, M.1; Lamers, C.1;
Schneider, G.2; Schubert-Zsilavecz, M.1
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9,
D-60438 Frankfurt, Germany
2 Institute of Pharmaceutical Sciences, ETH Zürich, Wolfgang-Pauli-Str. 10, CH-8093
Zürich, Switzerland
1
The ligand activated transcription factor nuclear farnesoid X receptor (FXR)
acts as a regulator in many metabolic pathways including lipid and glucose
homeostasis and pharmacological activation of FXR seems a valuable
therapeutic approach to treat several pathophysiological conditions. FXR
activation holds promise for the treatment of liver diseases such as primary
biliary cirrhosis and non-alcoholic fatty liver disease, metabolic disorders linked
to insulin resistance and certain forms of cancer. However, full FXR agonism
as it is exhibited by known FXR ligands can evoke undesirable effects which
hinder the clinical development of many known FXR agonists. FXR activation
may e.g. cause cholestasis and impaired lipid homeostasis. For long-term
treatment of metabolic disorders partial FXR agonists with balanced, moderate
FXR activating activity are therefore required, that avoid disadvantageous FXR
over-activation. We report the development and SAR of anthranilic acid
derivatives as potent FXR modulators in a reporter gene assay and on mRNA
level in liver cells. The most potent compound 1 of this scaffold exhibits partial
FXR agonism with an EC50 value of 8±3 nM.
Telukunta, K.K.; Lucas, X.; Günther, S.
Institute of Pharmaceutical Sciences, Research Group Pharmaceutical Bioinformatics,
Hermann-Herder-Strasse 9, 79104, Germany
SHP
*
CYP7A1
600
400
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200
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To optimize this technique and accelerate the virtual screening process we
have developed a more rational approach called dynamic virtual screening.
800
1
% mRNA expression (HepG2)
In the field of drug discovery, virtual screening has become an important
technique which may replace time consuming in vitro assays to obtain an
inhibitor for a protein. Current molecular libraries which can be used for virtual
screening may contain over 35 million drug like molecules [1]. Present
methods mainly apply brute force techniques i.e. all compounds are docked
one after the other.
References:
1. Düfer et al.: Diabetes. 2012, 6: 1479–1489.
2. Fiorucci, S. et al.: Future Med Chem. 2012, 7: 877–891.
3. Fiorucci, S. et al.: Curr Opin Gastroenterol 2009, 3: 252-259.
4. Lee, J.Y. et al.: Br J Cancer. 2011, 6: 1027–1037.
5. Merk, D. et al.: Bioorg. Med. Chem. 2014, 8: 2447–2460.
6. Merk, D. et al.: J Med Chem, submitted
7. Mudaliar, S. et al.: Gastroenterology. 2013, 3: 574-82.e1.
8. Richter, H.G. et al.: Bioorg. Med. Chem. Lett. 2011, 1: 191–194.
In this method we first obtain all the physicochemical properties of the
complete compound library with the help of the tool QikProp [2]. Subsequently
some arbitrary representative candidates of the complete library are taken. The
representative small molecules are scored using the Standard Precision (SP)
algorithm of Glide and sorted by their Glide score. From the sorted set of
compounds we take the top-ranked few compounds for the process of learning
appropriate physicochemical properties of putative binders. These learned
properties are used to filter the main compound library and generate a reduced
compound library space. The carved compound library is then applied for
virtual screening. The complete method can be reapplied to reduce the library.
We benchmarked the presented technique on existing virtual screening
campaigns and analysed if dynamic virtual screening technique could speed
up the search for inhibitors.
References:
1. John, J.I. et al.: J. Chem. Inf. Model. 2012, 52(7): 1757-1768.
174
MC.60
Pyridinol/Pyridinon-tautomerism determining activity at Farnesoid X Receptor: new agonistic or antagonistic ligands of FXR
Lamers, C.1; Merk, D.1; Flesch, D.1; Gabler, M.1; Carrasco-Gomez, R.1,2;
Schneider, G.2; Schubert-Zsilavecz, M.1
Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438 Frankfurt a.M.,
Germany
2 Swiss Federal Institute of Technology (ETH), Institute of Pharmaceutical Sciences,
Valdimir-Prelog-Weg 4, 8093 Zürich, Switzerland
1
Nuclear Farnesoid X Receptor (FXR) is a ligand-activated transcription factor
that is highly expressed in liver, intestine and kidney.[1] After activation it binds
to a DNA response elements either as monomer or as heterodimer with
Retinoid X Receptor (RXR) and regulates target gene expression. These
genes are involved in bile acid metabolism, lipid and glucose homeostasis.
Endogenous ligands are bile acids (most active: chenodeoxycholic acid) [2, 3,
4] as well as their metabolites and polyunsaturated fatty acids. Intense
research has been made to find potent synthetic ligands since FXR has gained
interest as potential pharmaceutical target [5, 6] due to its role in metabolic
pathways as well as in inflammation and tumorgenesis. Presently, FXR
agonists (e.g. 6-ECDCA, 6α-ethylchenodeoxycholic acid) are in clinical
development for treatment of primary billiary cirrhosis (PBC), non-alcoholic
fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
Besides FXR agonists the development of FXR antagonists has come into
focus in recent years since FXR knockout studies suggested positive impact of
FXR inhibition on insulin sensitivity and atherosclerosis in obese conditions.[7,
8]
Here we present the development of new potential ligands of FXR starting from
a dual PPAR/γ ligand previously developed in our workgroup which showed
slight FXR activity in our transactivation assay. Further improvement in activity
and selectivity at FXR was achieved by introduction of a substituted pyridinol
moiety. Therfore tautomerism occurs between pyridinol/pyridinon and
interestingly it distinguished between agonistic, antagonistic activity or
inactivity of the derivatives. We investigated the different binding modes of the
agonistic and antagonistic derivatives in docking studies and furthermore
optimized the reaction conditions to manage tautomerism.
set of diverse quinoxalinebisarylurea compounds that have been synthesized
and assayed for FLT3 kinase activity. The most promising compounds were
further evaluated in a zebrafish embryo phenotype assay.
References:
1. Tiesmeier, J. et al.: Leukemia Res. 2004, 28: 1069-1074.
2. Smith, C.C. et al.: Nature 2012, 485: 260-263.
3. Levis, M. et al.: Blood 2001, 98: 885-887.
MC.62
References:
1. Forman, B.M. et al.: Cell 1995, 81(5): 1866-1870.
2. Wang, H. et al.: Mol Cell 1999, 3(5): 543-553.
3. Makishima, M. et al.: Science 1999, 284(5418): 1362-1365.
4. Parks, D.J. et al.: Science 1999, 284(5418): 1365-1368.
5. Pellicciari, R. et al.: J. Med. Chem. 2005, 48(17): 5383-5403.
6. Modica, S. et al.: FEBS Lett. 2006, 580(23): 5492-5499.
7. Prawitt, J. et al.: Diabetes 2011, 60(7): 1861-1871.
8. Zhang, Y.: Arterioscler. Thromb. Vasc. Biol. 2006, 26(10): 2316-2321.
MC.61
Revival of an ancient tyrphostine scaffold: Computer-guided
design, synthesis and biological evaluation of quinoxalinebisarylureas as FLT3 inhibitors.
Bensinger, D.; Göring, S.; Schmidt, B.
Clemens Schöpf - Institute of Organic Chemistry and Biochemistry, Technische
Universität Darmstadt, 64287 Darmstadt, Germany.
Activating mutations of FLT3 are present in ~30% of patients with acute
myeloid leukemia (AML) and associated with poor prognosis.[1] Point mutations
in the Tyrosine kinase domain (TKD) are observed as primary mutations or
acquired as secondary mutations in FLT3 with internal tandem duplications
(FLT3-ITD) after treatment with Tyrosine kinase-Inhibitors (TKIs).
Although dozens of potent inhibitors against FLT3-ITD are reported in
literature, activating TKD point mutations especially at residues F691 and
D835 remain the leading cause for clinical resistance and failed studies
showing the steady need of new potent Inhibitors targeting specific resistance
patterns. [2]
Here we report on the discovery and characterization of novel quinoxaline
based FLT3 inhibitors. The known TK family III inhibitor AG1295 represents
one of the first “tyrphostins” showing activitiy in AML disease models but has
not been developed to clinical studies due to low inhibitory strength. We
discuss the pharmacophore features of a diverse set of known inhibitors as
starting point for a new optimization algorithm for type II TKI following
pharmacophore filtering and induced-fit docking of an in silico library to
homology models from different related kinases. This lead to the design of a
175
GPCR (G01-G14)
7 Department
G.01
Lack of evidence for the expression of the histamine H4-receptor
on human monocytes
Werner, K.; Neumann, D.; Seifert, R.
Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625
Hannover, Germany
The histamine H4-receptor (H4R) plays an important role in inflammation and
immune response. The H4R has been unequivocally identified in human
eosinophils [1,2], but the expression on human monocytes has only been
poorly studied so far [2,3]. The H4R is a Gi/o-protein-coupled receptor [1]. In
human myeloid cells, activation of Gi-coupled receptors canonically leads to
activation of phospholipase C (PLC) and subsequent increases in intracellular
calcium [Ca2+]i concentrations [4].
The aim of our present study was to clarify whether the H 4R is expressed and
functionally active on human monocytes. Therefore, we isolated monocytes
from peripheral blood of healthy human volunteers via density gradient
centrifugation and magnetic activated cell sorting. Additionally, we conducted
our experiments with U937 promonocytes. To assess H4R expression at the
RNA level, we performed quantitative real-time PCR. However, we did not
examine protein expression of H4R since serious concerns regarding specificity
of H4R antibodies were raised [5]. In order to investigate functional activity of
the H4R, we determined changes in [Ca2+]i concentrations using the Fura-2AM
method. Monocytes were stimulated with different concentrations of HA and
H4R agonists (5-methylhistamine and 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3[(2-phenylthio)ethyl]guanidine (UR-PI376)), whereas U937 promonocytes were
stimulated with HA alone and in combination with HxR antagonists.
We did not obtain evidence that the H4R is expressed at the RNA level on
human monocytes and U937 promonocytes. hH4R-transfected HEK-293 cells
were used as positive control cells. In human monocytes, adenosine 5’triphosphate (ATP) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)
induced large increases in [Ca2+]i concentrations, but stimulation with HA and
H4R agonists did not cause any increase in [Ca2+]i indicating that the H4R is not
present in monocytes. In U937 promonocytes, HA increased [Ca2+]i. However,
the effect was blocked by the H1R antagonist mepyramine, but not by the H2R
antagonist famotidine and the H4R antagonist 1-[(5-chloro-1H-indol-2yl)carbonyl]-4-methylpiperazine (JNJ7777120) proofing that in these cells
expression of the H1R is exclusively responsible for changes in [Ca2+]i caused
by HA. In conclusion, our study shows that the H4R is not expressed on human
monocytes and U937 promonocytes.
References:
1. Reher, T.M. et al.: Biochem. Pharmacol. 2012, 84(2): 192-203.
2. Seifert, R. et al.: Trends Pharmacol. Sci. 2013, 34(1): 33-58.
3. Dijkstra, D. et al.: J. Allergy Clin. Immunol. 2007, 120(2): 300-307.
4. Dillon, S.B. et al.: Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1988, 55(2): 65-80.
5. Beermann, S. et al.: Naunyn. Schmiedebergs Arch. Pharmacol. 2012, 385(2): 125-135.
of Pediatric Cardiology and Intensive Medicine, University Medical Center
Göttingen, Robert Koch Straße 40, 37075 Göttingen, Germany
8 Department of Cardiology and Pneumology, University Medical Center Göttingen,
Robert-Koch-Staße 40, 37075 Göttingen, Germany;
9 Clinic for Internal Medicine III, University Medical Center Saarland, Kirrberger Straße
100, 66421 Homburg/Saar, Germany
10 University of Kentucky School of Medicine, Department of Physiology, 900 South
Limestone, Lexington, Kentucky 40536, USA
11 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg,
Germany
Maladaptive cardiac hypertrophy leads to heart failure, one of the common
causes for hospitalization in the western world. Chronic β-adrenergic signalling
contributes to the pathogenesis of cardiac hypertrophy, as evidenced by the
therapeutic success of β-adrenoceptor antagonists. The cAMP Regulated
Transcriptional Coactivator 1 (CRTC1) is regulated by increases in cAMP and
calcineurin, as elicited by β-adrenergic signalling, both known to participate in
the development of cardiac hypertrophy.
An elevated protein content of CRTC1 was found in heart tissue of humans
and mice under two conditions of maladaptive hypertrophy by immunoblotting.
The CRTC1 protein content was neither elevated in hearts of humans under
conditions of dilated cardiomyopathy nor in hearts of mice under conditions of
physiological or dilated hypertrophy.
Treatment of cardiomyocytes with the β-adrenoceptor agonist isoprenaline
resulted in the activation i.e. dephosphorylation of CRTC1, as revealed by
immunoblot analysis. This effect was prevented by the addition of the βadrenoceptor antagonist propranolole.
To study the role of CRTC1 in the heart, mice deficient in CRTC1 were
investigated. These mice showed signs of hypertrophy compared to their
wildtype siblings, indicated by the increased ratio of heart weight to tibia length
and increased myocyte size. In addition, they showed reduced CRTC1-mRNA
levels but unchanged mRNA levels of CRTC2 and CRTC3 as measured by
quantitative real-time PCR.
In CRTC1 deficient mice the mRNA and protein level of the anti-hypertrophic
regulator of G-protein Signalling 2 (RGS2) was reduced. RGS4 and RGS5
showed no reduction in mRNA levels. Transient transfection assays of a
luciferase reporter gene under the control of the murine RGS2 promoter (-867
bp to +1 bp) showed that overexpressed CRTC1 stimulated RGS2 promoter
transcriptional activity. Mutation of the CRTC1-binding site, the cAMPresponse element, within the promoter prevented CRTC1-induced transcriptional activity.
Our data show that CRTC1 is elevated under conditions of maladaptive
cardiac hypertrophy and is activated through beta-adrenergic signalling. Also,
CRTC1 stimulates RGS2 gene expression and is thereby presumably
decreasing Gαq-mediated hypertrophic signalling. Thus, the increased CRTC1
protein levels in cardiac hypertrophy might represent a protective mechanism
and the loss of CRTC1 augments cardiac hypertrophy.
G.03
Investigations of biased signaling of the chemokine receptor
CXCR3 with small allosteric modulators
G.02
Influence of increased levels of the transcriptional ca-activator
CRTC1 in cardiac hypertrophy
Morhenn, K.1,2; Quentin, T.1; Schroeder, S.1; Pahl, A.1; Schlossarek, S.2,3;
Carrier, L.2,3; Eschenhagen, T.2,3; Cardinaux, J.-R.4; Lorenz, K.5; Lutz, S.6;
Zimmermann, W.H.6; Steinmetz, M.7; Kaul, A.8; Hasenfuss, G.8; Laufs, U.9;
Guo, Z.10; Oetjen, E.1,3,6,11
1 Department
of Clinical Pharmacology and Toxicology, Cardiovascular Research Center,
University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg,
Germany
2 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck
3 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research
Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246
Hamburg, Germany
4 Center for Psychiatric Neuroscience, Site Cery, 1008 Prilly-Lausanne, Switzerland
5 Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Straße 9,
6 Department of Pharmacology, University Medical Center Göttingen, Robert Koch Straße
40, 37075 Göttingen, Germany
176
Brox, R.; Bernat, V.; Admas, T.H.; Tschammer, N.
Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center,
Friedrich Alexander University, Schuhstraße 19, 91054 Erlangen, Germany
Anomalies in the regulation and response of the chemokine receptor CXCR3,
a rhodopsin-like G protein-coupled receptor that is mainly expressed on
activated T cells, are associated with various pathologies including autoimmune diseases, cancer, vascular diseases and transplant rejection. Therefore
CXCR3 is an attractive pharmacological target [1]. In the contrast to chemokines small synthetic ligands bind to the chemokine receptors at the allosteric
site(s) inside the hydrophobic pocket formed by transmembrane helices. While
allosteric compounds mediate their effects at sites that are topographically
distinct from the orthosteric binding site they gain advantages like probedependence or saturability of effect [2]. Furthermore it is well established that a
given ligand is able to induce signalling bias by the activation of G proteins or
β-arrestin-mediated pathways only. These ligands have a potential clinical
relevance by blocking harmful and maintaining beneficial stimuli [3]. Biased
allosteric modulation of G protein-coupled receptors represents thus an
attractive approach in drug design.
For the investigation of the molecular mechanisms of biased allosteric
modulation we used a small library of novel negative allosteric modulators
(NAMs), which were built on the 8-azaquinzolinone scaffold. Design of these
compounds and subsequent mutagenesis studies were based on predictions
from the CXCR3 homology modelling and docking. To estimate the binding
affinity of the NAMs, we performed an allosteric radioligand (RAMX3) [4]
displacement assay. This assay revealed that some of our novel compounds
bind to a second allosteric binding pocket in CXCR3, distinct from the binding
pocket occupied by RAMX3. In the next step the probe-dependent inhibition of
CXCR3 by novel NAMs was determined. The ability of NAMs to inhibit
CXCL11 and CXCL10 mediated activation of CXCR3 was determined in the
[35S]GTPγS incorporation assay, which monitors the activation of G proteins,
and in the β-arrestin 2 recruitment, which detects the recruitment of β-arrestin
2 to the activated receptor. Within our novel set of compounds we identified
two biased NAMs named BD064 and BD103, which display probe-dependent
inhibition of CXCR3. BD064 preferentially inhibit the CXCL11-mediated
recruitment of β-arrestin 2 to CXCR3 (pKb = 7.46 and αβ =0) over the
activation of G proteins (pKb = 6.05 and αβ = 0.11). BD103 preferentially
inhibits the activation of G proteins (pKb = 6.43 and αβ = 0.14) over the βarrestin 2 recruitment (pKb = 8.73 and αβ = 0.09). In contrast, the inhibition of
CXCL10-mediated activation of CXCR3 no pathway was preferred by
mentioned compounds.
To identify amino acid residue(s) important for the allosteric modulation of
CXCR3 and its interactions with ligands, we mutated ssveral residues within
the putative binding pocket. We identified the residue Lys300 as a molecular
switch that constrains the activation of G proteins. The mutation K300M
releases this molecular switch and increases the CXCR3-mediated cooperativity between CXCL11 and the G protein, but not between CXCL11 and the βarrestin 2.
Overall, we conclude that our novel class of compounds can serve as a useful
tool for the investigation of key receptor-ligand interactions and induction of
ligand-biased signaling. Furthermore the residue Lys300 is an important
anchor amino acid that can be explored for the design of biased allosteric
modulators of CXCR3.
References:
1. Wijtmans, M. et al.: ChemMedChem, 2008, 3: 861-872.
2. Christopoulos, A. et al.: Pharmacol Rev, 2002, 54: 352-354.
3. Rahmeh, R. et al.: PNAS, 2012, 109: 6733 .
4. Bernat, V. et al.: ChemMedChem, 2012, 7: 1481-1489.
G.04
Monovalent cations influence muscarinic M2 receptor spontaneous activity and agonist-dependent signaling
De Min, A.; Mohr, K.
Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, 53121
Bonn, Germany
G protein-coupled receptors (GPCRs) comprise the largest superfamily of cellsurface receptors, with key functions in physiology and drug targeting.
Recently, several class A GPCRs were crystallized and the presence of a
sodium binding site in the middle of the 7 transmembrane helical bundle was
identified in adenosine A2A, adrenergic β1, protease activated receptor 1 and δopioid receptors. The main residue involved in the coordination of the ion is a
highly conserved aspartate (D2.50) in the transmembrane helix 2 [1]. The
muscarinic acetylcholine receptors also belong to the family A of GPCRs, and
there are five different subtypes (M1-M5) expressed in many regions of the
central nervous system and in various peripheral tissues. The structures of M 2
and M3 receptors were crystallized, but no sodium binding pocket was
described so far, even though all the subtypes contain the D2.50 residue.
The aim of this study was to investigate whether sodium chloride (NaCl), had
an influence on M2 receptor activation, both in the presence or absence of an
agonist. The influence of potassium chloride (KCl) was studied for comparison.
Membrane homogenates prepared from CHO cells stably expressing the
human M2 receptor were used in [35S]-GTPγS binding studies to investigate the
influence of increasing concentrations of NaCl and KCl on basal G protein
activity, activation by the endogenous agonist acetylcholine (ACh) and
inhibition by the inverse agonist atropine. Besides, [35S]-GTPγS binding assays
were used to obtain dose-response curves of ACh and the superagonist
iperoxo [2] under control conditions and in the presence of 200 mM of NaCl or
KCl.
Both salts had the same influence on receptor-mediated G-protein activation:
[35S]-GTPγS basal binding and binding induced by the agonist and the inverse
agonist gradually decreased; furthermore, the spontaneous activity of the M2
receptor progressively diminished and was then completely abolished at the
highest salt concentration. Data from the dose-response assays showed that
both salts shifted the inflection point of the curves (pEC 50) to lower values,
indicating that these monovalent cations reduced the agonist potency.
Additionally, the effect of NaCl was stronger than that of KCl in decreasing the
pEC50 values, in the case of both ACh and iperoxo.
Taken together, M2 constitutive activity and agonist signaling were negatively
influenced by KCl and NaCl. The finding that NaCl had a greater effect on the
pEC50 of the compounds might suggest the presence of a sodium binding site
in this receptor.
References:
1. Katritch, V. et al.: Trends Biochem. Sci. 2014, 39: 233-244.
2. Schrage, R. et al.: Br. J. Pharmacol. 2012, 169: 357-370.
G.05
Discovery of a Highly Potent Biased Allosteric Agonist of the
Human Chemokine Receptor CXCR3
Milanos, L.; Brox, R.; Tschammer, N.
Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center,
Friedrich Alexander University, Schuhstraße 19, 91052 Erlangen, Germany
The human chemokine receptor CXCR3 is a rhodopsin-like Gi protein coupled
receptor, found on the surface of T–cells, natural killer cells and astrocytes. Its
endogenous agonists are soluble proteins called chemokines CXCL9, CXCL10
and CXCL11. The activation of CXCR3 upon binding of CXCL9, CXCL10, and
CXCL11 guides the trafficking of leukocytes during infection and inflammation.
Moreover CXCR3 and its chemokines seem to be involved in numerous
inflammatory and autoimmune diseases; therefore is a promising pharmaceutical target. Although the development of CXCR3 allosteric antagonist was the
main focus of medicinal chemists, recently the interest in the allosteric agonists
arose. Previously reported series of the non-peptidic allosteric agonists
VUF10661 and PS372424 namely showed anti-inflammatory activity [1, 2].
Furthermore it was shown that the endogenous agonists CXCL10 and CXCL11
promote wound healing properties [3]. This implies that the development of
small-molecule CXCR3 allosteric agonist might be of particular therapeutic
interest.
We have chosen VUF10661 as a starting point for our library of small-molecule
allosteric agonists. VUF10661 was reported to promote the CXCR3-mediated
β-arrestin2 recruitment with an EC50 of 1 μM and 160% efficacy compared to
CXCL11, and an activation of G proteins with an EC50 of 600 nM and the
efficacy comparable to CXCL11 (measured in the [35S]GTPγS incorporation
assay) [4]. With the aim to improve the potency and bias of VUF10661, we
synthesized a series of analogues based on this non-peptidic agonist. Our
efforts led us to the discovery of a very potent and strongly biased allosteric
partial agonist LMT36. LMT36 activates the CXCR3-mediated recruitment of βarrestin2 with an EC50 of 0.04 nM (25.000 fold increase compared to
VUF10661), an efficacy of 60% (compared to CXCL11) and has no effect on
the CXCR3-mediated activation of G proteins as detected in the [35S]GTPγS
incorporation assay. This unique highly potent and biased allosteric agonist will
be used as a chemical tool to dissect the complex molecular mechanisms
governing the allosteric modulation of CXCR3. The discovery of this allosteric
ligand that induces highly biased signaling expands the ways in which the
function of CXCR3 can be manipulated.
References:
1. Stroke, I.L. et al.: Biochem. Biophys. Res. Commun. 2006, 349: 221–228.
2. O’Boyle, G. et al.: PNAS 2012, 109(12): 4599-4603.
177
3. Davidson, J.M.: The Am. J. Pathol. 2010, 176(4): 1588-1591.
4. Scholten, D.J. et al.: Br. J. Pharmacol. 2012, 166: 898-911.
G.06
Synthesis of photoactivatable probes for labeling of the human
chemokine receptor CXCR3
Admas, T.H.; Bernat, V.; Brox, R.; Tschammer, N.
Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer center,
Friedrich Alexander university, Schuhstraße19, 91052 Erlangen, Germany
The chemokine receptor CXCR3, which belongs to the family of rhodopsin-like
G protein-coupled receptors, is mainly expressed on activated T lymphocytes
[1]. CXCR3 is involved in various autoimmune and inflammatory diseases,
which make this receptor an attractive therapeutic target. Although intense
efforts were dedicated to the development of small molecule antagonists of
CXCR3, the precise binding mode of these molecules remains to be elucidated. One of the most commonly used technique for the determination of
structural elements in the binding pocket of a receptor is photoaffinity labeling
followed by mass spectrometry. This method involves the design and synthesis
of ligands, which carry an appropriate photoactivatable functional group (such
as azide, diazirine or benzophenone). Upon the exposure to the UV light, these
functional groups yield reactive intermediates. Formed reactive intermediates
can than interact with amino acids in the binding pocket and form covalent
bond. The fragments can be later detected by appropriate mass spectrometry
technique.
This project aims at mapping of the allosteric binding site of CXCR3 receptor
with photoaffinity labeling followed by mass spectrometry. For this purpose
compound 1, a negative allosteric CXCR3 modulator with an azide functional
group was designed and synthesized first. As determined in a radioligand
RAMX3 [2] displacement assay, compound 1 and its non-biotinylated analogue
had comparable binding affinities of 0.8 nM and 9.8 nM respectively. This
assay indicated that the biotinylated group and the PEG linker are well
tolerated by CXCR3. Unfortunately, the azide derivative 1 was unstable upon
the UV light exposure at 254 nm, yielding undesired short fragments of the
compound. Hence we designed and synthesized another photoaffinity probe
that incorporated diazirine as a photoactivatable moiety and well characterized
8-azaquinazoline scaffold. The small size of diazirines and the possibility to
irradiate the probe at a longer UV wavelength (365 nm) makes diazirines
preferable choice over other photoactivatable moieties.
In a similar fashion to compound 1, the newly synthesized diazirine based
photoaffinity probe 2 and its non-biotinylated analogue have shown nanomolar
affinity towards CXCR3 in the RAMX3 displacement assay. The tendency of
the photoactivatable probe 2 to form the corresponding reactive carbene
intermediate upon UV irradiation was assessed by a phenomenon called
carbene trapping. Unlike compound 1, the photoaffinity probe 2 provides the
desired reactive intermediate on UV exposure. Studies on the irreversible
binding of the photolabeled ligand with the receptor and identification of the
residues, which directly interact with the ligand, are ongoing.
G.07
Synthesis of Quinazoline Derivatives as Adenosine A3 Receptor
Antagonists
Briel, D.1; Schäke, F.1; Schwan, G.1; Müller, C.E.2; Vielmuth, C.2; Lang, M.1
University of Leipzig - Institute of Pharmacy, Bruederstraße 34, 04103 Leipzig, Germany
Friedrich-Wilhelms-Universität Bonn, Pharmazeutisches Institut, An der
Immenburg 4, 53121 Bonn, Germany
1
2 Rheinische
Diversely aliphatic and aromatic-substituted quinazoline derivatives were
synthesized to evaluate their structure-activity relationships on the four
subtypes (A1, A2A, A2B and A3 [1]) of G-protein-coupled adenosine receptors
(ADOR).
The ADORA3 is a potential drug target [1,2], antagonists have been developed
and pharmacologically evaluated [3,4]. Apparently this receptor is involved in
the progression of several inflammatory diseases, as for instance plasma
extravasation [4], asthma bronchiale, chronic obstructive pulmonary disease or
idiopathic pulmonary fibrosis [5]. Moreover, ADORA3 antagonists may be
useful in the treatment of cancer to support chemotherapy [6]. The A3 receptor
is coupled to Gi proteins mediating inhibition of adenylate cyclase [7]. The
development of selective antagonists is required to achieve selective pharmacological effects. In the present study two different types of quinazoline
derivatives were investigated. For type I different aromatic amines were
coupled by nucleophilic substitution with a 2,4-chloroquinazoline bearing
hydrogen- or methoxy in position 6 and 7. In order to obtain type II derivatives
different alkyl- and aryl substituents were attached to a
6,7-dimethoxyquinazoline core. In a second step the hydroxyl function in
position 4 could be transformed into a chloride. The obtained quinazoline
derivatives were assayed for their potency and selectivity on the four ADOR
subtypes in vitro. Compound 1 was found to be potent at human A3: Ki = 30.6 ±
7.7 nM and showed high selectivity.
Thanks are due to J. Ortwein for HPLC analysis and Dr. L. Hennig for recording and
analysis of NMR data.
References:
1. Fredholm, B.B. et al.: Pharmacol. Rev. 2001, 53: 527–552.
2. Gessi, S. et al.: Pharmacology & Therapeutics 2008, 117:123–140.
3. Lee, J. et al.: Am. J. Pathol. 2013, 183: 1488-1497.
4. Mikus, E.G. et al.: Eur. J. Pharmacol. 2012, 699(1-3): 62–66.
5. Della Latta, V. et al.: Pharm. Res. 2013, 76: 182–189.
6. Merighi, S. et al.: Pharmacology & Therapeutics 2003, 100: 31–48.
7. Birnbaumer, L.: Biochim. Biophys. Acta 2007, 1768(4): 772–93.
References:
1. Baggiolini, M.: Nature. 1998, 392(6676): 565-568.
2. Bernat, V. et al.: ChemMedChem. 2010, 7(8): 1481-1489.
178
3 Pharmaceutical
G.08
Structural comparison of the allosteric binding pocket in muscarinic acetylcholine receptors.
Bermudez, M.; Wolber, G.
Institute for Pharmacy; Free University Berlin, Königin-Luise-Straße 2+4, 14195 Berlin,
Germany
G-protein coupled receptors (GPCRs) trigger multiple signal-switching
mechanisms, not only G-protein activation but also binding of ß-arrestin
proteins and activation of kinases [1]. Despite recent advances in GPCR x-ray
crystallography the understanding of conformational changes resulting in these
activations is still incomplete and remains a major challenge for the design of
specific GPCR modulating drugs. Due to the highly conserved orthosteric
binding pocket (figure 1) other binding sites are highly interesting for the design
of subtype selective drugs. The recently published crystal structures of the M2
and the M3 muscarinic acetylcholine receptor (PDB: 3UON [2], 4DAJ [3],
4MQS [4] and 4MQT [4]) provides the possibility to rationalize and understand
the binding of ligands to muscarinic acetylcholine receptors and to search for
novel binding subsites. We present the results of homology modeling for all 5
muscarinic acetylcholine receptor subtypes based on the available crystal
structures. The obtained 3D-models represent active and inactive receptor
conformations and indicate differences in their allosteric binding pockets.
These differences give new mechanistic insights in terms of subtype selectivity
and allosteric receptor modulation.
Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn,
Germany
4 CNS Research, UCB Pharma sprl, 1420 Braine l’Alleud, Belgium
GPR17 is an orphan G protein coupled receptor (GPCR), which is predominantly expressed in the oligodendrocyte-lineage cells of the central nervous
system. [1] Recently, inhibition of this receptor has been proposed as a novel
and promising therapeutic strategy for the treatment of demyelinating diseases
such as multiple sclerosis. [1, 2]
In spite of its attractivity as novel target to foster repair of demyelinated
neurons, the nature of its true endogenous ligands is still under debate. [3]
While uracil nucleotides and cysteinyl-leukotrienes have been proposed as
endogenous activators, [4] a number of independent studies did not confirm the
original deorphaning report. [2-3, 5-7] We further investigated whether GPR17
represents the elusive orphan receptor indeed responding to two ligand
classes or whether it may still require pairing with a yet unknown endogenous
modulator. To this end we employed a number of different functional assay
platforms based on label-free dynamic mass redistribution and label-free
bioimpedance, but also classical assays capturing defined intracellular events
such as Gαi, Gαq, and Gαs activation, β- arrestin recruitment, activation of
extracellular signal-regulated kinases 1 and 2, and binding of [35S]GTPγS to
Gα proteins. Notably, we were unable to demonstrate activation of GPR17 by
any of the purported endogenous ligands. Yet, robust activation was obtained
across all assays when the receptor was stimulated with the small molecule
MDL29,951.
Based on these data and the current literature, which does not support the
notion that GPR17 has been successfully matched with its correct endogenous
partners, we propose that GPR17 must still be regarded as orphan, awaiting
the identification of the true native ligands.
References:
1. Chen, Y et al.: Nat. Neurosci. 2009, 12(11): 1398–1406.
2. Hennen, S. et al.: Sci. Signal. 2013, 6(298):ra93.
3. Qi, A.D.; Harden, T. K.: Nicholas, R. A.: J. Pharmacol. Exp. Ther. 2013, 347(1):38-46.
4. Ciana, P. et al.: EMBO J. 2006, 25(19):4615-4627.
5. Heise, C.E. et al.: J. Biol. Chem. 2000, 275(39):30531-30536.
6. Maekawa, A. et al.: Proc. Natl. Acad. Sci. USA. 2009, 106(28):11685-11690.
7. Benned-Jensen, T.: Rosenkilde, M. M.: Br. J. Pharmacol. 2010, 159(5):1092-1105.
G.11
Molecular insights into the high constitutive activity of the
human histamine H4 receptor
Figure 1: Superimposition of the crystal structures of M2 AChR (yellow, first
number) and M3 AChR (blue, second number) with key residues for ligand
binding.
Wifling, D.1; Löffel, K.1; Nordemann, U.1; Bernhardt, G.1; Dove, S.1; Seifert, R.2;
Buschauer, A.1
1
2
References:
1. Jacoby, E., et al.: Chemmedchem, 2006, 1(8):760-782.
2. Haga, K. et al.: Nature 2012, 482(7386): 547-551.
3. Kruse, A. et al.: Nature 2012, 482(7386): 552-556.
4. Kruse, A. et al.: Nature 2013, 504(7478): 101–106.
G.09
G.10
GPR17: the elusive dual uracil nucleotide/cysteinyl-leukotriene
receptor or still an orphan?
Simon, K.1; Hennen, S.1; Merten, N.1; Schröder, R.1; Peters, L.1; Schrage, R.2;
Vermeiren, C.4; Mohr, K.4; Müller, C.E.3; Gillard, M.4; Gomeza, J.1; Kostenis,
E.1
1 Institute
for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn,
Germany
2 Pharmacology and Toxicology, University of Bonn, Gerhard-Domagk 3, 53121 Bonn,
Germany
Institut für Pharmazie, University of Regensburg, D-93040 Regensburg, Germany
Institut für Pharmakologie, Hannover Medical School, D-30625 Hannover, Germany
The histamine H4R belongs to class A of G-protein coupled receptors and is
considered as a promising drug target for the treatment of inflammatory
diseases like allergic asthma [1]. The validation of the H4R in translational
animal models is hampered by species-dependent differences. There are
considerable discrepancies in terms of ligand efficacies, potencies and
affinities, in particular, between the hH4R (human) and the rodent orthologs,
i.e. the mH4R (mouse) and rH4R (rat) [2,3].
In contrast to the mouse and rat H4R, the human H4R shows a high degree of
constitutive activity. Aiming at more detailed insights into the molecular
determinants of ortholog-dependent ligand-receptor interactions, we generated
and expressed (Sf9 cells) a series of H4R mutants to determine radioligand
binding and functional data ([35S]GTPγS assay) [2]. Apart from F169, which
was identified by Lim et al. as a key amino acid for distinct ligand binding
affinities at H4R orthologs [5], we mutated, based on molecular modeling
studies, S179, S330 and R341 to the corresponding amino acids of the rodent
H4Rs, resulting in hH4R F169V, hH4R S179M, hH4R S179A, hH4R
F169V+S179M, hH4R F169V+S179A, hH4R S330R and hH4R R341S [2].
Besides, the reciprocal mH4R mutants, mH4R V171F and mH4R
V171F+M181S served as control [2]. Moreover, to study the role of the
F168/F169 motif, which is also found in, e.g., the β2AR, H3R and the M2R, we
expressed the hH4R F168A mutant in Sf9 cells.
Whereas changes in ligand affinity and potency were only minor, the constitutive activity of the hH4R-F169V and the double mutants was significantly
reduced compared to the wild-type hH4R [2]. By contrast, an exchange of
S179 by M or A alone did not significantly affect constitutive activity. Strikingly,
179
the double mutants were comparable to the mH4R and to the rH4R, which are
devoid of constitutive activity. The inverse agonism of thioperamide decreased
from the hH4R via the hH4R F169V mutant to the hH4R F169V+S179M and
hH4R F169V+S179A double mutants, respectively [2]. The data for the hH4RF168A mutant revealed a major contribution of F168 to ligand binding with a
concomitant, up to over 100-fold decrease in ligand potencies and a complete
loss of constitutive activity, compared to the wild-type hH4R. Thioperamide
acted as a neutral antagonist and JNJ7777120 turned to partial agonism.
The results suggest that, in particular, F168 and F169 alone or F169 in concert
with S179 favor the switch from the inactive to the active state of the human
H4R.
Acknowledgments: This work was supported by the Graduate Training Programme
(Graduiertenkolleg) GRK 760 of the DFG and by the European Cooperation in Science
and Technology, COST Action BM0806.
References:
1. de Esch, I.J. et al.: Trends Pharmacol. Sci. 2005, 26: 462-469.
2. Wifling, D. et al.: Br. J. Pharmacol. 2014, in press, doi: 10.1111/bph.12801.
3. Schnell, D. et al.: Naunyn. Schmiedebergs Arch. Pharmacol. 2011, 383: 457-470.
4. Nordemann, U. et al.: PLoS ONE 2013, 8(9): e73961.
5. Lim, H.D. et al.: J. Pharmacol. Exp. Ther. 2008, 327: 88-96.
G.12
Label-free dynamic mass redistribution analysis of 5-oxo-ETE
receptor signaling via Gαi and Gβγ signaling routes
Büllesbach, K.1; Bautista, O.2; Blättermann, S. 1; Schröder, R.1; Weaver, C.D.3;
Guetschow, M.2; Kostenis, E.1
1 Molecular-,
Cellular- and Pharmacobiology Section, Institute of Pharmaceutical Biology,
University of Bonn, Bonn, Germany
2 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn,
Germany
3 Vanderbilt Institute of Chemical Biology, Department of Pharmacology, Vanderbilt
University, Nashville, Tennessee, United States
Dynamic mass redistribution (DMR) is a cutting edge label-free optical
biosensor technology to capture cellular responses in a holistic manner upon
exposure to extra- and intracellular stimuli [1]. This method has proven to be
particularly valuable for dissecting complex signaling behaviors in living cells
triggered via the family of G protein-coupled receptors, the largest family of
membrane signaling proteins. GPCRs respond to a wide array of stimuli and
serve to translate extracellular information to the inside of the cell via coupling
to heterotrimeric αβγ G proteins. G proteins are classified into four major
families, Gαq/11, Gαs, Gαi/o, and Gα12/13, and both Gα and free Gβγ subunit
complexes regulate diverse intracellular effectors. Most GPCRs couple to more
than one G protein heterotrimer, a feature referred to as pleiotropic signaling.
Therefore compounds that selectively interfere with individual G protein
heterotrimers are of great value to decipher contribution of defined signaling
branches to complex signaling networks. Among such compounds are
pertussis toxin (PTX), an invaluable probe to analyze Gαi-mediated cell
responses and FR900359 (UBO-QIC) as well as YM-254890 [2], two cyclic
depsipeptides that specifically silence signaling of Gαq family members. PTX
and FR900359 interdict signaling of entire heterotrimers, i.e. abrogate both Gα
and Gβγ–mediated cellular signaling.
We have recently identified a small molecule, Gue1654, that specifically
dampens Gβγ but not Gαi signaling of the chemoattractant receptor OXE-R,
thereby uncovering a novel mechanism for selective inhibition of Gβγ signaling
downstream of specific receptors [3]. Herein we wanted to take advantage of
this novel biased ligand to answer the following questions:
1) Do holistic DMR recordings visualize the contribution of Gβγ signaling?
2) To which extent do Gα and Gβγ effectors impact on complex whole cell
activation profiles?
To this end, signaling of OXE-R forcibly expressed in human embryonic kidney
(HEK293) cells and primary human neutrophils was investigated in cells pretreated with a) receptor-specific, βγ-biased inhibitor Gue1654, b) small
molecule Gβγ inhibitor Gallein [4], c) Gαiβγ inhibitor PTX, and d) novel indole
OXE-R antagonists recently disclosed in ref. [5] using DMR technology along
with classical endpoint assays.
Results show that 5-oxo-ETE-mediated DMR responses were entirely
abolished in cells pre-treated with PTX, partly diminished in cells pre-treated
with Gβγ inhibitor Gallein but essentially unaffected by OXE-R specific, Gβγ
biased Gue1654. However, when intracellular inositolmonophosphate (IP1)
accumulation was recorded to assess receptor activity both Gue1654 and
Gallein completely blunted the receptor-mediated IP1 response. The resulting
180
disparity between these assay results leads us to posit the intriguing concept
to target Gβγ-effector interaction in an effector- and receptor-specific way
potentially offering unprecedented precision in the control of cellular signaling
originating from cell surface GPCRs.
Acknowledgements: K.B. is a member of the DFG-funded Research Training Group RTG
1873
References:
1. Schröder, R. et al.: Nat. Protoc. 2011, 6(11): 1748-1760.
2. Nishimura, A. et al.: PNAS 2010, 107(31): 13666-13671.
3. Blättermann, S. et al.: Nat. Chem. Biol. 2012, 8(7): 631-638.
4. Lehmann, D.M. et al.: Mol. Pharmacol. 2008, 73(2): 410-418.
5. Gore, V. et al.: J. Med. Chem. 2014, 57(2): 364-377.
G.13
Comparison of Label-Free Methods with Conventional Assays for
the Functional Characterization of GPCRs: The Human Histamine
H1 Receptor as an Example
Lieb, S.1; Bernhardt, G.1; Wegener, J.2; Buschauer, A.1
1 Institute
of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany
of Institute of Analytical Chemistry, Chemo- and Biosensors, University of
Regensburg, D-93040 Regensburg, Germany
2 Institute
Optical- and impedance-based assays, as label-free techniques, exploiting a
holistic readout, enable the non-invasive study of cellular responses, e.g., upon
binding of a ligand to a transmembrane receptor. For instance, dynamic mass
redistribution (DMR) utilizes the refraction index as readout, whereas electric
cell-substrate impedance sensing (ECIS) records alterations of the impedance
signal caused by changes in the cell morphology [1,2]. Signals are recorded in
real time. In principle both holistic approaches are applicable to the study of Gprotein coupled receptors (GPRs), and the construction of concentrationresponse curves is possible, regardless of the involved signalling cascade(s).
With respect to deconvolution of these complex signals, we used the aforementioned label-free methods and compared the results with those obtained
from established functional assays, namely mobilization of intracellular
calcium, GTPase activity and luciferase gene reporter technology. Here we
present the results of investigation on the human histamine H1 receptor (hH1R),
a Gαq protein coupled GPCR, the stimulation of which results in phospholipase
C (PLC) activation, an increase in inositol trisphosphate (IP3) and subsequent
mobilization of intracellular Ca2+.
The endogenous ligand, histamine, and the H1R agonist KUM530 (N-[2-(1Himidazol-4-yl)ethyl]-2-[2-(3-bromophenyl)-1H-imidazol-4-yl]ethan-1-amine) [3]
as well as the selective hH1R antagonists mepyramine, diphenhydramine and
azelastine were studied on U-373 MG cells, which constitutively express the
hH1R and on genetically engineered HEK293T cells, which stably express the
hH1R and the firefly luciferase under the control of a cyclic AMP responsive
element (CRE). Generally, both agonists showed a 10 to 100-fold higher
sensitivity when studied with label-free methods compared to conventional
assays (Fura-2/AM calcium, GTPase, luciferase assay). The pEC50 values
determined with both label-free methods were in good agreement. The
investigated antagonists revealed comparable pKB values in all functional
assays.
As method-dependent differences became obvious from the investigations of
H1R agonists, it may be speculated that, in addition to the calcium signal, other
cellular signalling mechanisms contribute to the holistic readout. The dissection
of these pathways with appropriate molecular tools will be indispensable for
deconvolution.
Acknowledgments: A PhD fellowship by the Hanns-Seidel-Stiftung to S. L. is gratefully
acknowledged. The authors thank N. Kagermeier, Dr. A. Strasser and Dr. R. Robelek for
fruitful co-operation, as well as Dr. H.-P. Steffens (Perkin-Elmer) for kindly providing the
EnSpire multimode plate reader.
References:
1. Scott, C.W.; Peters, M.F.: Drug Discovery Today 2010, 15(17-18): 704-716.
2. Fang, Y., et al.: Comb. Chem. High Throughput Screening. 2008, 11(5): 357-369.
3. Strasser, A., et al.: Mol. Pharmacol. 2009, 75: 454-465.
G.14
Adenosine A2A receptors heterodimerize with A2B receptors
Hinz, S.1; Seibt, F.B.1; Schiedel, C.A.1; Franco, R.2; Müller, C.E.1
1 PharmaCenter
Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of
Bonn, 53121 Bonn, Germany
2 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of
Barcelona, Barcelona, Spain
It is now well accepted that G protein-coupled receptors are able to form
dimers or oligomers in intact cells, which may consist of identical receptor
proteins (“homomeric”) or of different receptors (“heteromeric”). Because of the
unique pharmacological properties of these complexes, in particular in the
case of heteromers, they represent novel targets for drug development.
Different biophysical techniques such as resonance energy transfer (bioluminescence or fluorescence energy transfer, BRET or FRET) or fluorescence
complementation techniques have been used to identify these complexes in
living cells. It is of great interest to analyze their structure, including different
possible interfaces that might be involved in receptor dimerization/oligomerization.[1] In the present study we examined potential heteromer
formation between the adenosine A2A and A2B receptor subtypes, which play
important (patho)physiological roles and are co-expressed in various cell types
and tissues, e.g. in heart.[2] Radioligand receptor-binding assays at membranes
of stably co-transfected CHO cells were performed to examine pharmacological properties of potential heteromers. Receptor function was investigated by
measuring agonist-induced cAMP production of the Gs-coupled receptors.
Furthermore, we used confocal laser scanning microscopy to show that the
ECFP and EYFP tagged receptor subtypes were co-localized at the plasma
membrane and in the ER/Golgi apparatus in stably co-transfected CHO cells.
Moreover with FRET studies in transiently transfected CHO cells expressing
both, A2A and A2B receptor subtypes, we were able to show that A2A/A2B
receptor heteromers were formed. Further studies using a C-terminal deletion
mutant of the A2A receptor showed that the C-terminus only plays a negligible
role for interaction of the receptors within heterodi- or oligomers. We report
here for the first time, that human A2A and A2BARs form heterodimers with
altered receptor pharmacology.
References:
1. Ferré, S. et al.: Pharmacol. Rev. 2014, 66(2): 413-434.
2. Zhan, E. et al.: Am. J. Physiol. Heart Circ. Physiol. 2011, 301(3): H1183-1189.
181
NATURAL COMPOUNDS (NC01-NC21)
NC.01
Discovery of new protease inhibitors from nature
Oli, S.1; Grün, J.1; Abdelmohsen, U.R.2; de Sousa, L.R.F.3, Wu, H.1, Hentschel,
U.2, Efferth, T.1, Schirmeister, T.1
1 Institute
of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099
Mainz, Germany
2 Julius-von-Sachs-Institute for Biological Sciences, University of Würzburg, Julius-vonSachs-Platz 3,Würzburg 97082, Germany
3 Departamento de Química, Universidade Federal de São Carlos, Rod. Washington Luís,
Km 235, 13565-905 São Carlos, SP, Brazil
Proteases catalyze the breakdown of proteins via catalytic hydrolysis of
peptide bonds [1]. Malfunction in the control of protease activity leads to
undesired and unregulated proteolysis which causes many diseases.
Therefore, inhibitors of proteases have the potential to provide successful
therapeutics for a wide range of diseases [2,3].
The abundance of compounds with different chemical functional groups has
renewed the interest in nature as a source for new leads for the next generation of drugs.
In this study, bioactive compounds with protease inhibiting properties were
discovered from sponges, plants and sponge-associated bacteria following
bioactivity-guided fractionation. For detection of protease inhibiting properties
we used fluorometric enzyme assays and microscale thermophoresis.
The bioactivity-guided fractionation of the crude extracts and the isolation,
identification and structural elucidation of novel and biologically active
secondary metabolites from marine sponge-associated bacteria, sponge
biomass and plants yielded e.g. plakortide E [4] from the sponge Plakortis
halichondroides and flavonoids from the Brazil plant Byrsonima coccolobifolia
Kunth.[5] as new protease inhibitors.
References:
1. Turk, B . et al.: Nat. Rev. Drug Discov. 2000, 6 (5): 785–799.
2. Powers, J.C . et al.: Chem. Rev. 2002, 102: 4639–4750.
3. Rawlings, N.D . et al.: Biochemie 2008, 90: 243–259.
4. Oli, S. et al.: Mar. Drugs. 2014, 12: 2614-2622.
5. Lorena R.F.de Sousa . et al.: J. Nat. Prod. 2014, 77 (2): 392–396.
NC.02
The war of invention: how the necessities of World War I promoted chemical drug development
Helmstädter, A.1,2; Siebenand, S.2
1
2
Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main
Govi Verlag Pharmazeutischer Verlag GmbH, Eschborn, Germany
Political crisis and the outbreak of World War I a hundred years ago let to
severe supply shortfalls in almost every economic area, including pharmacy.
So Germany soon fell into short supply of natural products imported from
abroad, including most alkaloid containing plant materials. So to give only one
example, Merck’s atropine production stopped in October 1916, as Hyoscyamus species usually imported from Africa were no longer available. As
chemical synthesis had already made some progress in the early 20th century,
it was obvious to look for synthetic routes based on domestic materials to
substitute natural products. Several of these substitution products and
procedures were abandoned after the war, but, as shown by Guy Hartcup [1],
there are also examples where necessities of war katalysed developments
also highly useful in times of peace. This study is intended to explore, if and
how far this approach also holds true for pharmaceutical substances.
So the well-known antipsoriatic agent Cignolin is a WW I development. It was
synthesized to substitute Chrysarobin, a compound derived from the South
American plant Andira araroba which was not available in times of war.
Cignolin was developed by the German Dermatologist Eugen Galewski (18641935) and the Bayer company in 1916 and turned out to have several
advantages over the mother compound.
In the early 20th century, extracts from the North-American medicinal plant
Lobelia inflata were frequently used as antiasthmatic agent and breathing
stimulant. Shortage of plant material promoted research regarding isolation of
the active ingredients and developing a synthetic procedure. Heinrich Wieland
(1877-1957) and two of his PhD students investigated possibilities of a lobelin
182
total synthesis but succeeded not before 1927. However, a technically suitable
synthesis procedure was not developed before 1937 when Boehringer started
to market synthetic lobelin right before World War II. The product, which was
still largely used as a medicine in the German army soon became the
company’s blockbuster and was marketed until the year 1980, being recommended as an agent for smoking cessation in the last decades.
One of the most important natural products unavailable in wartime was quinine
and the need for quinine substitutes clearly stimulated the active search for
synthetic antimalaria medicines which, however, did not succeed before the
1920s, when the Bayer company launched plasmoquine followed by chloroquine in 1934. In his study on the history of synthetic antimalarials, David
Greenwood stated: “During the war, the shortage of quinine impaired the
efficiency of the German forces especially in the East Africa Campaign. It is
doubtful if any of the synthetic antimalarials in use have been developed had
not Germany been deprived of all sources of quinine during the First World
War” [2].
References:
1. Hartcup, G.: The War of Invention. Scientific Developments, 1914-18, London 1988.
2. Greenwood, D.: J. Antimicrob. Chemother. 1995, 36: 857-872.
NC.03
Tripterygium wilfordii: how a Taiwanese medicinal plant found its
way to the West
Helmstädter, A.
Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main
Tripterygium wilfordii Hook. f. is a traditional medicinal plant originally found in
Taiwan. It has been known in Europe since the late 1850s but has not been
widely investigated in the West before the 1970’s. In the last few decades
increasing efforts have been made to elucidate its value as an antiinflammatory and immunosuppressive agent, in particular for the treatment of
rheumatoid arthritis. The plant is said to be already mentioned in the Chinese
herbal “Dian Nan Ben Cao” written in the 15th century by Mao Lan (13971476), and the ‘Compendium of Materia Medica” compiled by Li Shi-Zheu in
1578. The species later called Tripterygium wilfordii (German: “Wilfords
Dreifügelfrucht”) has been collected in Taiwan in June 1858 and the name
refers on the one hand to the the unusual three-winged shape of the fruits, on
the other hand to the man who had harvested the plant. “Pterygium” is said to
be the diminutive of “pteryx”, the Greek word for “wing”. The specific epithet
honours Charles Wilford, a botanist working for Kew Gardens, London, from
1857 to 1859. He was sent as botanical collector on the “splendid steam yacht
The Emperor” to India “for making botanical researches among the numerous
islands of the Japanese territories” and embarked May 2nd, 1857 to Hong
Kong [1]. The later “Tripterygium wilfordii” was collected in June 1858 and
labelled as has been found “on banks of the river Sanar, Formosa”. Wilford did
not care a lot about the instructions issued by his employer, according to which
the medicinal use of indigenous plants should be carefully monitored and
reported. This might be one of the reasons why the medicinal virtues of T.
wilfordii remained largely unknown in Western science until the end of the 20th
century. Early interest focused on the insecticidal use of T. wilfordii root
powder in the 1930’s. Around 1940 as well, first reports about the chemical
nature of active ingredients appeared in the literature (Schechter and Haller
1942), systematic investigations were undertaken in the early 1950’s. Most of
the work has been done by Morton Beroza (1917-2011), who did his PhD
thesis on the subject and eventually became a member of the Agricultural
Research Service's Science Hall of Fame. Further interest was triggered by the
cultural changes taking place in China after the World War II leading – among
many other developments – to an integration of Western style medicine into
Chinese healthcare. Western-trained and oriented physicians influenced
medical practice even in rural areas and vice versa, became familiar with local
traditions. As reports on beneficial, but also toxic effects of T. wilfordii were
manifold, trials in a number of autoimmune and inflammatory diseases were
organized and rapidly performed which led to a re-discovery of the plant’s
medicinal virtues. In 1982, already experiences with more than 2000 patients
with rheumatoid arthritis were reported [2]. Triptolide is now believed to be
responsible for most of the biological activities of T. wilfordii extracts [3] and
also serves as lead compound in drug development [4,5].
References:
1. Bretschneider, E.V.: History of European Botanical Discoveries in China. St. Petersburg
1898, pp. 539-544.
2. Lipsky, E., Tao, X.L.: Sem. Arthr. Rheum. 1997, 26: 713-723.
3. Brinker, A.M. et al.: Phytochemistry 2007, 68: 732-766.
4. Zhou, Z.L. et al.: Nat. Prod. Rep. 2012, 29: 477-475.
5. Werz, O.: Pharm. Ztg. 2012, 157: 404-415.
NC.04
Effectiveness of bulb extracts of Allium species on some
selected plant pathogenic fungi
Samadi, S.; Keusgen, M.
Institute of Pharmaceutical Chemistry, Philipps-Universität Marburg D-35037, Germany
The antimicrobial activity of Allium species has long been recognized, with
allicin, other thiosulfinates, and their transformation products having antimicrobial activity [1]. The sulfur chemistry of Allium species located in South West
and Middle Asia is much more complex and diverse than the chemistry of
those species which were traditionally used as vegetable, spice or medicine in
the Western World. Allium volatile compounds of the species from Asia seem
to be an excellent source for new sulphur compounds and aroma constituents.
This variety of sulphur compounds should also lead extracts with unknown
biological activities [2]. Fungal infections have increased over the past few
decades. There has been an intensive search for new drugs more effective
and less toxic than those already in use [3]. In vitro susceptibility testing of
filamentous fungi is becoming increasingly important because of the frequency
and diversity of infections caused by them [4].
Antifungal activities (MIC) of A. ampeloprasum L., A. atroviolaceum Boiss.,
A. cepa L., A. cepa L. var. aggregatum G. Don, A. darwasicum Regel,
A. hollandicum R.M.Fritsch, A. jesdianum Boiss. & Buhse, A. jesdianum Boiss.
& Buhse subsp. angustitepalum (Wendelbo) F.O.Khass. & R.M.Fritsch,
A. karataviense Regel, A. macleanii Baker, A. moly L., A. nevskianum Vved.,
A. oschaninii B.Fedtsch., A. rosenorum R.M.Fritsch, A. rotundum L.,
A. sativum L., A. scorzonerifolium Redouté, A. stipitatum Regel, A. talassicum
Regel and miconazole (positive control) on Aspergillus flavus, A. niger,
Penicillium digitatum, P. italicum and Mucor hiemalis were investigated.
Dilution series of ethyl acetate extracts obtained from Allium bulbs were tested
on all the above mentioned fungi using PDA micro-dilution susceptibility testing
method, disk diffusion method and double-dish chamber.
Extracts of A. stipitatum showed the highest antimicrobial effect against all the
tested fungi (MIC ≥ 0.53g/ml; related to the weight of the fresh bulbs) followed
by A. sativum (MIC ≥ 0,54g/ml), A. ampeloprasum (MIC ≥ 0,85g/ml) and then
A. karataviense (MIC ≥ 1,53g/ml). The MIC of miconazole as a control was
≥0.27mg/ml. From the fungal point of view, P. italicum showed the highest
susceptibility, while M. hiemalis and A. flavus demonstrated more resistancy
towards Allium extracts and miconazole.
The results indicate that extractions of Allium spp. have antifungal activity and
might be promising, at least, in ‘biological’ treatment of fungal-associated plant
diseases. Raw extracts were investigated. Identification of active substances is
ongoing.
1 Institute
of Pharmaceutical Biology, Goethe-University Frankfurt, Biocenter, Max-vonLaue-Str. 9, 60438 Frankfurt/Main, Germany
2 Department of Pharmacy, Center for Drug Research, Pharmaceutical Biology, University
of Munich, Butenandtstr. 5-13, 81377 Munich, Germany
3 Preclinical Research, Dr. Willmar Schwabe Pharmaceuticals, Willmar-Schwabe-Str. 4,
76227 Karlsruhe, Germany
4 Institute of Pharmacology and Toxicology, Division of Nephropharmacology, GoetheUniversity Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany
Haemanthus coccineus (Amaryllidaceae) extracts (HCEs) have been used in
traditional African medicine against febrile colds and asthma. The main
ingredient of HCE, the non-basic alkaloid narciclasine, was recently reported to
induce apoptosis in tumor cell lines [1]. Beyond this anti-cancer action, we
hypothesized that HCE and narciclasine could exhibit an anti-inflammatory
potential.
Dried bulbs of H. coccineus were extracted using 60 % (w/w) ethanol. The
ethanol was largely removed and the remaining solution was partitioned with
ethyl acetate. The organic phase was separated and dried (DER 50:1). The
resulting HCE contained 2.2 % narciclasine. In vitro, HCE (3 ng/ml to 10 μg/ml)
concentration-dependently inhibited the proliferation of lymphocytes and the
synthesis of pro-inflammatory cytokines (TNF-α, IL-6, IL-1 β) in murine
macrophages without inducing cytotoxicity. Moreover, HCE decreased the
TNF-α-induced adhesion of leukocytes to human endothelial cells (ECs) and
the surface expression of EC adhesion molecules (ICAM-1, VCAM-1, Eselectin) without affecting EC viability. Extract fractions containing basic
alkaloids did not display any activity, whereas the non-basic main alkaloid
narciclasine (1 nM to 10 µM) clearly reduced adhesion molecule expression.
HCE as well as narciclasine attenuated the TNF-α-triggered expression of NFκB-dependent genes (reporter gene assay) without influencing NF-κB DNAbinding activity (gel shift assay), IκB-α degradation (Western blot), or p65
nuclear translocation (microscopy). In vivo, HCE (450 mg/kg, orally) was found
to clearly reduce edema formation and leukocyte infiltration in a dermal ear
edema model by croton oil or arachidonic acid (AA). Also in a renal injury
model we could demonstrate that HCE (50 µg/animal, i.p.) strongly attenuates
leukocyte infiltration and cytokine expression.
In conclusion, our study highlights that the use of HCE/narciclasine could
represent a novel interesting anti-inflammatory approach.
Acknowledgments: HCE and narciclasine were kindly provided by Dr. Willmar Schwabe
GmbH & Co. KG. This work was supported by the German Research Foundation (DFG,
SFB 815, project A5, SFB 1039, project B2) to L.S. and by GRK1172 (cooperation
contract with Merck Serono) to L.T.H.
References:
1. Ingrassia, L. et al.: J. Transl Oncol. 2008, 1(1): 1-13.
NC.06
Antischistosomal activity of derivatives of caffeic acid bornyl
ester isolated from Valeriana wallichii rhizomes
Glaser, J.1*; Schurigt, U.2*; Suzuki, B.3, Caffrey, C.R.3#; Holzgrabe, U.1#
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074
Würzburg, Germany
2 Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str.
2/D15, 97080 Würzburg, Germany
3 Center for Discovery and Innovation in Parasitic Diseases, Department of Pathology,
University of California, San Francisco, California, United States of America
*shared first authorship
#shared senior authorship
1
Acknowledgments: Many thanks to Dr Mehrdad Abbasi, Head of department of Botany at
Iranian Research Institute of Plant Protection, for providing us with fungal specimens, and
to Prof. Reinhard M Fritsch, Das Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), for the wild Allium samples.
References:
1. Kyung, K.H.. Current Opinion in Biotechnology, 2012, 23(2): 142–147.
2. Keusgen, M. Volatile Compounds of the Genus Allium L. (Onions). In Volatile Sulfur
Compounds in Food (pp. 183–214). American Chemical Society. 2011.
3. Pinto, E. et al.: Industrial Crops and Products. 2007, 26(2): 135–141.
4. Meletiadis, J.; Meis, J.F.G.M.; Mouton, J.W.: Journal of Clinical Microbiology. 2001,
39(2): 478–484.
NC.05
Characterization of Haemanthus coccineus extract and its
bioactive component: a novel anti-inflammatory approach
Fuchs, S.1,2; Hsieh, L.T.4; Saarberg, W. 3; Schaefer, L.4; Erdelmeier, C.A.J.3;
Koch, E. 3; Fürst, R.1
Schistosomiasis is a parasitic disease infecting over 200 million people that
continues to pose a public health hazard in developing countries. Schistosoma
blood fluke parasites are prevalent in Africa, Asia and South America, and
utilize amphibious or aquatic snails as vector hosts. Infection of humans occurs
during contact with contaminated water bodies. Free-swimming larvae
(cercariae) penetrate the skin to eventually reach the bloodstream where they
develop into adult worms [1]. The parasites can infest the human body for
decades and their eggs induce various organ pathologies leading to a range of
chronic and morbid physical and psychological consequences.
Treatment and control of schistosomiasis relies on just one drug, praziquantel
[2-3], and the danger of resistance by the parasite to the drug cannot be
ignored [4-5]. Research needs to concentrate on finding new and better drugs
to fight this disease.
183
We recently discovered caffeic acid bornyl ester with antileishmanial activity
from the rhizomes of Valeriana wallichii and subsequently synthesized a small
compound library of systematically varied derivatives [6]. The library was
screened for schistosomizidal activity against Schistosoma mansoni postinfective larvae (schistosomula) at a 5 µM concentration. Especially eugenyl
cinnamate (1) and thymyl cinnamate (2) showed good activity with compound
1 leading to the death of the schistosomula after 72 hours. Additionally both
compounds induced the formation of vacuoles, an interesting new phenotype.
NC.08
Targeting virulence: Inhibition of a streptococcal key pathogenic
mechanism by bacterial natural compounds
Rox, K.1,2,3; Gerth, K.4; Rohde, M.1,3; Chhatwal, G.S.3; Müller, R.2
1 Central
facility for microscopy, Helmholtz Centre for Infection Research, Inhoffenstraße
7, 38124 Braunschweig, Germany
2 Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical
Research Saarland, Saarland University Campus, Building C2.3, 66123 Saarbrücken,
Germany
3 Department of Medical Microbiology, Helmholtz Centre for Infection Research,
Inhoffenstraße 7, 38124 Braunschweig, Germany
4 Department of Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstraße
7, 38124 Braunschweig, Germany
Thanks are due to the SFB 630 for financial support.
References:
1. Gryseels, B. et al.: Lancet 2006, 368: 1106-1118.
2. WHO: Schistosomasis Fact sheet N°115.
3. WHO: Weekly epidemiological record 2014, 21-28.
4. Doenhoff, M.J., Pica-Mattoccia L.: Expert Rev. Anti Infect. Ther. 2006, 4(2): 199-210.
5. Doenhoff, M.J., Cioli D., Utzinger J.: Curr. Opin. Infect. Dis. 2008, 21: 659-667.
6. Glaser, J. et al.: Molecules 2014, 19: 1394-1410.
NC.07
Humulus lupulus derived bitter acids - potential antidepressant
and anxiolytic effects mediated by TRPC6 channel activation
Freigang, M.1,2; Pischetsrieder, M.2; Friedland-Leuner, K.1
Department of Chemistry and Pharmacy, Molecular and Clinical Pharmacy, FriedrichAlexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
2 Department of Chemistry and Pharmacy, Emil-Fischer-Center, Henriette SchmidtBurkhardt Chair of Food Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg
(FAU), Erlangen, Germany
1
Humulus lupulus extracts are used in folk medicine to treat anxiety, depression
and sleeping disorders. In animal models, sedative, anxiolytic and antidepressant-like effects could be demonstrated. However, the molecular mechanism of
action and the active constituents of the extract are still discussed. Interestingly, the hop bitter acid lupulone and its analogs have strong structural resemblances with hyperforin, the antidepressant active constituent of St. John’s wort
extract. Both show acyl- and prenyl-modifications on a phloroglucinol core
structure. Results from in vivo tests on rats suggest an anti-depressant effect
of this class of hop bitter acids. Hop β-acid fractions (5mg/kg) reduced
immobility time in the forced swimming test without achieving a sedative effect.
The purpose of this study was to investigate if hop ß-acids might also activate
the molecular target of hyperforin, the classical transient potential channels
TRPC6. TRPC6 channel activation by hyperforin leads to an influx of sodium
and calcium ions. This shift of the electrochemical gradient indirectly inhibits
monoamine transporters and directly improves synaptic plasticity, two
important mechanisms involved in antidepressant activity. First, we investigated if hop ß-acids lead to a non-selective cation influx in PC12 cells. Hop βacids showed a hyperforin-like activity profile in PC12 cells. Based on our
findings we next wanted to elucidate if hop ß-acids also activate TRPC6
channels. To address this question, several pharmacological tools known to
interfere with non-selective cation channels were used such as SK&F 96365,
2-APB, flufenamic acid, lanthanum, gadolinium and ACA as well as ruthenium
red, which blocks TRPV, TRPM6, TRPM8, and TRPA1 channels. All inhibitors
except ruthenium red strongly impaired hop ß-acid induced calcium influx in
PC12 cells. These results suggest that hop ß-acids might also activate TRPC6
channels and thereby mediate their antidepressant effects. However, further
experiments are needed to further support this hypothesis.
Every year millions of people all over the world are suffering from streptococcal
diseases. Whereas common throat infections can be treated and cured quite
effectively with penicillin, sepsis or necrotizing fasciitis can become lifethreatening.[1] Moreover, post-infection sequelae represent a serious health
hazard especially in developing countries.[2] Antibiotic resistance is emerging
– therefore, it is inevitable to find new antibiotics targeting key pathogenic
mechanisms.[3],[4] Such a mechanism is invasion of eukaryotic cells: after
invasion of streptococci dissemination is possible as well as persistence which
can cause recurrent infections.[5] Consequently, several compounds and
extracts derived from myxobacteria were tested with the aim to block the
invasion process.
Two different Group A streptococcal strains representing two important
invasion mechanisms were used for cell infection assays.[6] In every assay
compounds or extracts were added to analyse possible inhibitory effects. To
estimate the range of inhibition of invasion, double-immunofluorescence
staining and examination via electron microscopy after an infection experiment
were used. By using a bacterial invasion assay intracellular surviving bacteria
were determined. Moreover, cytotoxicity was assessed by using an MTT assay
under infection conditions.
Two compounds inhibit invasion of Group A streptococci into two different
types of epithelial cells. As two different invasion mechanisms are addressed
in the infection assays, it can be shown that these different pathways can be
inhibited quite effectively. Additionally, the compounds do not show cytotoxic
effects under infection conditions.
Inhibition of invasion will be the first step to encounter persistence and
dissemination: if streptococci are disabled to invade cells, the reservoir
required to cause recurrent infections is reduced. Moreover, dissemination will
be hampered as streptococci will only be able to adhere to cells. Additionally,
killing of streptococci using antibiotics such as penicillin will be facilitated as
the bacteria cannot hide in an intracellular compartment anymore.
Acknowledgments: Katharina Rox acknowledges the President’s Initiative and Networking
Fund of the Helmholtz Association of German Research Centers (HGF) (contract number
VH-GS-202) for supporting her research project.
References:
1. Lamagni, T.L. et al.: J. Clin. Microbiol. 2008, 46(7): 2359-2367.
2. Carapetis, J.R. et al.: Lancet. Infect. Dis. 2005, 5(11): 685-694.
3. Cantón, R. et al.: J. Antimicrob. Chemother. 2002, 50(S1): 9-24.
4. Capoor, M.R. et al.: Jpn. J. Infect. Dis. 2006, 59(5):334-336.
5. Rohde, M., Chhatwal, G.S.: Curr. Top. Microbiol. Immunol. 2013, 368:83-110.
6. Nitsche-Schmitz, D.P., Rohde, M., Chhatwal, G.S.: Thromb. Haemost. 2007, 98(3):488496.
NC.09
Rice Bran Extract administration improves age-related brain
mitochondrial dysfunction in NMRI mice
Hagl, S.1; Berressem, D.1; Grebenstein, N.2; Frank, J.2; Eckert, G.P.1
1 Department
of Pharmacology, Biozentrum Niederursel, Goethe University, Max-vonLaue-Str. 9, 60438 Frankfurt, Germany
2 Institute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstr. 28,
70599 Stuttgart, Germany
In the last few decades, life expectancy was rising constantly due to improvements in health care and technology which lead to increased incidence of agerelated diseases including Alzheimer’s disease. Research revealed that
184
mitochondria are significantly involved in aging processes that ultimately lead
to neurodegeneration. A healthy lifestyle including a diet rich in antioxidants
and polyphenols represents one strategy to protect the brain and to prevent
neurodegeneration.
Key components of Rice Bran Extract (RBE) are tocopherols, tocotrienols and
γ-oryzanol. RBE has been reported to have anti-inflammatory, antioxidant,
cholesterol-lowering and anti-diabetic activities. Since we could recently show
that RBE feeding increased brain mitochondrial function in young guinea pigs
[1] we now tested the effect of RBE administration on brain mitochondrial
function in aged mice.
Aged (18 months old) NMRI mice were fed with RBE (340mg RBE/kg body
weight) via oral gavage once a day for 3 weeks. 3 and 18 months old NMRI
mice fed with vehicle served as control groups. We assessed mitochondrial
function by measuring mitochondrial respiration in isolated brain mitochondria
and mitochondrial membrane potential (MMP) and ATP levels in dissociated
brain cells (DBC). Additionally, we determined levels of mitochondrial marker
proteins using Western Blot analysis and examined blood plasma and brain
tissue concentrations of key components of RBE.
ATP levels, complex I respiration, the respiratory control ratio and protein
levels of PGC1α were significantly decreased in aged NMRI mice. RBE
administration was able to entirely compensate this age-related mitochondrial
dysfunction by elevating PGC1α protein levels and citrate synthase activity and
by improving the resistance of DBC against sodium nitroprusside induced
nitrosative stress. According to these results, RBE is a very promising
candidate neutraceutical in the prevention of age-related neurodegenerative
diseases like Alzheimer’s disease.
Reference:
1. Hagl, S. et al.: Pharmacol. Res. 2013, 76: 17-27.
NC.10
Effect of polyphenol-rich grape extract on mitochondrial dysfunction in PC12-cells and memory and motor performance in
C57BL/6 mice
Asseburg, H.1; Plank, C.1; Borchiellini, M.2; Mueller, M.3; Pohland, M.1;
Berressem, D.1; Eckert, G.P.1.
1 Department
of Pharmacology, Biozentrum Niederursel, Goethe University, Max-vonLaue-Str. 9, 60438 Frankfurt am Main, Germany
2 Department of Pharmaceutical Science, University of Perugia,Via del Liceo 1, 06123
Perugia, Italy
3 Provadis School of International Management and Technology, Industriepark Höchst,
65926 Frankfurt am Main, Germany
The increasing life expectancy is accompanied by several age-related
neurological changes which may lead to neurodegenerative diseases. One key
factor of aging and neurodegenerative diseases is mitochondrial dysfunction
(MD) which includes decreased mitochondrial membrane potential (MMP) and
ATP levels [1,2]. Polyphenol-rich fruit like grapes and berries have been shown
to have neuroprotective potential in rodents and human pilot studies [3,4],
however, information on the effects on MD is scarce. Therefore, this study
aims to investigate the effects of polyphenol-rich grape extract (PGE) on
mitochondrial function and behavioral performance in aging.
PC12 cells were pre-incubated with PGE (50µg/ml) for 1 h and subsequently
incubated with 0.5mM sodium nitroprusside (SNP) leading to MD. Parameters
of mitochondrial function in vitro and ex vivo included ATP levels (luciferasecatalysed bioluminescence), MMP (rhodamine 123 fluorescence) and activity
of mitochondrial respiratory complexes (Oxygraph-2k). SNP-treatment of PC12
cells decreased ATP levels and MMP to 40.±2.4% and 83.4±1.3% of
untreated controls, respectively. PGE pre-incubation reduced the SNP-induced
MD resulting in ATP levels of 54.6±1.3% and MMP of 90.0±2.0% of untreated
controls. In vivo effects of PGE were assessed in C57BL/6 mice. For shortterm treatment in aged mice (19-22 months), PGE (200mg/kg b.w.) was
administered via oral gavage over a period of 3 weeks. Mitochondrial function
was measured in isolated brain mitochondria and dissociated brain cells.
Short-term PGE treatment did not have an effect on mitochondrial function.
The reduced ATP levels and impaired activity of the mitochondrial respiratory
chain in aged mice compared to young mice were not ameliorated by PGE
treatment. However, as mitochondrial function was assessed after short –term
treatment in aged mice, a longer treatment period starting at middle age might
be more effective to protect mitochondria und therefore prevent or delay the
age-related decline in brain function. For long-term treatment of mice, PGE
dissolved in water (2g/L, eq. to 200mg/kg b.w.) served as sole source of liquid
from the age of 6 months. Motor performance (Rotarod) and short-term
memory (Social Recognition Test) were assessed in a 3-month interval. PGE
treatment for 6 months but not for 9 months led to an improvement in motor
performance and short-term memory. The long-term effect of PGE on MD
needs further investigation.
References:
1. Schaffer, S. et al.: Mol. Neurobiol. 2012, 46(1): 161-178.
2. Asseburg, H.; Hagl, S.; Eckert, G.P.: Pharma Nutrition - An Overview (Springer) 2014,
in press.
3. Cherniack, E.P.: Br. J. Nutr. 2012, 108(5): 794–800.
4. Joseph, J.A.; Shukitt-Hale, B.; Willis, L. M.: J. Nutr. 2009, 139(9): 1813S-1817S.
NC.11
Nematicidal activity of some Allium spp extracts against rootknot nematode Meloidogyne incognita
Jivishova, S.; Jivishov, E.; Keusgen, M.
Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Philipps University of
Marburg, Marbacher Weg 6, 35037, Marburg, Germany
To investigate nematicidal activity against root-knot nematode Meloidogyne
incognita, 17 ethyl acetate extracts of Allium species were tested. 8 Concentrations of bulb and flower extracts were prepared and nematicidal activity was
determined after 48 hours of exposure. Responses varied with test material
and concentration. Good nematicidal activity against infective second-stage
juveniles (J2) was achieved with A. sativum, A. karataviense, A. zebdanense,
A. aflatunense and A. stipitatum bulb extracts. The highest nematicidal activity
was shown by A. sativum and A. karataviense at LD50 concentrations of
0.07425 mg ml-1 and 0.1891 mg ml-1, respectively (values related to amount of
extract). Lannate 20 L was used as a positive control with LD50 of 0.1249 mg
ml-1. Other bulb extracts of Allium spp showed weak nematicidal activity, while
nematicidal activity of flower extracts was not significant.
Acknowledgments:
Institut für Pflanzenschutz in Freising Mr. Andreas Hermann, Julius Kühn-Institut Dr.
Johannes Hallmann, Institute of Pharmaceutical Chemistry, Philipps University of
Marburg, Mr. Floris van Elsäcker, DuPont de Nemours (Deutschland) GmbH, Dr. Martin
Lechner.
References:
1. Nguyen, D.-M.-C. et al.: Nematology 2009, 11(6): 835-845.
2. Park, I.-K. et al.: Nematology, 2005, 7(5): 767-774.
3. Danquah, W.B. et al.: Nematology, 2011, 13(7): 869-885.
4. Choi, I.-H. et al.: Nematology, 2007, 9(2): 231-235.
5. Amaral, D.F. et al.: Nematology, 2003, 5(6): 859-864.
NC.12
Qualitative and quantitative analysis of cysteine sulfoxides in
flowers of some Allium species
Jivishov, E.; Neumann, S.; Keusgen, M.
Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6, D-35037,
Marburg, Germany
Since ancient times, onions, garlic and some other species of the genus Allium
L. (onions) have been used as phyto-pharmaceutics, seasonings, and
vegetables. Most prominent are common onion (A. cepa L.) and garlic (A.
sativum L.). The medicinal benefits of these two species were intensely
investigated during the last decades and lipid lowering, antibiotic, antiatherosclerotic and anti-diabetic effects were described. Also, a canceroprotective effect was proven by a number of ethnic studies. The health benefits of
Allium vegetables are mainly related to sulphur containing compounds as well
as saponins.
The species-rich genus Allium has a main centre of distribution reaching from
Southwest Asia to the high mountains of Central Asia. In this area, several wild
species are used by the local population, as one can be concluded from casual
remarks in some floras. The so-called cysteine sulphoxides of these plants are
believed to be mainly responsible for these health benefits. These compounds
are converted to thiosulphinates like allicin, when plant material is disrupted.
This reaction is catalysed by the action of the enzyme alliinase.
185
Flowers of 22 Allium species, most of them for the first time, were analyzed for
their cysteine sulfoxide(CSO) content. While all of the flowers possessed
methiin, only a few of flowers carried any of the following CSOs: propiin,
isoalliin, alliin, marasmin, S-(2-pyrrolyl)-cysteine sulfoxide, S-(2-pyridyl)cysteine N-oxide and butiin (Figure). Total CSO content of analyzed flowers
varied from 0.01% to 0.5% relatively to fresh material weight. A few compounds, which possibly can be γ-glutamyl derivatives of some aminoacids
were observed in mass chromatograms. No homoisoalliin, penthiin, ethiin or
known γ-glutamyl derivatives of CSOs were detected in any of the flowers.
O
NH2
O
O
NH2
S
S
S
COOH
COOH
Alliin
Methiin
O
NH2
S
COOH
NH
N
O
NH2
COOH
NH2
S
COOH
Homoisoalliin
COOH
COOH
Marasmin
Figure: Most important cysteine sulphoxides as well as an N-oxide of the
genus Allium
Acknowledgements: Institute of Pharmaceutical Chemistry, University of Marburg, van
Elsäcker F, Brauschke M, Gercke N, Pipp K
References:
1. Kusterer, J., Keusgen, M.: J. Agric. Food Chem. 2010, 58: 1129–1137.
2. Kusterer, J. et al.: J. Agric. Food Chem. 2011, 59: 8289–8297.
3. Kubec, R. et al.: J. Agric. Food Chem. 2011, 59: 5763–5770.
NC.13
Stereochemistry of the cysteine sulphoxide marasmin in the
South African plant Tulbaghia violacea Harv.
Neumann, S.; Keusgen, M.
Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6-10,
D-35032 Marburg, Germany
Cysteine sulphoxides (CSO) are well known sulphur-containing aroma
precursors of plants belonging to the family of Alliaceae. When a cell is
disrupted, cysteine sulphoxides deposited in the cytoplasm are cleaved by an
alliinase stored in the cell vacuole. This enzymatic reaction results in the
formation of volatile sulphenic acids, which condensate spontaneously to
thiosulphinates. These substances do have a strong alliaceous smell and
interesting bioactivities like antimicrobial effects [1].
The thiosulphinate derived from the CSO marasmin was named marasmicin
and has been shown to be the main flavour compound of some fungi and
plants mentioned below. Biological activity against fungi and tuberculosis has
been recently demonstrated [2].
The first record of the CSO marasmin was for some basidiomycota of the
genus Marasmius [3]. These fungi contain the γ-glutamylised form of marasmin
with (S) being the absolute configuration at the sulphur atom [(SS,RC)marasmin; Figure]. The first detection in a plant was 11 years later in the
Malaysian tree Scorodocarpus borneensis Becc.. Its fruits contain the free
sulphoxide with converse orientation at the sulphur atom ((RS,RC)-marasmin)
[4]. From then on marasmin was found in numerous genera of the Alliaceae
especially Tulbaghia, Allium, Ipheion and Leucocoryne [5,2,6].
The investigation of the CSO content of T. violacea, known as ‘sweet garlic’ or
‘society garlic’, showed something very unusual. All parts of the investigated
plant did not only contain (RS,RC)-marasmin), even the (SS,RC)-marasmin)
could be detected in relevant amounts. These findings are rather unusual,
because the appearance of both stereoisomers of the same CSO in a plant
has been rarely reported so far and previous research did not reveal the
(SS,RC)-marasmin in T. violacea nor in any other plant.
186
NC.14
Biological active compounds from Streptomyces living in
symbiosis with Arnica montana
Butiin
O
S
S
References:
1. Kusterer, J.: Neue Erkenntnisse der Schwefelchemie und Chemotaxonomie in Arten
des Genus Allium. (Universitätsbibliothek Marburg) 2010
2. Kusterer, J.; Fritsch, R.M.; Keusgen M.: J. Agric. Food Chem. 2011, 59(15):8289–8297.
3. Gmelin, R. et al.: Phytochem. 1976, 15(11):1717–1721.
4. Kubota, K.; Hirayama H.; Sato Y.: Phytochem. 1998, 49(1):99–102.
5. Kubec, R.; Velísek, J.; Musah, R.A.: Phytochem. 2002, 60(1):21–25.
6. Kubec, R. et al.: J. Agric. Food Chem. 2013, 61(6):1335–1342.
NH2
S
Propiin
O
COOH
O
NH2
S
O
COOH
Isoalliin
NH2
S
NH2
Acknowledgements: Philipps-Universität Marburg, Floris van Elsäcker
Wardecki, T.1; Brötz, E.2; Ebeling, S.1; von Löwenich, F.3; Häcker, G.3;
Luzhetskyy, A.2; Merfort, I.1
1 Institute
of Pharmaceutical Science, Department of Pharmaceutical Biology and
Biotechnology, University of Freiburg, 79104 Freiburg, Germany
2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C 2.3, 66123
Saarbrücken, Germany
3 Institute of Microbiology and Hygiene, University Hospital Freiburg, 79104 Freiburg,
Germany
Arnica montana L. is a well-known traditional medicinal plant. In modern
therapy extracts of Arnica montana flowerheads are used externally to treat
several injuries as bruises, distortions, sprains, hematoma and inflamed insect
stings. Moreover, the extracts are used as supportive treatment of rheumatic
diseases. As active compounds, the sesquiterpene lactones helenalin, 11α,13dihydrohelenalin and their esters have been identified [1]. Beside their
pronounced anti-inflammatory effects these sesquiterpene lactones also
showed antibacterial activity against gram-positive bacteria [2].
Streptomyces are gram-positive bacteria that are frequently found in soil and
also in plant roots. They have come into focus due to their ability to produce
important biological active compounds, such as antibiotics and antitumorals
[3]. Endophytic Streptomyces have already been discovered in other members
of the Asteraceae family, for example in the medicinal plant Artemisia annua L.
[4]. Therefore, it was attractive to search for Streptomyces in Arnica montana
as well. We observed that the roots of these plants indeed were inhabited by
Streptomyces. Intriguingly these organisms are able to grow in such a hostile
environment, where antibacterial sesquiterpene lactones are present. We
cultivated these bacteria and analyzed their extracts. Several secondary
metabolites were identified. The extracts were shown to exhibit cytotoxic
activity and antibacterial properties against gram-negative bacteria.
References:
1. Merfort, I.: Z. Phytother. 2010, 31: 188-192.
2. Lee, K.-H. et al.: Phytochemistry 1977, 16(8): 1177-1181.
3. Procópio, R.E. et al.: Braz J Infect Dis. 2012, 16(5): 466-471.
4. Lin, L. et al.: Planta 2012, 236(6): 1849-1861.
NC.15
Inhibition of SDF-1-induced tumor cell activation and migration
by purified fucoidan fractions
Ehrig, K.; Schneider, T.; Alban, S.
Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118
Kiel, Germany
Fucoidans from brown algae exhibit numerous biological effects relevant for
anti-tumor activity. Currently, the SDF-1(CXCL12)/CXCR4 axis is considered a
promising target for tumor therapy, since recent results showed that this
chemokine/receptor axis plays a central role in tumor progression, angiogenesis, metastasis and therapy resistance [1].
The aim of this study was to characterize the fucoidan from Saccharina
latissima (S.l.) and to investigate whether it influences the SDF-1/CXCR4 axis.
Extraction of S.l. from Faroe Islands provided > 5% alginic acid-free sulfated
glycans, which were purified and separated into two distinct fractions by anion
exchange chromatography. The more active one was identified as a sulfated
branched galactofucan (SGF) with a degree of sulfation of 0.8.
SGF exhibited no cytotoxic activity, but showed a variety of effects relevant for
anti-metastatic and anti-angiogenic activity such as inhibition of heparanase,
elastase and chemotaxis of tumor cells in migration and scratch assays.
Investigations by flow cytometry, Western blotting, ELISA and fluorescence
microscopy revealed that SGF interferes with the SDF-1/CXCR4 axis in Raji
cells (Burkitt lymphoma) by inhibiting the SDF-1-induced CXCR4 activation.
Compared to fucoidan from Sigma and heparin, it was much more active. In
contrast to the CXCR4 receptor antagonist AMD3100, SGF antagonizes the
chemokine. In gene expression analysis, SGF additionally reduced the
expression of SDF-1 and several further genes important for tumor progression
and metastasis.
In conclusion, SGF from S.l. is obtained in reproducible quality by an optimized
isolation method. The pharmacological profile of SGF indicates potent antitumor effects. Based on our results, SGF should now be examined in vivo.
Acknowledgement: This study is part of the project “Algae against cancer”, financially
supported by the Federal Ministry of Research and Education (BMBF).
Reference:
1. Cojoc, M. et al.: OncoTargets and Therapy 2013, 6: 1347–1361.
NC.16
Triterpenes from birch bark and their influence on cutaneous
cells derived from healthy and diabetic patients
Werner, P.1,4; Wardecki, T.1,4; Thomas, M.2; Zanger, U.M.2; Brandner, J.3;
Merfort, I.1
1 Institute
of Pharmaceutical Science, Department of Pharmaceutical Biology and
Biotechnology, University of Freiburg, 79104 Freiburg, Germany
2 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, 70376 Stuttgart,
Germany
3 Clinic for Dermatology and Venereology, University Hospital Hamburg-Eppendorf, 20246
Hamburg, Germany
4 These authors contributed equally to this work
One of the major health problems in industrial countries is diabetes mellitus [1].
The prevalence in Germany has been estimated at 7.2 % and is predicted to
increase further. This high number of patients suffering from diabetes mellitus
leads to enormous costs in public health system [2]. One problem which many
diabetic patients have to deal with is impaired wound healing. Therefore,
efficient remedies are urgently needed. Besides conventional drugs phytomedicines may be an interesting alternative. An extract from birch bark (Betula
pendula Roth), named as TE1 and enriched in triterpenes with betulin as the
main constituent, has been clinically proven to benefit wound healing [3].
Previously we have shown that TE1 and betulin transiently upregulate proinflammatory mediators, such as cytokines and COX-2 in keratinocytes and
promote keratinocyte migration by altering the actin cytoskeleton [4].
In continuation of our studies we here focus on the effects of TE1 and betulin
on human dermal fibroblasts from healthy and diabetic donors. TE1 and betulin
treatment led to increased levels of IL-6, IL-8, Rantes, and TNF-α mRNA
expressions which were higher compared to those ones in keratinocytes.
Additionally, some factors were only upregulated in fibroblasts, e.g. CCL2.
Presumably fibroblasts are more susceptible to the used stimuli than keratinocytes, although it has to be considered that a high variability was observed
between the different donors. Expression of these genes were similar in
fibroblasts from healthy and diabetic patients.
Moreover, we concentrated on cytoskeletal changes of migrating fibroblasts
from healthy patients. Treatment with TE1, betulin, and also with the minor
constituents of TE1, lupeol and betulinic acid, promoted the reorganization of
actin cytoskeleton in fibroblasts, a prerequisite for migration into the wound site
and creation of granulation tissue [5].
Acknowledgement: Funding by Aif and providing of TE1 and the triterpenes by Birken AG
is gratefully acknowledged.
References:
1. Rothenbacher, D. Diabetes aktuell 2011, 9(8):340–343.
2. Heidemann, C. et al.: Bundesgesundheitsbl 2013, 56:668–677.
3. Metelmann, H. et al.: J Craniomaxillofac Surg. 2012, 40(5):e150-4.
4. Ebeling, S. et al.: PLoS One 2014, 9(1):e86147.
5. Gurtner, G.C. et al.: Nature 2008, 453(7193):314-321.
NC.17
Expeditious Syntheses of Rutaecarpine and Analogs and
Preliminary Evaluation for Cytotoxic Activity
Huang, G.1; Roos, D.1; Heilmann, J.2; Decker, M.1
1 Institut
für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg,
Am Hubland, D-97074 Würzburg, Germany
2 Institut für Pharmazie, Universität Regensburg, Universitätstrasse 31, D-93053
Regensburg, Germany
Rutaecarpine (1) is a natural quinazolino carboline-type alkaloid first isolated
from fruits of Evodia rutaecarpa (Juss.) Hook. f. et Thoms (Rutaceae) which
are used in Traditional Chinese Medicine as herbal remedy [1]. Subsequently,
1 and derivatives bearing hydroxy and methoxy groups at ring A or E were
isolated also from other plant genera. Accumulating research has proven
useful biological properties of 1 and its natural and synthetic analogs, such as
anti-platelet aggregation, vasorelaxing properties, and cyclooxygenase-2
inhibition [2], often exhibiting in-vitro activity in the lower micromolar range [3].
Rutaecarpine and derivatives were described as templates for anti-cancer
drugs [4, 5].
Its multiple biological activities intrigued many efforts on the syntheses of 1 and
its analogs [6 and references therein]. During our search for new inhibitors of
cholinesterases, we have developed a novel and expeditious procedure for
preparation of rutaecarpine and its analogs. Two starting materials, isatoic
anhydride and 3,4-dihydroisoquinoline or 4,9-dihydro-3H-pyrido[3,4-b]indole,
respectively, are mixed and heated overnight. Afterwards, after simple
precipitation good to excellent yields of the heterocycles are obtained (69 96%). This novel, short and versatile method gives high yields while devoid of
tedious purification procedures [7]. Unlike previously described procedures,
neither multi-step syntheses nor special reagents are necessary, and no
special starting materials have to be prepared prior to the synthesis [6, 7].
Applying this reaction, 20 novel heterocyclic compounds were obtained. In a
preliminary in vitro study, selected compounds were evaluated for cytotoxicity
on a HeLa cell line. Several derivatives exhibit pronounced potency compared
to rutaecarpine.
References:
1. Wang, L.; Argade, N.P.: Planta Med. 2005, 71(5): 416–419.
2. Mhaske, S.B. et al.: Tetrahedron 2006, 62(42): 9787–9826.
3. Baruah, B. et al.: Bioorg. Med. Chem. 2004, 12(9): 1991–1994.
4. Yang, L.-M.: Bioorg. Med. Chem. Lett. 1995, 5(5): 465-468.
5. Dong, G. et al.: J. Med. Chem. 2012, 55(17): 7593-7613.
6. Richers, M.T. et al.: Synthesis 2013; 45(13): 1730-1748.
7. Huang, G. et al.: Tetrahedron Lett. 2014, 55(26): 3607-3609.
NC.18
Horse chestnut fruit extract: Criteria for cell established use in
chronic venous insufficiency are met
Kraft, K.1; Kelber, O.2; Müller, J.2
1
Chair of Natural Medicine, University of Rostock, Rostock, Germany;
Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany
2 Scientific
Purpose: In the therapy of chronic venous insufficiency (CVI) compression
treatment is the most popular option, despite it often causes discomfort and
has been associated with poor compliance. Oral drug treatment with standardized extracts from horse chestnut seed (HCSE) is therefore an attractive
treatment option. For an overview a systematic evaluation of the evidence
187
(available clinical studies, Cochrane reviews, monographs, medical guidelines
in this indication) concerning standardization, efficacy and safety was
conducted.
Results: Standardized HCSE leads to a clear improvement in CVI related signs
and symptoms compared with placebo. This is reflected in all reviews and
monographs as well as in clinical guidelines. Adverse events were usually mild
and infrequent. Study durations of 12 weeks were appropriate and sufficient to
demonstrate clinical efficacy in comparison to compression treatment. Adherence was 90%, compared to 47% with compression treatment.
Conclusion: HCSE is an efficacious and safe treatment for CVI, as studies of
up to 12 weeks duration have shown. Long-term compliance is key factor in
CVI treatment and is higher for treatment with HCSE than for compression
therapy. As long-term tolerability is good according to pharmacovigilance data,
HCSE can be recommended for long-term application in CVI. The therapeutic
usefulness of HCSE is therefore well established also in-long term treatment.
NC.19
Isothiocyanate Sulforaphane inhibits expression of the central
iron regulator hepcidin in an inflammatory cell model
Martin, J.; Fischer, B.; Steinhilber, D.; Stein, J.; Ulrich-Rückert, S.
Institute of Pharmaceutical Chemistry Goethe-University Frankfurt, Max-von-Laue-Straße
9, 60438 Frankfurt a.M., Germany
Hepcidin, a regulatory peptide of the iron metabolism, is regarded as the
central mediator of anaemia of chronic inflammation (ACI), a common
complication in chronic inflammatory disease [1]. Via the activation of STAT3
signal transduction, inflammatory stimuli invoke an increase in hepcidin
expression, leading to the impairment of intestinal iron absorption [2]. The antiinflammatory properties of the isothiocyanate sulforaphane (SFN), primarily
found in cruciferous vegetables, have been confirmed in numerous in vitro and
in vivo models [3, 4] . The aim of this experiment was therefore to achieve
hepcidin regulation using SFN.
Caco-2, Huh7 and HepG2 cells were cultivated in standard conditions and
incubated for 16 hours with SFN [10µM]. An inflammatory situation was
imitated by stimulation of the cells with Oncostatin M [10ng/ml] (OncM).
Quantitative real-time PCR was performed to determine mRNA expression.
For reporter gene assays, calcium chloride was used to transfect plasmid and
luciferase activity was measured luminometrically. Proteins were detected by
Western blot analysis.
SFN significantly inhibits not only OncM-induced hepcidin promoter activation
(***p<0.001), but also hepcidin mRNA expression (HepG2 *p<0.05; Huh7
**p<0.01). Furthermore, in the co-culture model, an increase of intracellular
ferritin levels in intestinal cells was achieved (**p<0.01). Whereas OncMinduced effects are mediated via activation of the transcription factor STAT3,
which was confirmed using a specific STAT3 inhibitor [50µM] (***p<0.001), the
effects of SFN are apparently not induced by inhibition of this signal transduction pathway, since it was not possible to achieve a reduction of OncM-induced
STAT3-phosphorylation (**p<0.01).
The study data show for the first time that SFN counteracts the induction of
hepcidin by proinflammatory stimuli, although further research is necessary to
discover the exact mechanisms involved. SFN may therefore be an option for
improving iron absorption in patients with chronic inflammatory disease.
References:
1. Means, R.T.Jr.: Am J Med Sci 2013, 345(1): 57-60.
2. Poli , M. et al. : Front Pharmacol 2014, 28(5): 86.
3. Li, B. et al.: Exp Neurol 2013, 250: 239-249.
4. Lin, W. et al.: Biochem Pharmacol 2008, 76(8): 967-973.
NC.20
188
NC.21
Joint action of herbal components influence the effects of STW 5
on inflammatory processes and disturbed motility
Hoser, S.1; Winkelmann, V.1; Baumgärtel, A.1; Mishenzon, N.1; Abdel-Aziz, H.2;
Weiser, D.2; Okpanyi, S.N.2; Nieber, K.1; Kelber, O.2
1 University
of Leipzig, Institute of Pharmacy, Pharmacology, Leipzig, Germany;
Arzneimittelwerk GmbH, Scientific Department, Darmstadt, Germany
2 Steigerwald
The herbal drug STW 5 (Iberogast®) contains nine individual plant extracts
which affect inflammatory processes and disturbed motility in the gut. The aim
of the study was to investigate the effect of individual ethanolic extracts (in
concentration used in STW 5) and selected combinations on targets related to
inflammation.
The cytotoxicity was examined in CaCo-2 cells using the lactate dehydrogenase (LDH) assay whereas, THP-1 cells were used to determine the TNFα
release using an ELISA. ACh-induced isometric contractions of a rat small
intestinal preparations were measured in an commercial organ bath setup.
STW 5 (500.05μg/ml), STW 5-II (5.11μg/ml, containing only 6 of the 9 extracts)
and lemon balm (59.9μg/ml) reduced LPS (10ng/ml)-induced LDH release from
CaCo-2 cells by 74-75%. Iberis amara (29.1μg/ml), peppermint (38.9μg/ml),
chamomile (116.3μg/ml), angelica (84.7μg/ml) and milk thistle (15.2μg/ml)
inhibited LDH release by 25-37%. Liquorice (84.9μg/ml), caraway (30.9μg/ml)
and celandine (61.9μg/ml) had no effect. The combinations of Iberis amara and
peppermint as well as peppermint and milk thistle revealed synergistic or
additive effects, whereas the combination of chamomile and angelica evoked
additive or antagonistic effects depending on their compositions. STW 5, STW
5-II, Iberis amara, peppermint, liquorice, caraway, milk thistle and lemon balm
in concentration used in LDA assay inhibited LPS (100ng/ml)-induced TNF
release to 51-67%. The combinations of Iberis amara and peppermint as well
as chamomile and liquorice had additive or antagonistic effects depending on
their compositions. STW 5 and STW 5-II reduced the acetylcholine (ACh)induced contractions in rat ileum preparations to 81-83%. The individual
extracts inhibited the contractions to 83-91% except lemon balm. The
combination of Iberis amara and peppermint as well as liquorice and caraway
had additive or antagonistic effects depending on the ratios. Our results allow
insight into the interactions between the extracts of STW 5 and confirm the
concept of multi-target actions.
Disclosure: Funding for experimental studies was provided by Steigerwald
Arzneimittelwerk GmbH.
Reference:
1. Hoser, S. et al.: Planta Med 2013, 79: 1258.
NEUROACTIVE DRUGS (ND01-ND13)
ND.01
N-phenyl substituted thiazolidine-2,4-diones as neuroprotective
agents
Kraus, A.L.1; Oppermann, S.2; Elsässer, K.2; Schrader, F.C.1; WegscheidGerlach, C.1; Culmsee, C.2; Schlitzer, M.1
1 Institut
für pharmazeutische Chemie, Fachbereich Pharmazie, Philipps-Universität
Marburg, Marbacher Weg 6, D-35032 Marburg, Deutschland
2 Institut für Pharmakologie und Klinische Pharmazie, Fachbereich Pharmazie, PhilippsUniversität Marburg, Karl-von-Frisch-Str. 1, D-35032 Marburg, Deutschland
Mitochondrial damage is a result of glutamate-induced excitotoxicity or
oxidative stress and plays an important role in neuronal death, e.g. after
cerebral ischemia or in neurodegenerative diseases. In the underlying intrinsic
death pathway of programmed cell death, the protein Bid is a well-established
key factor in the process of mitochondrial demise, characterized by loss of
mitochondrial membrane potential, mitochondrial fission and increased
mitochondrial ROS formation. Therefore, Bid was chosen as target for
neuroprotection. A range of N-phenyl substituted thiazolidine-2,4-diones were
synthesized and tested for their neuroprotective effects in a model of Biddependent neural cell death. Some of the initially synthesized compounds
showed pronounced protection in vitro and are basis for further modification
and development of novel structures.
The synthesis was carried out using thioglycolic acid methyl ester and either
substituted phenylisocyanates or substituted aniline and 1,1’carbonyldiimidazole. The most promising structures contained orthosubstitutents on the aromatic moiety, leading to a rotational barrier and
reduced flexibility.
References:
1. Becattini, B. et al.: Proc. Natl. Acad. Sci. U.S.A. 2006, 103: 12602-12606.
2. Becattini, B. et al.: Chem. Biol. 2004, 11: 1107-1117.
3. Oppermann, S. et al.: J. Pharmacol. Exp. Ther. 2014, 350: 273-289.
ND.02
Interaction between TRPC6 channels and FKBP51 – implications
for the pathophysiology of mood disorders
Ye, L.1; Hausch, F.2, Friedland, K.1
Department of Molecular and Clinical Pharmacy, University of Erlangen, 91052
Erlangen, Germany
2 Max Planck Inst. for Psychiatry, 80804 Munich, Germany
active constituent of St. John’s wort extract, which is used to treat mild to
moderate depression. These findings might contribute to a better understanding how FKBP51 and TRPC6 channels are involved in the pathogenesis of
mood disorders.
ND.03
Nutritive Modulators of Mitochondrial Function in Neurodegenerative Diseases
Denzer, I.1,2; Pischetsrieder, M.2; Friedland-Leuner, K.1
1 Department
of Chemistry and Pharmacy, Molecular and Clinical Pharmacy, FriedrichAlexander-Universität Erlangen-Nürnberg (FAU), Cauerstr. 4, 91058 Erlangen, Germany
2 Department of Chemistry and Pharmacy, Emil Fischer Center, Henriette SchmidtBurkhardt Chair of Food Chemistry,Friedrich-Alexander-Universität Erlangen-Nürnberg
(FAU), Schuhstr. 19, 91052 Erlangen, Germany
In the wake of increasing life span of humans, aging-associated neurodegenerative diseases have become a major health problem in society [1]. Agerelated mitochondrial dysfunction is an early event in the pathology of many
common neurodegenerative diseases, including Alzheimer’s disease (AD)
[2,3]. Diet and lifestyle seem to have a major impact on neuronal function and
are considered to be important preventive key-players in healthy aging [4].
Therefore, mitochondria seem to be an interesting pharmacological target for
nutritive modulators of brain functionality and activity in aging and AD. Several
constituents of food such as L-sulforaphane (SFN) from broccoli, S-allyl-Lcysteine (SAC) from garlic or isoliquiritigenin (iso) from licorice are known to
activate the nuclear factor erythroid-2-related factor 2 (Nrf2) [5]. Importantly,
Nrf2 expression is downregulated in AD and this might result in reduced
mitochondrial biogenesis and finally in impaired production of ATP [6]. The
purpose of this study was the screening of food items for mitochondrial
protection. Therefore, we first investigated the effects of iso, SAC and SFN in a
neuronal cell model on basal mitochondrial function and cell viability and
afterwards looked for protective effects in a cell model of aging and AD. 20-h
preincubation with iso (1 µM), SAC (5 µM) or SFN (1 µM) showed an increase
of the mitochondrial membrane potential of PC12 cells, which were harmed
with the complex I inhibitor of the respiratory chain rotenone (3 µM, 24 h) as a
cellular model of aging. Similar results could be observed for the mitochondrial
membrane potential and the cell viability of PC12 cells which were preincubated with iso (1 µM), SAC (5 µM) or SFN (1 µM) for 20 h and stressed with the
nitric oxide donor sodium nitroprusside (SNP) (350µM or 400 µM, 24 h), a
generator of nitrosative stress and a complex IV inhibitor. These results show
that the nutritive Nrf2 activators iso, SAC and SFN improve mitochondrial
function and cell viability in a cell model for aging and AD.
Funding Acknowledgments:
This study is implemented within the framework of the Neurotrition Project, which is
supported by the FAU Emerging Fields Initiative.
References:
1. Hampel, H. et al.: Prog. Neurobiol. 2011, 95: 718-728.
2. Leuner, K. et al.: Front. Neurosci. 2010, 4: 44.
3. Leuner, K. et al.: Antioxid. Redox Signaling 2012, 16 (12): 1421-1433.
4. Eckert, G.P. et al.: Mol. Neurobiol. 2012, 46 (1): 136-150.
5. Surh, Y. J. et al.: Planta Med. 2008, 74 (13): 1526-1539.
6. Tufekci, K.U. et al.: Parkinsons‘s disease 2011, 2011: 314082.
1
The FK506 binding protein 51 (FKBP51) is a member of the immunophilin
superfamily. FKBP51 acts as an Hsp90-associated co-chaperone regulating
responsiveness of steroid hormone receptors. Genetic association studies
revealed an association of FKBP51 with emotional processing and affective
disorders such as major depression. Until now, its molecular effects were
mainly considered to be mediated via steroid hormone receptors. Here, we
demonstrate that part of its effects in neuronal cells might be induced by the
interaction with the classical transient receptor potential channel TRPC6.
TRPC6 channels are non-selective cation channels permeable for mono- and
divalent cations such as calcium. In the CNS, TRPC6 channels are discussed
to be involved neuronal differentiation and proliferation, in axon pathfinding and
synaptogenesis during development but also in the adult brain. Furthermore,
TRPC6 channels are the molecular target of hyperforin, the antidepressant
ND.04
189
ND.05
Novel analogues of strychnine as potent inhibitors of glycine
receptors
Zlotos, D.P.1; Mohsen, A.Y.1; Holzgrabe, U.2; Jensen, A.A.3
The German University in Cairo, Dept. of Pharmaceutical Chemistry, New Cairo City,
11835 Cairo, Egypt
2 Institut für Pharmazie and Lebensmittelchemie, Universität Würzburg, Am Hubland,
3 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
1
Strychnine (1), the major alkaloid from the plant Strychnos nux vomica,
exhibits pharmacological activity at several neurotransmitter receptors,
including a number of ligand-gated ion channels. Its most pronounced action is
a strong antagonistic activity at glycine receptors (GlyRs), often referred to as
‘strychnine-sensitive glycine receptors‘, which are anionic chloride channels
linked to hyperpolarisation and inhibition of neuronal firing [1-3]. In the course
of our studies on Strychnos alkaloids, we have recently identified the lactam
group and the C19-C20 double bond of strychnine as essential structural
features required for strong activity at glycine α1 and α1β receptors [4].
Moreover, the only structure modification that did not impair the activity of
strychnine was the (E)-configurated hydroxyimino group at C22 (compound 2)
[5]. Here, we describe the synthesis and pharmacological evaluation of a
series of oxime ethers 3a-f obtained by O-alkylation of 2. Interestingly, most of
the new ligands 3a-f display high antagonistic potency at glycine α1 and α1β
receptors, comparable to that of strychnine.
N
N
H
20
H
19
H
N H
H
N H
H
O
O
O
H
O
H
N
H
OR
3a-f
1
N
R
H
H
N H
H
O
O
N
H
OH
a
b
c
d
e
f
Me
nPr
nBu
allyl
benzyl
-(CH2)2Ph
2
Acknowledgments: Deutscher Akademischer Austauschdienst (DAAD), The Novo Nordisk
Foundation
References:
1. Lynch, J.W.: Physiol. Rev. 2004, 84: 1051.
2. Laube, B. et al.:Trends Pharmacol Sci., 2002, 23: 519.
3. Jensen, A.A.; Kristiansen, U.: Biochem. Pharmacol. 2004, 67: 1789.
4. Mohsen, A.M.Y. et al.:Chemistry & Biodiversity, 2014, accepted
5. Jensen, A.A. et al.:Eur. J. Pharmacol. 2006, 539: 27.
ND.06
Activation of SK channels prevents ER stress-induced neuronal
cell death.
Richter, M.1, 2; Dolga, A.M.1; Dodel, R.2; Culmsee, C.1
1 Institute
for Pharmacology and Clinical Pharmacy, University Marburg, 35032 Marburg,
Germany
2 Department of Neurology, University Marburg, 35043 Marburg, Germany
190
Stress of the endoplasmic reticulum (ER stress) is involved in the pathogenesis of various neurodegenerative diseases. Contribution of the unfolded protein
response (UPR) in Parkinson’s and Alzheimer’s disease has been shown in
post-mortem human brains and different rodent models in vivo. Thus, drugs
that cope with neuronal ER stress are supposed to have broad therapeutic
potential. Recent studies emphasized the importance of small conductance
calcium-activated potassium (SK) channels in neuronal calcium homeostasis.
Cytoprotection by activation of mitochondrial SK channels has been described
in conditions of mitochondrial dysfunction in neuronal cells. Here, we addressed the question whether pharmacological activation of SK channels can
affect the ER unfolded protein response, and protect against ER stressassociated cell death.
Pharmacological activation of SK2 channels by use of CyPPA (N-CyclohexylN-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine) rescued HT-22
neurons from apoptosis induced by the ER stress evoking compound brefeldin
A as analyzed by AnnexinV/PI staining and flow cytometry. Brefeldin A
treatment affected intracellular calcium levels, which was compensated by SK
channel activation. Furthermore, cleavage of caspase 12 and caspase 3 was
attenuated as detected by Western Blot. SK channel activation altered the
level of UPR proteins, i.e. further increased CHOP levels and reduced ATF4
levels compared to brefeldin A-treated cells. Knockdown of ATF4 by siRNA
approaches showed similar protective effects like SK channel activation,
pointing for an important role of ATF4 protein attenuation in CyPPA-mediated
protection against brefeldin A. Oxidative stress (assessed by H2DCFDA and
MitoSOX), as a late result of ER stress, was diminished by positive modulation
of SK channels. In the absence of extracellular calcium as well as in the
presence of the extracellularly acting SK channel blocker apamin, pharmacological activation of SK channels was still protective against ER stress.
Therefore, we concluded that intracellularly located SK channels are important
for CyPPA-mediated protection.
Overall, these data show that pharmacological SK channel activation restores
disturbed calcium levels and reduces neuronal cell death associated with ER
stress. Intracellularly located SK channels are essential mediators of protection
against ER stress. Collectively, it is proposed that SK channels could be a
therapeutic target in neurodegenerative diseases related to ER stress.
ND.07
Bid links ferroptosis to mitochondrial cell death pathways in
neurons
Neitemeier, S.; Laino, V.; Oppermann, S.; Ganjam, G.K.; Dolga, A.; Culmsee,
C.
Institut für Pharmakologie und Klinische Pharmazie, Biochemisch-Pharmakologisches
Centrum Marburg, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg
Germany
Ferroptosis is considered to be an iron-dependent form of nonapoptotic cell
death first described in cancer cells induced by the small-molecule erastin
which inhibits the cystine/glutamate antiporter (Xc-). The subsequent increase
in lipid peroxides is a major hallmark in the death pathways of ferroptosis in
contrast to other forms of apoptotic cell death [1]. The inhibition of the X c- is
also the initiator of a cell death pathway called oxytosis in which toxicity is
achieved by high extracellular glutamate concentrations. Oxytosis plays a
pivotal role in neurodegeneration [2]. Besides the formation of reactive oxygen
species (ROS) the activation of the proapoptotic BH3-only protein Bid is crucial
in this cell death mechanism and leads to mitochondrial demise [3]. In the
present study we investigated whether pathways of ferroptosis and oxytosis
may be linked through the activation of Bid and according mitochondrial
damage in neural cells.
In order to elucidate the role of Bid in ferroptosis we induced cell death with
erastin in a mouse hippocampal HT-22 cell line and investigated the effects of
the Bid-inhibitor BI-6c9 on cell viability and mitochondrial integrity and function.
Further, we analyzed the effects of ferrostatin-1 in glutamate-induced
mitochondrial damage and neural cell death. The inhibition of Bid by BI-6C9
prevented erastin-induced morphological changes of HT-22 cells and the loss
of cell viability detected by the MTT assay. Similar protective effects were
observed by ferrostatin-1 in the glutamate model of oxytosis. Because
lipoxygenation is crucial for erastin-dependent cell death lipid peroxidation was
further investigated by Bodipy staining and subsequent FACS analysis. BI-6c9
fully abolished lipid peroxide formation in the erastin model, and also ferrostatin-1 inhibited lipid peroxidation after the glutamate challenge. Mitochondrial
ROS production was also significantly decreased by BI-6c9 and ferrostatin-1 in
the particular model systems assessed by MitoSOX staining and FACS
analysis. Next, mitochondrial morphology and function were analyzed to gain
more insights into the changes in mitochondrial integrity after the respective
challenges associated with oxytosis and ferroptosis. BI-6c9 significantly
reduced mitochondrial fission and also preserved the mitochondrial membrane
potential in erastin-treated cells detected by flow cytometric analysis after
TMRE staining. Further, luciferase-based measurements of ATP revealed that
BI-6c9 prevented erastin-induced loss of ATP. Ferrostatin-1 reduced mitochondrial fission, preserved mitochondrial membrane potential and ATP levels
after glutamate exposure as well. To further confirm the role of Bid in both cell
death pathways HT-22 cells were co-transfected with a red-labeled Bid and a
mitochondrial-targeted GFP vector to investigate the translocation of Bid to the
mitochondria in response to erastin and glutamate-induced stress, respectively. Both BI-6c9 and ferrostatin-1 were able to inhibit the translocation of Bid to
mitochondria as detected by fluorescent confocal microscopy.
Overall, these results show that ferroptosis and oxytosis share common
features and that Bid links both pathways to intrinsic mechanisms of mitochondrial demise.
References:
1. Dixon, S.J. et al.: Cell 2012, 149(5): 1060-1072.
2. Tan, S.; Schubert, D.; Maher, P.: Curr. Top. Med. Chem. 2001, 1(6): 497-506.
3. Tobaben, S. et al.: Cell Death Differ. 2011, 18(2): 282-292.
ND.08
Sustained neuroprotective effects after pharmacological inhibition of p53 compared to siRNA-mediated p53 silencing
Neitemeier, S.; Diemert, S.; Ganjam, G.K.; Culmsee, C.
Centrum Marburg, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg
Germany
The tumor suppressor p53 is regarded as the guardian of the genome playing
crucial roles in sensing DNA damage and cell cycle regulation. Depending on
the nature and the extent of cellular stress and associated DNA damage, p53
can either mediate DNA repair or cell death.
The inhibition of p53 by the drug pifithrin-alpha (PFT) has been shown to be
protective against oxidative stress and subsequent mechanisms of programmed cell death in neuronal cells. This suggests that the selective
inhibition of p53 is a potential target to mediate neuronal survival. Therefore, in
the present study we used a model of glutamate induced cell death in a mouse
hippocampal HT-22 cell line to further investigate p53-dependent pathways in
neuronal cell death.
Specific siRNA (20 nM) targeting p53 was applied in the HT-22 cells for
silencing of the tumor suppressor protein. The p53 knock down was confirmed
at mRNA levels and protein levels by RT-PCR and Western blotting, respectively. Gene silencing of p53 protected HT-22 cells against glutamate induced
damage detected by the MTT assay. However, in contrast to the sustained
protective effects of PFT, the neuroprotection by the p53 siRNA was transient
and lasted only for two hours as detected by real time impedance measurements (xCelligence system, Roche). Both, PFT and p53 siRNA blocked the
transcriptional activity of p53 after glutamate treatment and under control
conditions as shown by a luciferase reporter assay. Notably, p53 siRNA did not
preserve mitochondrial morphology and integrity, as detected in fluorescent
microscopic analysis of mitochondrial fission and measurements of the
mitochondrial membrane potential at 7 h and 14 h after onset of the glutamate
challenge by FACS analysis. In contrast, PFT significantly reduced mitochondrial fission and also preserved the mitochondrial membrane potential in
glutamate treated cells.
Overall, these results suggest that p53 siRNA mediated neuroprotective effects
via transcriptional effects while PFT may exert additional effects at the level of
mitochondria, which was essential to provide sustained neuroprotection.
ND.09
Improvement of mitochondrial dysfunction by modulators of the
human γ-secretase modulators/PPARγ agonists with a dual
mechanism of action
Pohland, M.1; Hagl, S.1; Wurglics, M.2; Schubert-Zsilavecz, M.2; Eckert, G.P.1
1 Department
of Pharmacology, Goethe-University Frankfurt am Main, Max-von-LaueStraße 9, 60438 Frankfurt, Germany
2 Instiute of Pharmaceutical Chemistry, Goethe-University Frankfurt am Main, Max-vonLaue-Straße 9, 60438 Frankfurt, Germany
Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder leading
to dementia. Deposits of beta amyloid protein (Aβ) and intracellular neurofibrillary tangles are pathological hallmarks of AD. Increasing evidences indicate
mitochondrial dysfunction as an early event in AD pathogenesis. Mitochondria
are essential for the supply of energy but are also involved in oxidative stress
and apoptosis. Current drugs act merely symptomatic and new disease
modifying drugs against AD have almost failed in human clinical trials recently.
We investigated the efficacy of a novel class of acidic γ-secretase modulators/PPARγ agonists with a dual mechanism of action against mitochondrial
dysfunction in HEK293-APP695 cells expressing neuronal APP. Dimebon,
DAPT, and Pioglitazone were used as controls.
The basic structure of the new compounds, derivated of pirinixic acid, is
thiobarbituric acid. Optimization of pharmacological activities leaded to new
molecules with a significantly increased activity at both pharmacological
targets. All examined compounds were active and influenced the cell biology of
the studied HEK293-APP695 cells. First, the cell viability was measured by
MTT assay in the range of 0,03 - 30 µM. For the non-toxic concentrations we
measured changes in mitochondrial membrane potential (MMP) and levels of
adenosine triphosphate (ATP) to determine alterations of mitochondrial
efficiency and function. The compounds MH49, MH73, MH84 and MH163 were
able to exhibit significant protective effects against NO-releasing drugs like
sodium nitro prusside (SNP).
Aβ-induced changes in mitochondrial enzyme activities are leading to oxidative
stress and enhanced apoptosis. The rate of mitochondrial respiration was
investigated and especially in the complex-IV respiration, which is decreased
by Aβ, we were able to show a significant improvement after treatment with
compound MH84 compared to control group.
To get a closer look insight, we measured the citrate synthase (CS) activity,
used as a marker enzyme for the mitochondrial mass. CS activity in HEK293APP695 cells was increased by the GSM MH84, indicating an increased
mitogenesis.
In summarizing review substances MH84 and MH73 in our in-vitro experiments
were the most convincing compounds in the nM range. Further experiments
will reveal the molecular consequences of γ-secretase/PPARγ modulation and
to test the compounds in mouse models of Alzheimer`s disease.
This project is supported by the Doktor Robert Pfleger-Stiftung
ND.10
Caspase inhibition prevents alpha synuclein-toxicity in human
dopaminergic neurons
Ganjam, G.K.1; Dolga, A.M.; Neitemeier, S.1; Bolte, K.4; Höllerhage, M.2;
Oertel, W.E.3; Hoeglinger, G.U.2,3; Culmsee, C.1
Centrum Marburg, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032
Marburg, Germany
2 German Center for Neurodegenerative Diseases (DZNE), Max Lebsche Platz 30, 81377
Munich, Germany.
3 Department of Neurology, Philipps-Universität Marburg, Rudolf-Bultmann-Strasse 8,
35033 Marburg, Germany.
4 Department of Biology, Faculty of Biology,Philipps-University Marburg, Karl-von-FrischStraße 8, 35043 Marburg, Germany.
1
191
Parkinson’s disease is a common neurodegenerative movement disorder
characterized by midbrain dopaminergic neuronal loss in the substantia nigra
that has been linked to alpha-synuclein toxicity. However, the molecular
mechanisms underlying alpha-synuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. The goal of this study was to
investigate the deleterious effects of alpha synuclein in particular mitochondrial
toxicity in human dopaminergic cells. Therefore, we have generated neuron
specific, adeno associated virus type 2 (AAV2) expressing cytosolic as well as
mitochondrial targeted alpha synuclein and EGFP expressing viruses used as
respective controls.
Overexpression of both, the cytosolic and the mitochondrial variants of alpha
synuclein severely disrupted the dendritic network, induced loss of cellular
ATP, enhanced mitochondrial ROS production, and was associated with
activation of caspases and dopaminergic cell death in a time-dependent
manner. In addition, real-time analysis of mitochondrial bioenergetics using the
Seahorse Bioscience system following AAV infection elicited a complete
damage to mitochondrial respiration capacity in the dopaminergic neurons. Our
results suggested that mitochondrial targeted expression of alpha synuclein
appeared to be more toxic than the cytosolic form of alpha synuclein. In
addition, ultrastructural mitochondrial morphological analysis by transmission
electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and
massively swollen mitochondria following the expression of mitochondrial
targeted alpha synuclein in the human dopaminergic neurons.
In addition, by pharmacological approaches, we found that inhibition of
caspases by QVD significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. Interestingly, inhibition of caspases preserved neuronal
network integrity, ATP levels and mitochondrial respiration capacity in both
paradigms of cytosolic and mitochondrial alpha synuclein overexpression.
Overall, our findings show that cytosolic as well as mitochondrial targeted
expression of alpha synuclein is detrimental to human dopaminergic neurons,
while inhibition of caspases amend alpha synuclein toxicity. Thus, caspase
inhibitors provide promising therapeutic potential to prevent dopaminergic
neuronal death in Parkinson’s syndromes that are associated with alpha
synuclein toxicity.
ND.11
Dynamic changes in extracellular ATP levels during status
epilepticus as monitored by a novel microdialysis probe
Lietsche, J.; Imran, I. ; Hardt, S. ; Klein, J.
Department of Pharmacology, Biocenter N260, Max-von-Laue Str. 9, Goethe University
Frankfurt, 60438 Frankfurt, Germany
In the neuroscience research environment microdialysis is a widely applied
technique to collect extracellular fluid of many tissues, including brain.
Samples of the extracellular space are proteinfree and can be used for
chemical analysis without any further purification [1]. The major element of
microdialysis is the probe which contains a semi-permeable membrane.
Substances in extracellular fluid surrounding the probe pass the dialysis
membrane by passive diffusion along their concentration gradient [2].
In our lab a custom-made microdialysis probe with a 10 kDa molecular weight
cut-off (MWCO) is applied. The assembly was developed by Santiago and
Westerink in 1990 [3]. This probe is equipped with a polyacrylonitrile membrane (AN69 HF; Hospal-Gambro, Planegg-Martinsried, Germany) which is no
longer available. Therefore a newly developed probe containing a semipermeable membrane (FXCorDiax 600; Fresenius Medical Care, Bad Homburg,
Germany) with a MWCO of 30 kDa had to be constructed with new parameters
because the diameter of the dialysis membrane was smaller than that of
polyacrylonitril membrane (210 µm vs. 280 µm). In the course of probe
manufacture, several parameter such as tubing diameters, silica diameters and
type of glue had to be optimized.
The 30 kDa custom-made probe (exchange length 2 mm) yields significantly
higher recoveries for ATP (22.4 ± 0.7 %) than the 10 kDa custom-made probe
(13.1 ± 0.7 %). The improvement of ATP recovery allowed measurement of
ATP in the extracellular space in distinct brain areas with the method of
microdialysis.
Here, we show dynamic changes of hippocampal ATP release during lithiumpilocarpine induced status epilepticus in rats. Administration of pilocarpine
(30mg/kg s.c.) to rats pretreated with litium chloride (127 mg/kg i.p.) caused a
decrease of ATP levels when rats start to develop tonic seizures. The present
study reveals that basal ATP levels (1.12 nM; recovery corrected) decrease
under detection limit (0.1 nM) immediately with the beginning of status
192
epilepticus and remain low when seizures were stopped by administration of
diazepam (10mg/kg i.p.).
Various studies have shown that the purinergic system is involved in the
pathophysiology of epilepsy. During epileptic seizures adenosine concentrations are increased which are considered to have an anticonvulsant effect [4].
Adenosin originates from the catabolism of ATP by ectonucleotidases and
ecto-5´-nucleotidase [5] which leads to the assumption that, when adenosine
concentration increases, ATP concentration has to decrease. This hypothesis
is confirmed by our findings described above.
Acknowledgments:
The authors are grateful to G. Barka (SunChrom, Friedberg, Germany) who provided the
epoxy glue, and to Fresenius Medical Care (Bad Homburg, Germany) for the supply of the
Capillary Haemodiafilter FX CorDiax 600. Funding was obtained from Goethe University,
Frankfurt.
References:
1. Benveniste, H.: J. Neurochem. 1989, 52(6): 1667-1679.
2. Bourne, J.A.: Clin. Exp. Pharmacol. Physiol. 2003, 30(1): 16-24.
3. Santiago, M., Westerink, B.H.: Naunyn-Schmiedebergs Arch. Pharmacol. 1990, 342,
(4): 407-414.
4. Boison, D.: Neuroscientist. 2005, 11(1): 25-36.
5. Zimmermann, H.: Neurochem. Int. 2008, 52(4): 634-648.
ND.12
A kinetic study of triheptanoin fed mice in a middle cerebral
artery occlusion model
Koch, K.A.; Konietzka, J.; Berressem, D.; Eckert, G.P.; Klein, J.
Department of Pharmacology, Goethe University, Max-von-Laue Str. 9, 60438 Frankfurt
am Main, Germany
Stroke is a major medical emergency and the second most frequent cause of
death in the world. While drug treatment of stroke remains unsatisfactory,
dietary approaches to stroke prevention and regeneration, such as anaplerotic
diet, have recently attracted much interest. Our anaplerotic approach is
triheptanoin, which is cleaved into glycerol and three heptanoate moieties. In
the liver, heptanoate is metabolized via β-oxidation in two mol of acetyl-CoA
and one mol of propionyl-CoA. Our group hypothesized that propionyl-CoA
could act as an anaplerotic agent being metabolized to succinyl-CoA which
feeds into the citric acid cycle. In the present study, we have used a common
experimental stroke model, middle cerebral artery occlusion (MCAO), in
triheptanoin-fed mice to investigate the levels of odd chain fatty acids before
and after brain ischemia. In female CD-1 mice, ischemia was induced in the
left hemisphere with the MCAO method. After 90 min of ischemia, mice were
sacrificed, blood was withdrawn and liver and brain tissue were harvested and
extracted for odd-chain fatty acid measurements by GC-MS. Brain and liver
homogenates were extracted in a biphasic manner with methanol/water/chloroform. The residue of the water soluble phase was derivatized
with BSTFA/TMCS (99:1). In liver homogenate of mice fed triheptanoin ad
libitum for two weeks, odd chain fatty acids were: propionate (sham group)
25.66 ± 8.65 µM, propionate (stroked group) 37.18 ± 10.69 µM, pentanoate
(sham group) 14.70 ± 4.28 µM and pentanoate (stroked group) 29.52 ± 8.41
µM (data ± SEM, N=6) (all data calculated for intracellular water). In the
control group, which was fed with soja oil instead of triheptanoin, the odd-chain
fatty acids were below limit of detection. In brain homogenates, the following
data were obtained: propionate (sham group) hemisphere 32.67 ± 5.64 nM,
propionate (stroked group) left hemisphere 60.52 ± 6.40 nM, right hemisphere
70.05 ± 18.76 nM; pentanoate (sham group) 60.10 ± 13.04 nM; pentanoate
(stroke group) left hemisphere 109.20 ± 23.00 nM, right hemisphere 100.8 ±
21.92 nM. Plasma levels were: propionate (sham group) 5.79 ± 1.47 µM and
propionate (stroked group) 6.51 ± 2.08 µM; pentanoate (sham group) 7.59 ±
4.31 µM and pentanoate (stroked group) 4.31 ± 0.30 µM; heptanoate (sham
group) 15.45 ± 5.43 µM and heptanoate (stroked group) 2.24 ± 0.21 µM.
In conclusion, we found a characteristic pattern of distribution in triheptanoinfed mice. While control mice, which were fed with soja oil, did not show any
detectable levels of odd-chain fatty acids in plasma or brain, triheptanoin-fed
mice had levels of propionate and pentanoate in the micromolar range. Oddchain fatty acids in triheptanoin-fed mice were highest in the liver, lower in in
plasma and even lower in brain homogenates. Current projects are aimed at
measuring extracellular levels of these metabolites in the brain by microdialysis.
ND.13
Dynamics of acetylcholine and metabolites release during
lithium-pilocarpine-induced status epilepticus in rats: a microdialysis study
Imran, I.; Hillert, M.; Klein, J.
Department of Pharmacology, Max von Laue Str. 9, Goethe University Frankfurt, Germany
Status epilepticus (SE) is a life threatening condition which requires an
intensive therapeutic management. The lithium-pilocarpine model is an
epilepsy model in rats inducing status epilepticus with low mortality rate. In this
study we measured changes of acetylcholine (ACh) and metabolites released
in the hippocampus before, during and after status epilepticus as monitored by
microdialysis in unanesthetized rats. After 90 minutes of status, rats were
decapitated for the isolation of mitochondria from brain to evaluate the
respiratory functions. Administration of pilocarpine (30 mg/kg s.c.) to rats
which were pretreated with lithium chloride (127 mg/kg) caused a massive, sixfold increase of hippocampal ACh release concomitant with the development
of tonic seizures. When seizures were terminated with diazepam (10 mg/kg) or
ketamine (75 mg/kg) after 90 minutes, ACh levels returned to normal.
Administration of atropine (1 mg/kg) 2 h after pilocarpine caused a further
increase of ACh but did not affect seizures whereas injection of mecamylamine
(5 mg/kg) reduced ACh levels. Local infusion of tetrodotoxin (TTX, 1 µM) or
hemicholinium (HC-3, 10 µM) strongly reduced ACh release, but had only a
minor and delayed effect on seizures. Among the metabolites quantified;
glucose remained unchanged, lactate increased up to 4-6 fold. Glycerol an
indicator of membrane damage increased up to 6-10 folds. Surprisingly,
mitochondrial respiration was found to be absolutely normal after 90 minutes of
SE. Taken together, our results demonstrate that seizure development in SE
model is accompanied by massive increases of extracellular ACh, lactate and
glycerol whereas glucose levels and mitochondrial integrity remain unaltered.
Inhibition of ACh release or blockade of cholinergic receptors, however, does
not terminate seizures.
I am thankful to DAAD (German academic exchange service) for stipend and other
financial support.
193
BIOPHARMACEUTICS (BP01-BP09)
BP.01
BP.02
How to address the variety of eating habits in children in the
development of biorelevant dissolution test methods simulating
fed state conditions in the paediatric GI tract
Kersten, E.; Blank, S.; Klein, S.
University of Greifswald, Department of Pharmacy, Institute of Biopharmaceutics &
Pharmaceutical Technology
Eating and drinking habits of children can be very heterogeneous and
especially in the first years of life, they differ significantly from adult eating
behaviour [1]. Since food and fluid consumption may have a tremendous
impact on drug dissolution in the upper gastrointestinal tract, it is of vital
importance to address the special eating habits in children when developing of
biorelevant dissolution methods mimicking the fed state in children of different
age groups.
We performed a survey on the food and fluids consumed by children at the age
of 1 - 6 years. Parents of infants and pre-school children were asked to
document the respective data over 4 days in a questionnaire. 97 completed
questionnaires were received back. The reported meals and fluids were
grouped according to the age of the child. Since the morning is the most
important time of drug intake, the focus of the present study was set on the
composition of the breakfast.
When evaluating the data set, it became obvious that there are huge inter-, but
also intraindividual differences when comparing the meal and fluid composition, as can be seen in table 1. This is particular true for children at the age of
1 – 2 years. The table shows the breakfast consumed by 3 individuals at four
days. Whereas one of the infants still consumed formula milk without any solid
food, breakfast composition of the other two children was similar to that of
other children or adults and also varied from one day to another.
This observation clearly indicates that there will not be a “one-fits-all” test
design but that rather the variability observed in our survey with extreme
conditions whereas food was administered without any fluid or meals with high
osmolality and/or fat content were administered will need to be properly
addressed to obtain predictive in vitro results
Individual
1
Food/Drink
Individual 2
Individual 3
Food
1 roll with
strawberry jam
235 mL
½ roll with
Day
formula
spread meat
1
milk Pre
1 clementine
1 toast with
spread meat
1 toast with
butter
235 mL
1 toast with
Day
formula
liver sausage
2
milk 1
1 yoghurt
(strawberry
flavour)
1 toast with
liver sausage
235 mL
½ roll with liver
Day
formula
sausage
3
milk 1
1 piece of
cucumber
1 clementine
1 toast with
spread meat
235 mL
1 toast with
Day
formula
liver sausage
4
milk 1
1 toast with
margarine
1 clementine
Table 1: Breakfast of three children at
four different days
Drink
20 mL
water
-
30 g
cornflakes
150 mL milk
(3.8 % fat)
4
cherry
tomatoes
2 mini-salami
15 g
cornflakes
50 mL milk
(1.8 % fat)
2
cherry
tomatoes
1 mini-salami
Drink
50 mL
fennel
tea
100 mL
milk
(1.8 %
fat)
-
½ rolls with
butter and
strawberry
jam
2
small
tomatoes
150 mL
fennel
tea with
apple
juice
-
1 toast
1 slice of
salami
2
small
tomatoes
100 mL
milk
(3.5 %
fat)
the age of one year on
Reference:
1. Mesch, C. et al.: Appetite, 2014, 76: 113-119.
194
Food
Structural Dynamics of Novel Gastrointestinal Model Fluids by
combined SANS and DLS
Khoshakhlagh, P.1a; Johnson, R.1a; Nawroth, T.1a; Langguth, P.1a; Schmueser,
L.1b; Decker, H.1b; Hellmann, N.1b; Szekely, N.2
1 Gutenberg-University,
a) Pharmacy; b) Molecular Biophysics; Staudingerweg 5, D-55099
Mainz
2 JCNS Outstation at FRM II, Instrument KWS2, Lichtenbergstr.1, D-85747 Garching
Oral administration of drugs is considered as the preferred route in drug
delivery. Solubility and bioavailability of drugs are depicted by the Biopharmaceutics Classification System (BCS). Lipophilic drugs of the BCS classes II and
IV are subject of drug formulation development during the last decade.
However, many of these drugs do not have sufficient solubility and that makes
them undesirable for in vivo studies.
In vivo, the chyme containing the drug from stomach emptying undergoes in
the first half of the duodenum a pH shift. After subsequent addition of bile and
pancreatic juice it shows a slightly acidic pH (6.5), while the concentration
varies depending on the composition of the meal and fluid intake. In parallel
intestinal nanoparticles (micelles and liposomes) develop from the bile
components, food and drug in a dynamic process. These native nanoparticle
systems can be used and modified for lipophilic drug solubilization and uptake.
In vitro biorelevant media such as fasted state simulated intestinal fluid
(FaSSIF) and fed state simulated intestinal fluid (FeSSIF) are established tools
to predict the absorption of several drugs in vivo.
We have developed physiologically similar intestinal fluids, which differ from
the former FaSSIF/FeSSIF by the presence of cholesterol. The lipid composition in the novel FaSSIF-C fluids was systematically varied, according to the
lipids occurring in the physiological composition of the human bile: FaSSIF-7C
modelled female, FaSSIF-10C male, and FaSSIF-13C people with diseases
leading to gall stones.
Time resolved Dynamic Light Scattering (DLS) and Small Angle Neutron
Scattering (SANS) were used to investigate the structural development
(kinetics) of the fluids with and without Fenofibrate (BCS class 2) in a
gastrointestinal simulator device (GIMod). The development of micelles to
liposomes with intermediate size structures was observed. In the time regime
1-60 min after the modelled bile influx to the intestine (FeSSIF to FaSSIF
dilution), two micellar intermediates were detected, which could influence the
resolution and delivery of the hydrophobic drug.
Acknowledgments: We are thankful for the funding by the German ministry of science and
education BMBF, grant 05KS7UMA; the Forschungszentrum Jülich FZJ, Jülich Centre of
Neutron Science JCNS, outstation at the FRM2 reactor Munich Garching; and support by
the Dr. Georg-Scheuing Stiftung, Mainz. This work was contributed to the OrBiTo project
(http://www.imi.europa.eu/content) as side ground.
References:
1. Amidon, G. L. et al.: Pharmaceut. Res. 1995, 12(3): 413-420.
2. Nawroth, T. et al.: Mol. Pharm. 2011, 8,(6): 2162-2172.
3. Kleberg, K. et al.: J. Pharm. and Pharmacol. 2010, 62(11): 1656-1668.
BP.03
Use of Simulated Intestinal Fluid Solutions in Integrated Dissolution/Permeation Models for Poorly Soluble Drugs with Rat
Intestine
Meinhardt, K.1; Khoshakhlagh, P.1; Konerding, M.2; Nawroth, T.1; Langguth, P.1
1 Johannes
Gutenberg University Mainz, Staudingerweg 5, 55099, Germany
Medical Center of Johannes Gutenberg University Mainz, Johann-Joachim
Becher Weg 13, 55099, Germany
2 University
Biopredictive media as simulated intestinal fluids (SIFs) are often used in
dissolution experiments of poorly soluble drugs, making it reasonable to also
apply them in integrated dissolution/permeation models. Until now they are
considered incompatible with rat intestine1). This incompatibility has only been
shown for rat ileum2). The attempted explanation as to why SIFs are compatible with Caco-2 cells but not with rat ileum has been the different experimental
set-up: Rat ileum has been used in the Ussing Chamber where the abrasion
could have been higher than in the experimental set-up used for the Caco-2
cell system. We suggest that the incompatibility is rather region specific and
can be circumvented using upper parts of the intestine as excised sheets.
The viability of three parts of rat intestine (duodenum, jejunum and ileum) was
investigated in various media: Hank’s Balanced Salt Solution (HBSS), Modified
Fasted-State Simulated Intestinal Fluid (FaSSIFmod 6.53), Fasted-State Simulated Intestinal Fluid including 7 % Cholesterol (FaSSIF-7C), Modified Fed-State
Simulated Intestinal Fluid (FeSSIFmod 6.53) and Fed-State Simulated Intestinal
Fluid including 7 % Cholesterol (FeSSIF-7C). The potential difference across
the excised sheet was constantly measured for 2 hours as indicator of the
intestinal viability. At the end of the experiment the morphological state of the
corresponding intestinal region was monitored via scanning electron micrograph.
The results indicate the viability of duodenum in all of the investigated media
throughout the duration of the experiments. This applies to some extent to the
jejunum. The ileum, as the lowest investigated part of the intestine, is not
viable in simulated intestinal fluids.
This study shows that an integrated dissolution/permeation model using both,
simulated intestinal fluids as dissolution media and rat intestine as permeation
model, is feasible provided that the intestinal segment is chosen adequately.
Acknowledgments: We thank Mrs Kerstin Bahr for her great technical assistance with the
scanning electron micrograph.
References:
1. Kleberg, K. et al.: Journal of Pharmacy and Pharmacology. 2010, 62(11): 1656–1668.
2. Patel, N. et al.: Drug Development and Industrial Pharmacy. 2006, 32(2): 151–161.
3. Kataoka, M. et al.: Journal of Pharmaceutical Sciences. 2006, 95(9): 2051–2061.
BP.04
Ionic liquid versus prodrug strategy – overcoming biopharmaceutical challenges
Wiest, J.; Balk, A.; Meinel, L.; Holzgrabe, U.
Institute of Pharmacy and Food Chemistry, Am Hubland, University of Würzburg, DE97074 Würzburg, Germany
The main problems of drug discovery and development are compounds of high
molecular weight and high lipophilicity combined with poor water solubility [1].
A poorly water soluble acidic active pharmaceutical ingredient (API) against
migraine attacks is prepared as an ionic liquid (IL) and compared to a prodrug
strategy. An ionic liquid is defined as a salt with a melting point below 100°C.
The IL approach leaded to a significantly longer duration of API supersaturation, a 700 fold faster dissolution rate, an 8 fold faster reach of the maximum
plasma concentration and a twofold increased bioavailability as compared to
both, the free acid and the prodrug. The underlying mechanism was studied by
NMR spectroscopy, X-ray crystallography and Sirius experiments.
BP.05
A combined doxorubicin and doxorubicinol pharmacokinetic
model to evaluate dosing regimens in infants and older children results of the EPOC-MS-001 study
Völler, S.1; Boddy, A.V.2; Boos, J.3, Kontny, N.E.1; Krischke, M.4; Würthwein,
G.4.; Hempel, G.1
Westfälische Wilhelms-Universität, Institut für pharmazeutische und medizinische
Chemie, Klinische Pharmazie, Corrensstraße 48, 48149 Münster, Germany
2 Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School,
Framlington Place, Newcastle upon Tyne, NE2 4HH, UK
3 Universitätsklinikum Münster, Klinik und Poliklinik für Kinderheilkunde, Pädiatrische
Hämatologie und Onkologie, Funktionsbereich Pädiatrische Hämatologie und Onkologie,
Albert-Schweitzer-Straße 33, 48149 Münster, Germany
4 Universitätsklinikum Münster, Centre for Clinical Trials, ZKS Münster, Münster, Germany
1
Objectives: Although almost 60% of children diagnosed with cancer receive
anthracyclines as part of their treatment, the knowledge on the pharmacokinetics (PK) of the drug in children, especially in the very young, is limited.
Empirical dose reduction in infants is performed in almost all clinical trial
protocols. However, dose reduction strategies differ widely across studies due
to the unavailability of clinical PK data. As doxorubicin was included in the
European Medicines Agency priority list for studies on off-patent paediatric
medicinal products, a multicentre, multinational phase II PK study investigating
a possible age-dependency in the clearance (CL) of doxorubicin in children
with solid tumours and leukaemia was conducted. The data were analysed
using a population PK model generated in NONMEM® 7.2.
Methods: Samples from 2 doxorubicin administrations in 101 patients treated
according to the tumour-specific national or European therapeutic trial were
collected with a particular focus on recruiting children less than 3 years. PK
data of doxorubicin and its main metabolite doxorubicinol from 94 patients
were analysed using NONMEM® 7.2, R, Xpose4 and a predefined stepwise
strategy. A large number of covariates including patient characteristics,
laboratory values (e.g. bilirubin, serum creatinine, alanine aminotransferase,
serum albumin, haematocrit) and 17 single nucleotide polymorphisms were
available in the study. Covariate modelling was performed using the stepwise
covariate modelling strategy (SCM) integrated in PsN® with the linearisation
option.
Results: A three compartment model for doxorubicin and one additional
compartment for doxorubicinol was most suitable to characterise the PK of
doxorubicin and its metabolite. All parameters of the model were scaled to
body surface area. The inclusion of age on the CL of doxorubicin yielded a
significant improvement of the model. No other patient-related covariate,
including liver function, was found to influence the parameters of the model.
Pharmacogenetic variants, including those in transporters and drug metabolising enzymes, had no influence on pharmacokinetic parameters. Using the
mean estimated CL value for each individual, children less than 3 years had a
lower CL (21.1 ± 5.8 l/h/m2) than older children (26.6 ± 6.7 l/h/m2) (p=0.0004),
even after correcting for body size. These results indicate that the empirical
dose reduction of doxorubicin in infants is justified.
Conclusion: This study demonstrates an age-dependency in the clearance of
doxorubicin in children. The results may be useful for refining dosage regimens
in this patient group.
Acknowledgments: This project was funded by the European Community’s Seventh
Framework Programme (FP7/2009-2013) under grant agreement n° 222910.
Acknowledgements:
2 Novartis Pharma AG, Lichtstraße 35, CH-4002 Basel, Switzerland, Widmer, T;
Berghausen, J.; Galli, B.
3 Institute for Inorganic Chemistry, Am Hubland, University of Würzburg, DE-97074
Würzburg, Germany, Matthes, P; Müller-Buschbaum, K., Bertermann, R.
This study was funded by Novartis Pharma AG, Basel.
References:
1. Lipinski, C.A et al.: Advanced Drug Delivery Reviews 1997, 23: 3-25.
BP.06
Investigation of the hydrodynamics in the Ph. Eur. disintegration
test device
Kindgen, S.1; Wachtel, H.2; Langguth, P.1
1 Institute
of Pharmacy and Biochemistry, Staudingerweg 5, 55128 Mainz, Germany
Ingelheim Pharma GmbH & Co.KG, Binger Straße 173, 55216 Ingelheim am
Rhein, Germany
2 Boehringer
Disintegration of oral solid dosage forms is a key step in drug liberation. In vitro
disintegration testing according to the Ph. Eur. is indispensable in the
195
development of solid oral dosage forms. For such in vitro tests it is essential to
simulate the in vivo conditions accurately to predict the in vivo performance
appropriately. The main factors influencing in vivo disintegration of solid oral
dosage forms are food composition, fluid viscosity, water diffusivity, film
precipitation on tablet surface, hydrodynamics around the dosage form and
mechanical stress on the dosage form [1]. Recently, Radwan et al. studied the
impact of food composition, viscosity and water diffusivity on tablet disintegration and found a delayed disintegration rate and decreased water uptake in
viscous media [1, 2, 3]. But to our knowledge, there is only limited information
available on the hydrodynamics and mechanical stress in the disintegration
test device and the impact of these factors on disintegration.
The aim of this work was to experimentally and computationally characterize
the hydrodynamics in the disintegration test device using Particle Image
Velocimetry (PIV) and Computational Fluid Dynamics (CFD), respectively.
PIV is an optical method to determine velocity fields. Therefore the medium is
enriched with tracer particles which are photographed on small timescales.
The velocity vectors can be calculated from the particle position at the photo
series. The results show, that when entering the tube the particles follow a
laminar pattern. The tablet forces the fluid aside and right above the tablet
some turbulences occur. At a certain height the fluid flow returns to a laminar
pattern.
CFD uses numerical methods and algorithms to solve fluid flow problems.
Using the CFD software SolidWorks, the geometry of the basket rack
assembly and the beaker were reconstructed. A tablet was fixed at the bottom
of one of the tubes of the basket rack assembly and the simulations were run
for media with different viscosities to examine the influence of viscosity on fluid
flow and velocity magnitude.
The fixed tablet represents an obstacle for the fluid which is forced aside.
Although, the basket rack assembly is moved in a sinusoidal profile with
maximum speed of 80 mm/s, the calculated maximum speed in the liquid is
much higher, increasing with increasing viscosity.
From our experiments and CFD calculations we conclude that the flow velocity
in the tubes of the disintegration test device is much too fast compared to the
in vivo situation. The hydrodynamics are depending on the viscosity of the test
medium and do not reflect the in vivo situation. Thus, the compendial
disintegration test device and the test conditions are not suitable to predict the
in vivo performance of solid oral dosage forms and need to be modified.
This work was contributed to the OrBiTo project (http://www.imi.europa.eu/content/) as
sideground.
References:
1. Radwan, A. et al.: European Journal of Pharmaceutical Science. 2014, 57: 273-279.
2. Radwan, A., Amidon, G.L., Langguth, P.: Biopharm. Drug Dispos. 2012, 33: 403-416.
3. Radwan, A. et al.: Mol. Pharmaceutics. 2013, 10: 2283-2290.
assess pulmonary processes independent of systemic distribution characteristics. As a second step, various absorption models were investigated to
characterise the absorption process. Oral bioavailability of swallowed drug was
considered negligible due to virtually no bioavailability of olodaterol after oral
dosing.
Results: A PK model that characterised the pulmonary absorption of
olodaterol with three parallel absorption processes best described the clinical
data after drug inhalation. The three pulmonary absorption processes were
characterised by different first-order absorption rate constants (ka) and different
fractions that were associated with one of the three ka values (fast, intermediate, slow). Only a small fraction of 3.90% of the lung dose was fast absorbed
with an absorption half-life of 18.9 min and 8.35 min in non-smokers and
smokers, respectively. This fast absorbed fraction mainly contributed to the
early maximum plasma concentration. The slowly absorbed fraction of 74.8%
was absorbed with an absorption half-life of 27.1 h and mainly contributed to
the terminal shape of the plasma concentration-time profiles after drug
inhalation and also contributed to higher plasma trough concentrations after
multiple dose inhalation compared to trough concentrations after single dose
inhalation. The lung dose was estimated with 51.7% of the nominal dose with a
wide range of individual estimates ranging from 21.5% to 83.5%.
Discussion/Conclusions: The PK characteristics of inhaled and intravenously
administered olodaterol solution were successfully described by a population
PK model with three parallel absorption processes. The determined lung dose
of approximately 50% was in agreement with in vitro deposition data indicating
a lung dose of 67% [3]. The large range of individual estimates for the lung
dose emphasised the high variability of inhaled drugs. Although in vitro assays
and deposition models indicated that the Respimat has a pronounced
deposition in the peripheral airways and the alveoli [3], where absorption of
dissolved drug is assumed to be fast, an unexpected small fast absorbed
fraction of the lung dose was determined. In addition it was unexpected that a
large fraction of the dissolved drug was slowly absorbed to the plasma with a
half-life of approximately one day. Both findings raised the question if the
frequently used assumption of fast absorption of dissolved drug especially in
the alveolar space holds true. For inhaled olodaterol, the results indicate that
additional aspects than the pulmonary deposition patterns determine the
absorption characteristics. One mechanistically plausible aspect for olodaterol
might be lysosomal trapping.
References:
1. Ruge, C., Kirch, J., Lehr, C.: Lancet Respir Med. 2013, 1(5): 402-413
2. Patton, J.S., Byron, P.R.: Nat Rev Drug Discov. 2007, 53(1):67–74.
3. Ciciliani, A.M., Wachte, l.H., Langguth, P.: Respiratory Drug Delivery 2014, 2: 453-456.
BP.08
BP.07
Systemic and pulmonary pharmacokinetics of inhaled olodaterol
For abstract see short lectures SL.12.
Borghardt, J.M.1,2; Weber, B.2; Staab, A.2; Kunz, C.2; Schiewe, J.3; Kloft, C.1
of Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet
2 Translational Medicine and Clinical Pharmacology, Boehringer Ingelheim Pharma GmbH
& Co. KG, Birkendorfer Str. 65, 88397 Biberach, Germany
3 Respiratory Drug Delivery, Boehringer Ingelheim Pharma GmbH & Co. KG, Binger Str.
173, 55216 Ingelheim am Rhein, Germany
BP.09
1 Dept.
Objectives: Up to now, the quantitative, mechanistic understanding about
pulmonary deposition, dissolution and absorption of inhaled drugs is limited
and based on many assumptions, such as fast absorption of dissolved drugs
[1,2]. With the aim to increase the quantitative understanding about these
processes and to evaluate several assumptions about the pharmacokinetics
(PK) after drug inhalation, clinical data of inhaled olodaterol (a long-acting sympathomimetic drug approved for the treatment of COPD) was analysed
with a mathematical modelling approach.
Methods: Plasma concentration-time profiles after inhalation and intravenous
(IV) administration and urine data after IV administration were available for
population PK modelling from three trials in healthy volunteers. In addition to
single dose profiles, one trial also contained plasma concentration-time profiles
after once daily inhalation of an olodaterol solution with the Respimat® over two
weeks. The PK analyses were performed using NONMEM 7.2.0 and R 2.14.2.
As a first step, a systemic PK disposition model was developed with IV data
only. Subsequently this IV model was assumed constant for further analyses to
196
Structural Dynamics of Liposomal Amphotericin B in FaSSIF by
combined SANS and DLS
Johnson, R.1a; Khoshakhlagh, P.1a; Nawroth, T.1a; Langguth, P.1a; Schmueser,
L.1b; Decker, H.1b; Hellmann, N.1b; Szekely, N.2
Gutenberg-University, a) Pharmacy; b) Molecular Biophysics; Staudingerweg 5, D-55099
Mainz
2 JCNS Outstation at FRM II, Instrument KWS2, Lichtenbergstr.1, D-85747 Garching
1
Amphotericin B remains the drug of choice for the treatment of systemic fungal
infections. However, it is still administered parenterally due to its poor solubility
and permeability (BCS IV) as well as instability in gastric fluid. Current
research is directed towards the development of an appropriate oral dosage
form with improved aqueous solubility and intestinal permeability. Particle size
plays a crucial role in the solubilisation and uptake of drugs in the gastrointestinal tract. The release of bile acids into the duodenum influences the size and
structural development of dosage forms thus affecting the extent to which
drugs permeate the mucosa of the small intestines(1). Additionally, intestinal
lymphatic transport has been considered a minor pathway of drug absorption
except for highly lipophilic molecules and other lipoidal molecules. For these
molecules, of which Amphotericin B is part, the intestinal lymphatics represent
an alternate route to the portal blood by which they can gain access to the
systemic circulation [2].
With this insight, small unilamellar vesicles of amphotericin B were prepared by
the film method, swelling, vortexing and subsequent sonication using DOPC
with and without cholesterol. The nanoparticle size (100 nm (MLV), 30 nm
(SUV)) and structure were monitored by dynamic light scattering DLS and
neutron small angle scattering SANS. The resolution and kinetics of Amphotericin B nanoparticles in gastrointestinal model fluid (FaSSIF) was estimated with
stopped flow technology for SANS and DLS in a simulator of the human
gastrointestinal system GIMod [3]. According to SANS (fig.1) the sonified PCCholesterol-AmB liposomes (SUV) were unilamellar depicting a size of 34 nm
and evidence of a lateral phase separation. This investigation gives an insight
into whether the drug would be in an uptake competent form for efficient
permeation.
Fig.1: Neutron small
angle scattering SANS of
liposomes (SUV) from
lecithin
(DOPC),
cholesterol and Amphotericin B. The evaluation by a
Guinier plot above yields
a radius of Gyration Rg =
14.51 ± 0.07 nm; i.e a
size of s = 34.02 ± 0.2 nm
(with a membrane span d
= 5 nm).
Acknowledgements: We are grateful for the funding by the German ministry of science
and education BMBF, grant 05KS7UMA, The Forschungszentrum Jülich FZJ, outstation at
the MLZ, FRM2 reactor Munich Garching, the government of Ghana and the DAAD. This
work was contributed to the OrBiTo project.
References:
1. Dangi, J.S. et al.: Drug Dev. Ind. Pharm. 1995, 21 (17):2021-2027.
2. Charman, W.N. et al.: Adv. Drug Dev. Rev. 1991, 7: 1-14.
3. Nawroth, T.et al.: Mol. Pharm. 2011, 8(6), 2162-2172.
197
PHARMACEUTICAL TECHNOLOGY AND DRUG DELIVERY (PT01-PT34)
PT.01
PT.03
Advanced Formulation for Solid Crystal Suspensions Reaching
Supersaturation
Cationic nanoparticles as a promising drug delivery system? In
detail characterization of surface properties
Reitz, E.; Thommes, M.
Gossmann, R.1; Mulac, D.1; Hummel, M.2; Brockmeyer, J.2; Langer, K.1
Institute of Pharmaceutics and Biopharmaceutics, University of Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany
1 Institute
Solid dispersions are one approach to enhance the dissolution rate of poorly
water soluble drugs. These systems are frequently characterised by a physical
instability, which is not found in terms of solid crystal suspensions. Thereby a
crystalline drug is dispersed in a crystalline carrier, consisting of a sugar
alcohol mixture. In this study sodium lauryl sulphate (SLS) added as solubilizer
to the formulation in hot melt extrusion process using different quantities
between 0.1 and 10 %. The final tablets were characterized with respect to its
solid state properties by powder x-ray diffraction (XRPD), differential scanning
calorimetry (DSC), dissolution and confocal laser scanning microscopy
(CLSM).
The solid crystal suspensions were prepared by hot melt extrusion (Leistritz
Micro GL 27 – 28D). The tableting was performed on a rotary die press (IMA
pressima) using the tableting mixture introduced by Reitz et al. (2014). The
extrudate as well as the tablets were characterized by DSC (DSC 1, Mettler
Toledo) and by XRPD (X’Pert Pro, Panalytical). Drug dissolution was
determined in accordance to the Ph.Eur. using a paddle apparatus and water
as dissolution media. In CLSM the drug (405 nm, griseofulvin) and carrier
(559 nm, sugar alcohol mixture) were excited specifically and the fluorescence
were detected at 436-486 nm (griseofulvin) and 573-633 nm (sugar alcohol
mixture).
The solid state investigation by XRPD and DSC indicate no amophization or
polymorphic change of the drug in the formulations, which is consistent with
the concept of solid crystal suspensions. However supersaturating was
obtained in dissolution studies and also found after storage for several weeks
in stability tests (figure 1). These observations were unexpected because it is
in contrast to the general concepts of solid crystal suspension. Therefore,
different reasons were investigated. The formulations containing SLS showed
a higher dispersity, found in CLSM investigations (figure 2). Presumably, the
SLS supports a drug desagglomeration in the carrier melt during extrusion at
levitated temperatures.
In conclusion a physically stable solid dispersion was prepared allowing
supersaturation during dissolution. This could be a general approach for
several drugs with low aqueous solubility.
mit SDS
Figure 1: Drug release of
tablets containing a SCS
before and after storage of
6 weeks (av ± CI, n = 6).
ohne Trägermaterial
mit Trägermaterial
ohne SDS
of Pharmaceutical Technology and Biopharmacy, University of Muenster,
Corrensstr. 48, 48149 Muenster, Germany
2 Institute of Food Chemistry, University of Muenster, Corrensstr. 45, 48149 Muenster,
Germany
The preparation of cationic nanoparticles for intracellular drug delivery enters
the focus of many research groups, because of their promising increased
cellular uptake based on the electrostatic interaction between the cationic
surface and the negatively charged lipid membrane [1]. The aim of the present
study was to establish an analytical characterization of cationic didodecyldimethylammonium bromide (DMAB) stabilized poly(lactic-co-glycolic acid) (PLGA)
nanoparticles based on examination of size, zeta potential, electrolyte
sensitivity, and cellular uptake.
The preparation of DMAB-stabilized nanoparticles was performed using
emulsion-diffusion method leading to nanoparticles with a diameter of
approximately 80 nm, a PDI below 0.1, and a positive zeta potential of about
+50 mV [2]. Modification of the nanoparticles was performed by altering the
surface using the hydrophilic polymers poly(vinyl alcohol) (PVA) and polyethylene glycol (PEG).
The DMAB-stabilized nanoparticles appeared to be sensitive to electrolyte
influence due to compression of the electrical double layer in conjunction with
a decrease in zeta potential. This resulted in particle agglomeration which was
shown by increased size and PDI. In contrast the modifications with PVA or
PEG enabled steric stabilization and avoided agglomeration.
Uptake studies performed with Caco-2 cells showed that compared to
negatively charged nanoparticles both modified nanoparticle systems were
taken up more effectively, confirming the expected effect of cell mebrane
interaction.
In addition to the characterization of the particle system and cellular uptake,
the identification of the protein corona, which occurs by applying the nanoparticles into biological systems, was a special focus of this research project.
Identification of adsorbed serum proteins was performed by in solution
digestion and subsequent high resolution LC-MS analysis. The adsorbed
protein corona gives the nanoparticles a kind of new biological identity, which
determines the physiological response including agglomeration, cellular
uptake, circulation lifetime or toxicity [3].
In conclusion this study offers a closer and critical point of view in preparation,
in-vitro and analytical evaluation of DMAB-stabilized nanoparticles for the
physiological use. Furthermore this work succeed in identifying the adsorbed
serum proteins, which could give a first hint on possible physiological
response.
References:
1. Xu, A., et al.: Int. J. Nanomedicine 2012, 7: 3547–3554.
2. Bhardwaj, V. et al.: Pharm Res 2009, 26(11): 2495–2503.
3. Rahman, et al.: Protein-Nanoparticle Interactions (Springer Berlin Heidelberg) 2013.
Figure 2: CLSM picture of the surfactant free
(left) and surfactant containing extrudate
(right).
Reference:
1. Reitz, E. et al.: Pharm. Ind., 2014, 76(2): 286-296.
PT.04
The Effect of Different Salts on the Disintegration Properties of
Enteric-Coated Soft Gelatin Capsules
Al-Gousous, J.1; Penning, M.2; Langguth, P.1
Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger
Weg 5, 55128 Mainz, Germany
2 Pennconsult, Mainz, Germany
1
PT.02
198
The purpose of this investigation was to characterize the effect of using
different salts of shellac on the properties of enteric-coated soft gelatin
capsules. Oval size 4 placebo capsules were coated with four shellac-based
enteric coating solution formulations differing from each other by the salt of
shellac used. One formulation was based on an ammonium salt, one the
sodium salt, one on the potassium salt and one on a composite ammoniumsodium salts containing ammonium and sodium ions in a 1:1 mole:mole ratio.
The coating was performed to the same level for all formulations using a Glatt ®
GC-300 drum coater (Glatt, Germany). Disintegration testing was performed
according to the European Pharmacopoeia (Ph Eur) and disintegration times in
the pH 6.8 buffer were noted. The disintegration testing results have shown
that alkali metal salts promote faster disintegration compared to ammonium
salts. The reasons behind this phenomenon were investigated by comparing
the solubility of dried films of the different shellac salts and measuring their
FTIR, NMR, DSC and MALDI-TOF spectra. It was shown that ammoniumcontaining films are water-insoluble while alkali metal salt films are watersoluble which can explain the differences in the disintegration behaviour. The
explanation behind that can be offered by the spectral findings which suggest
that, in the presence of ammonium, the degree of ionization of the shellac
carboxyls is lesser due to the protonation of the carboxylate ions by the
ammonium ions and loss of ammonia during drying. In addition, in the
presence of ammonium ions, oxidation of the shellac’s aldehyde groups into
carboxylic acid groups was promoted leading to stronger solute-solute
interactions. In addition, the potentially greater extent of the partial hydrolysis
of the high molecular weight fractions of shellac in the case of sodium and
potassium salts may be a contributing factor. Therefore, the choice of the salt
used to prepare a shellac-based enteric coating solution is an important
formulation parameter that can affect the release properties of the product.
Acknowledgments: We would like to thank the German Academic Exchange Service
(DAAD) for their support, Catalent Pharma Solutions for providing us with the placebo
capsules, the group of Professor M. Karas at University of Frankfurt for performing the
MALDI-TOF measurements, and the group of Professor T. Schirmeister at University of
Mainz for performing the NMR measurements.
PT.05
Hyperbranched thermosensitive polyglycerol nanogels – new
tools for efficient skin delivery
Obst, K.1; Witting, M.2; Soler, M.M.3; Friess, W.2; Calderon, M.3; Küchler, S.1
Institute for Pharmaceutical Sciences, Freie Universität Berlin, 14195 Berlin, Germany
of Pharmaceutical Sciences, Ludwig-Maximilians-Universität, 80539 Munich,
Germany
3 Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany
1
2 Department
To enhance the efficiency and to reduce systemic side effects, new delivery
systems for topical applications particularly for biomacromolecules such as
proteins are needed. In this study, we investigated the suitability of hyperbranched polyglycerol (hPG) based nanogels as new potential protein delivery
system [1, 2]. Thermosensitive (TS) polyglycerol based nanogels are able to
maintain the stability of proteins and allow for a triggered drug release at
temperatures higher than 32°C [1, 2]. To assess the efficacy of the nanogels
for topical drug delivery, we studied the particle properties, protein release and
activity as well as skin absorption in reconstructed skin models using the
model proteins bovine serum albumin and L-asparaginase.
For characterisation, the particle size and dispersity were measured by
dynamic light scattering (DLS) applying a temperature ramp from 25 – 40°C (1
C°/min). In addition, the protein release was evaluated by size exclusion
HPLC. To determine the enzymatic activity of L-asparaginase after loading and
release, a specific asparaginase activity assay was performed [3]. To assess
the dermal drug delivery efficiency, we applied asparaginase-loaded nanogels
onto reconstructed skin models (normal and barrier-impaired) [4] applying a
temperature ramp from 32°C to 37°C for 3 hours. Subsequently, the protein
amount in the epidermis was determined with an asparaginase activity assay
or skin constructs were embedded in tissue freezing medium, cryosections
were prepared and immunostaining with a monoclonal antibody against
asparaginase was performed.
The particle size of BSA-loaded TS hPG nanogel was 207 ± 12 nm (PDI 0.5).
Following temperature increase to 34°C – 35 °C, the TS hPG-nanogel
exhibited a sharp decrease in particle size to 170 ± 3 nm (PDI 0.1), but the
nanogels remained unchanged at temperatures below the trigger point of
34°C. At temperatures ≥ 35°C the TS hPG nanogel instantly released 85% of
the protein payload. Furthermore, the activity of L-asparaginase was preserved
after loading and release (specific activity 93.2 ± 4.6 %).
In terms of protein delivery efficiency, skin absorption studies in reconstructed
skin models showed significantly enhanced amounts of asparaginase (9.5 ±
0.1 µg) in the epidermal layers of the skin models following the application of
protein-loaded nanogels. In contrast, the application of the aqueous asparaginase solution resulted in significantly lower amounts (6.3 ± 0.1 µg).
Furthermore, we studied the penetration of L-asparaginase into normal and
barrier-deficient skin constructs by immunohistochemistry. Data analysis
showed significantly enhanced skin absorption of L-asparaginase in the viable
epidermis after application of the thermoresponsive nanogels. This effect was
most pronounced in barrier-deficient skin models. The application of the
control solution did not result in dermal penetration of L-asparaginase.
In conclusion, our data indicate that thermosensitive hPG-nanogels are
suitable and promising carrier systems for labile drugs such as biomacromolecules. Despite harsh chemical conditions efficient protein encapsulation and
release from the hPG nanogels were achieved with good preservation of
protein activity. Furthermore, our results clearly indicate skin penetration
enhancing effects which are most pronounced in barrier-impaired skin. In the
next step, the model protein will be replaced by the therapeutic protein
transglutaminase-1 in order to provide the proof-of-concept that topical protein
substitution might be an interesting therapeutic approach for the treatment of
severe skin diseases.
Acknowledgments:
This work was supported by a grant from the German Research Foundation (DFG; KU
2904/2-1 S.K.).
References:
1. Calderón et al.: Journal of Controlled Release 2011, 151: 295-230
2. Cuggino et al. Soft Matter 2011, 7:11259-11266.
3. Mashburn, L.T. and Wriston, J.C.: BBRC 1963, 12(1): 50-55.
4. Küchler et al.: ATLA 2011, 39:471-480.
PT.06
Preparation and characterization of solid adsorbates based on
semisolid SNEDDS
Hassan, T.H.; Metz, H.; Mäder, K.
Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy, Martin Luther
University, Wolfgang-Langenbeckstr. 4, D-06120 Halle (Saale), Germany.
Self-nanoemulsifying drug delivery systems (SNEDDS) have created
considerable interest and ultimately therapeutical and commercial success in
the oral delivery of poorly water-soluble drugs. They provide the drug in the
form of solubilized nanodispersions. Accordingly, the rate-limiting step of drug
dissolution is bypassed. However, SNEDDS are typically filled in soft gelatine
capsules, which might cause the following problems: interaction with the
capsule shell, instability, higher production cost and possible drug precipitation
[1,2]. Therefore, alternative formulation strategies, e.g. the inclusion of
SNEDDS into a solid dosage form are desirable; nevertheless, very challenging.
Neusilin® US2 (N-US2) is an amorphous, synthetic, neutral grade of magnesium aluminium-metasilicate. It occurs in nanoporous, ultra-light granules
prepared by spray drying. N-US2 is non-toxic and do not form gel upon contact
with aqueous fluids. Furthermore, N-US2 has an excellent flowability, very high
specific surface area and good compressibility [3]. N-US2 was recently used to
solidify liquid self-emulsifying systems [4,5].
Semisolid SNEDDS was incorporated (20 % - 90 %) in N-US2. The physical
appearance and flow properties were evaluated. Differential scanning
calorimetry (DSC) and benchtop nuclear magnetic resonance (BT-NMR) were
used to study the interaction between the semisolid SNEDDS and the
nanoporous carrier. Furthermore, the adsorbates were moulded or compressed into tablets and their dispersibility was evaluated in 0.1 N HCl and
phosphate buffer pH 6.8 USP. In addition, Lumogen ® F305 florescence dye
was incorporated in the SNEDDS to help visualizing the release process.
At least 40 % of N-US2 was required to obtain a freely flowable solid. At lower
level, gummy solids to plastic pastes were obtained. DSC study showed a shift
in the melting behaviour of the SNEDDS with the increase of N-US2 level. BTNMR study showed a strong interaction between SNEDDS and N-US2 with the
presence of different mobility states. Moulded adsorbates showed complete
dispersibility within 2 - 3 hr, while compressed ones were non-dispersible. A
disintegrant was required to provide a fine dispersion of the compressed
adsorbates. Tablets with 10 % disintegrant gave a fine dispersion with good
SNEDDS release.
Acknowledgments: We would like to thank the Egyptian Ministry of Higher Education and
Scientific Research and the Deutscher Akademischer Austauschdienst (DAAD) for the
financial support as a PhD scholarship to Mr Tamer H. Hassan, Assistant Lecturer,
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal
University, Ismailia, Egypt. We would like to thank Mrs. Kerstin Schwarz for the DSC
measurements. Abitec Corporation, BASF AG, Croda GmbH, and Fuji Chemical Industry
Co. Ltd are gratefully acknowledged for the excipient donation.
199
References:
1. Pouton, C.W.: Eur. J. Pharm. Sci. 2000, 11: S93–S98.
2. Rahman, M.A., et al.: Drug Dev. Ind. Pharm. 2013, 39(1): 1–19.
3. Sander, C. and Holm, P.: AAPS PharmSciTech 2009, 10(4): 1388–1395.
4. Williams, H.D., et al.: J. Pharm. Sci. 2014, 103(6): 1734–1746.
5. Gumaste, S.G., Dalrymple, D.M. and Serajuddin, A.T.M.: Pharm. Res. 2013, 30(12):
3186–3199.
PT.09
Pig ear skin penetration of azithromycin nanocrystals for Lyme
Borreliosis infection
Jin, N.1; Staufenbiel, S.1; Keck, C.M.1,2; Müller, R.H.1
PT.07
PT.08
Small scale production of nanocrystals in drug discovery and
development phase
Romero, G.B.1; Keck, C.M.2; Müller, R.H.1
1 FU
Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology &
NutriCosmetics, Kelchstr.31, 12169 Berlin, Germany
2 Fachhochschule/University of Applied Sciences Kaiserslautern, Applied Pharmacy,
Campus Pirmasens, Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany
The process of drug discovery and development has increasingly become
more costly and longer during the last decades. New drugs approvals are
interrupted due to lack of efficacy, toxicity, poor absorption. Some of these
issues are related to poor solubility and dissolution velocity, which can be
overcome by nanonization [1]. However, during pre-formulation stage and late
discovery, the new chemical moiety is normally available at very low amounts.
Therefore, in this study, the miniaturization of nanocrystals production was
evaluated using an accessible, cheap, simple approach.
Milling was performed in a 2 mL glass vial and the grinding media was yttria
stabilized zirconium oxide beads (sizes varying from 0.05 mm to 0.6 mm). The
trick of the set up was the use of a triple arrangement of 3 magnetic stirrers
(one above the other). Various model drug were used, e.g. cyclosporin A,
ascorbyl palmitate, hesperidin. The coarse suspension was composed of 5%
(w/w) drug powder and 1% (w/w) stabilizer. Stirring was performed on a
magnetic stirring plate RCT basic (IKA-Werke GmbH & Co. KG, Germany) at
1,200 rpm. Particle size and polydispersity index (PI) were assessed by photon
correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, UK) and light microscopy (LM). Samples were drawn from 1 to 120
hours.
Nanonization of drug crystals can be industrially achieved by different
techniques and different equipments. Some equipments on the market are
able to produce nanosuspensions in a small scale, such as the Bühler PML-2
(150 g batch) or the APV LAB 40 homogenizer (40 mL batch). Even smaller
batches can be obtained with the Nanomill System® (10 mL batch) or the
Avestin EmulsiFlex-B3 (3.5 mL batch). However, this is still not sufficiently
small to perform screening tests. Therefore, the batch size of nanosuspension
in the present study was 0.5 g (25 mg of drug). For all tested drugs, particle
size reduced as a function of time until it reached a lower size limit from where
further milling either did not lead to particle size reduction or resulted in crystal
growth or aggregation. It was also observed that the smaller the milling beads
size, the more efficient particle size reduction occurred. The smallest PCS
diameter was about 90 nm and polydispersity index was 0.14 for cyclosporin A
after 72 hours milling (0.05 mm milling beads). Moreover, the weight of the
magnet stirrers was assessed before and after milling and no corrosion was
observed (unlike other setups in which corrosion occurred).
Although very simple and unexpensive, this method can successfully produce
sub-micron crystals and nanocrystals in the lower nanorange (<100 nm). This
is an accessible method since it requires commonly used equipments found in
every laboratory, unlike previously described methods which require specific,
costly equipments. It allows the screening of different stabilizers/stabilizer
concentrations, as well as production parameters i.e. milling beads size, using
minute amounts of the drug.
Acknowledgements: the authors would like to thank PhamaSol GmbH and the Brazilian
ministry of education through the CAPES/DAAD doctoral program (Grant no. 12416/12-6)
for R&D and financial support.
Reference:
1. Shegokar, R., Müller, R.H.: Int. J. Pharm. 2010, 399: 129-139.
200
1 FU
NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany
2 FH Kaiserslautern, Applied Pharmacy Division Campus Pirmasens, Carl-Schurz-Str. 1016, 66953 Pirmasens, Germany
For more than one century, many people in North America and Europe are
predisposed to Lyme Borreliosis infection after tick bite. Antibiotic treatment
has proven to be effective in clearing the infection when used early. However,
bacterial resistance to administered antibiotic or malaise and toxicity has to be
considered when using antibiotics systematically. These side effects can be
reduced when drug concentrations are kept at a minimum and the antibiotic is
administrated dermally. Azithromycin is preferred in this study due to its lower
minimum inhibitory concentration, fewer adverse effects and quicker onset
time compared to other popular antibiotic agents. Considering the difficult
diagnosis in the early stage of Lyme Borreliosis and serious clinical signs in its
late stage, quick initiation of a dermal azithromycin treatment after tick
exposure could pave the way for preventing Lyme Borreliosis. Even though
there is a dermal formulation, applying ethanol to dissolve the raw drug powder
[1], to overcome solubility problems, according to Fick’s first law, nanocrystals
are assumed to have improved skin penetration. Thus the object of this study
was to develop a dermal gel with azithromycin nanocrystals.
10% azithromycin nanosuspension was produced by bead milling (Bühler;
PML 2), stabilized with 1% tocopheryl polyethylene glycol succinate (TPGS).
The saturation solubility of the nanosuspension compared to raw drug powder
formulation including TPGS was determined in water by shaking for 8 hours in
vials. The obtained concentrations of dissolved drug were determined by
HPLC. Nanocrystals and raw drug powder with 0.5% TPGS was incorporated
into 5% hydroxypropylcellulose (HPC) to get 5% azithromycin-nanocrystal gel
and 5% azithromycin-raw drug powder gel, respectively. 10% azithromycinethanol-solution gel (azithromycin raw drug powder 10%; (94%) ethanol
77.5%; polyacrylate 0.5%; HPC 5%; mygliol 7%) was selected as a comparison which demonstrated effectiveness in clinical studies [1]. Subsequently a
penetration study (n=3) via tape stripping was performed in the pig ear skin
model and the AZ amount in the different strips was measured by HPLC.
Azithromycin nanocrystals with particle size of 189 nm (z-ave) were produced
by bead milling in only 10 minutes. The nanosuspension had an about 2 times
higher saturation solubility in water (227 µg/ml) compared to the raw drug
powder. 5% azithromycin-nanocrystal gel showed higher penetration ability
compared to raw drug powder, penetration was even higher than for the
“ethanol-solution gel” with 10% azithromycin. This can be seen by considering
e.g. the azithromycin amounts found in the strips of the 6th and the 7th layer:
10% ethanol solution gel (12.3 µg±2.2 / 10.7 µg±2.0), azithromycin-raw drug
powder gel (19.9 µg±1.9 / 13.3 µg±2.4) and azithromycin-nanocrystal gel
(47.8 µg± 4.3 / 27.3 µg± 4.9). This may be due to both the volatility of ethanol
[2] leading to re-crystallization in the solution formulation, and increased
saturation solubility of nanocrystals.
In summary, a 5% azithromycin-nanocrystal gel was developed in this study. It
showed higher saturation solubility and higher penetration than 5% raw drug
powder and penetration was even higher than the reported 10% ethanol
azithromycin dermal formulation.
Acknowledgments: PharmaSol GmbH Berlin, Germany and China Scholarship Council.
References:
1. Knauer, J. et al.: J. Antimicrob. Chemother. 2011, 66 (12): 2814-2822.
2. Belsey, N.A.; Rojas, L.R.C.; Guy, R.H.: Clinical Dermatology (Springer Berlin/Heidelberg) 2014.
PT.10
Azithromycin nanocrystals for i.v. drug targeting – effect of
stabilizers on surface hydrophobicity and protein corona
References:
1. Owens Iii, D.E. and N.A. Peppas: International Journal of Pharmaceutics, 2006, 307(1):
p. 93-102.
2. Petri, B., et al.: Journal of Controlled Release, 2007, 117(1): p. 51-58.
Staufenbiel, S.; Jin, N.; Müller, R.H.
FU Berlin - Institute of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology &
The use of nanocrystals as drug carriers for i.v. administration is a promising
approach to overcome application problems of poorly soluble drugs, whereby
adsorbed blood proteins determine their biodistribution. Beside others (e.g.
size, shape, charge etc.) surface hydrophobicity is one of the key parameters
determining the protein corona, whereby hydrophilic surfaces can lead to a
decreased liver uptake due to a decreased protein (opsonin) adsorption and
subsequently to an increased blood circulation [1]. Thus one can select
nanocrystal coatings to reduce surface hydrophobicity. Besides analyzing
hydrophobicity, additionally the protein adsorption pattern has to be analyzed,
because the adsorption of some specific proteins can lead to drug targeting,
e.g. into the brain. Thus it was the aim of this study to analyze hydrophobicity
and protein corona from differently coated azithromycin nanocrystals, because
cerebral infections are still challenging until today.
Azithromycin nanosuspension (10% w/w) was produced by bead milling (PML
2, Bühler) with 4 different stabilizers (1% w/w; Poloxamer 188, Poloxamer 407,
Tween 80, TPGS) leading to approx. 250 nm particles (photon correlation
spectroscopy, Zetasizer Nano ZS). Surface hydrophobicity was determined by
hydrophobic interaction chromatography (HIC) and two phase partitioning
(TPP). HIC was performed using a C 10/20 column (Pharmacia Biotech) filled
with octyl-agarose. Elution (2 ml/min) profiles were recorded by UV reading.
TPP was performed by partitioning particle suspensions in a system containing
a 12% (m/v) polyvinyl alcohol phase and a 16% (m/V) dextran phase.
Furthermore, samples were incubated with pooled human plasma and total
amount of adsorbed protein was determined with the bicinchoninic acid (BCA)
assay. Subsequently, the protein adsorption pattern was analyzed by twodimensional-polyacrylamide-gel-electrophoresis (2D-PAGE) and spots were
quantified with the MELANIE software. All results are the mean of three
independent measurements.
The HIC elution time (peak maxima) from the TPGS, Tween 80 and Poloxamer
188 sample were close to the void volume (6.4 min, 6.5 min, 7.4 min). This
indicates a low hydrophobicity whereby polypropylene glycol (PPG) blocks in
the Poloxamer 188 sample lead to a slight increase in elution time. The
Poloxamer 407 sample was even eluted at 18.7 min reflecting the increased
PPG content. TPP results confirming these observations, because large
amounts of those samples were found in the more hydrophilic dextran phase
([c dextran]/[c PVA] = 3.08; 1.03; 0.69 / TPGS; Tween 80; Poloxamer 188),
whereby this ratio of the Poloxamer 407 sample was only 0.18. The TPGS
sample seems to be even more hydrophilic here compared to HIC results,
reflecting the fact that this is the only investigated stabilizer with only one
polyethylene glycol (PEG) chain increasing dextran interactions due to
decreased intra-molecular PEG/PEG interactions. Additionally the Tween 80
and the TPGS sample showed low BCA amounts (8 and 8.5 mg protein/g
particles), whereby the Poloxamer samples had approx. 17 mg of total
adsorbed protein, reflecting the avoiding of protein adsorption by hydrophilic
surfaces, whereby also other parameters (like e.g. steric orientation of the
stabilizer) influence protein adsorption. This can be seen from the Poloxamer
188 sample. 2D-PAGE results revealed that the Poloxamer samples had a
high adsorption of the opsonin fibrinogen (about 50% of all detected protein)
whereby apolipoprotein-AI (Apo-AI) adsorption was only about 10%. This was
changed on the TPGS samples where fibrinogen was decreased to 37.4% and
Apo-AI was increased to 31.8%. In contrast especially to the Poloxamer
samples, the Tween 80 sample showed only 14.5% fibrinogen and 52.3% ApoAI.
In summary, azithromycin nanocrystals with different coatings were produced
whereby HIC results were in accordance with the structures of these stabilizers
(respectively with their HLB values) and these results were additionally
confirmed and supplemented by TPP. The low surface hydrophobicity of the
TPGS and Tween 80 samples was in agreement with a low amount of total
adsorbed protein and simultaneously with a low amount of detected opsonins.
This can lead to an increased systemic circulation. Additionally the Tween 80
sample showed a particularly high adsorption of Apo-AI, which opens the
option for a certain brain targeting potential [2].
PT.11
ARTcrystal®-technology for nanocrystal production: Comparison of continuous and discontinuous premilling step
Scholz, P.1,2; Müller, R.H.2; Keck, C.M.1,2
1
2
University of Applied Sciences Kaiserslautern, Campus Pirmasens, Germany
Free University of Berlin, Germany
Introduction:
The ARTcrystal®-technology is a combination process aiming for an economically production of drug nanocrystals. It combines a high speed pre-milling step
and high pressure homogenization (HPH) at reduced pressure and cycle
numbers [1]. The pre-milling step is performed with an ART MICCRA D27
rotor-stator system, allowing a continuous high speed stirring, i.e. pre-milling,
at up to 36,000 rpm. After pre-milling, the suspension is subjected to HPH at
300-500 bar for up to 5 cycles in order to form a nanosuspension [2].
Aim:
In order to further understand and to optimize the pre-milling step, pre-milling
was performed in continuous mode as well as in discontinuous mode. In a
discontinuous process every particle is forced to pass the milling chamber
while continuous processing allows faster processing, but turbulences in the
product container can lead to an unequally number of passages of every
particle through the milling chamber.
Materials and Methods:
Coarse material (rutin, Denk Ingredients, Munich, Germany) was used as
testing material. Pre-milling was performed in an ART MICCRA D27 (ART
Prozess- und Labortechnik, Müllheim, Germany) at 24,000 rpm for 30 minutes
(continuous) or 100 cycles (discontinuous). Cooling at -10°C was provided.
Particle size was analyzed by laser diffraction (Mastersizer 2000, Malvern, UK)
and light microscopy (DM 1000, Leica, Wetzlar, Germany).
Results and Discussion:
The continuous process provided an efficient reduction in particle size. After
only 5 minutes of processing a d(v) 0.50 of 3.74 µm and a d(v)0.99 of 26.51
µm was obtained. Further processing of up to 30 minutes led to a further
diminution of the larger particles (d(v)0.50: 3.03 µm, d(v)0.99: 15.07 µm).
Within the discontinuous process similar results to 5 minutes of processing in
continuous mode where obtained after 20 cycles (d(v) 0.50: 3.47 µm, d(v)0.99:
20.81 µm). 60 cycles led to sizes similar to processing in continuous mode for
30 min (d(v)0.50: 3.09 µm, d(v)0.99: 15.52 µm). Further processing to up to
100 cycles did not lead to a further size reduction (d(v)0.50 3.02 µm, d(v)0.99
16.27 µm). Thus 60 cycles represent the processing condition at which the
maximum dispersity of the material is reached. Since one cycle in discontinuous mode takes about 6 seconds, it can be followed that by discontinuous
processing (60 cycles) the optimum could theoretically be reached after 6min,
while this optimum was only reached after 30 minutes (i.e. about 300
passages) by continuous processing. Results, i.e. lower milling efficacy with
continuous processing, can be explained by turbulences inside the product
container, which lead to an inhomogeneous number of passages for each
single particle.
Conclusion:
Both process modes lead to a similar dispersity of the material, either after 30
minutes (continuous mode) or after 60 cycles of processing (discontinuous
mode). While processing in discontinuous mode was more effective, the
processability in continuous mode is much better. Thus, further developments
will focus on combining the advantages of each processing mode.
References:
1. Keck, C.M., US20130095198 A1, 2011.
2. Scholz, P., Keck, C.M., ARTcrystal®-technology: Influence of starting material size on
final particle size. DPhG - Tag der Pharmazie, July 4th, 2014, Berlin.
Acknowledgments: The authors would like to thank PharmaSol GmbH for financial and
scientific support.
201
PT.12
PT.13
Dermal delivery systems – nanoemulsion vs. nanostructured
lipid carriers (NLC)
Glass transition temperature of poly(D,L-lactic-co-glycolic acid)
nanoparticles
Pyo, S.M.1; Meinke, M.C.2; Keck, C.M.3; Müller, R.H.1
Lappe, S.; Langer, K.
1 FU
Institute of Pharmaceutical Technology and Biopharmacy, Westfälische WilhelmsUniversität Münster, Corrensstraße 48, 48149 Münster, Germany
2 University Hospital Charité Berlin, Department of Dermatology, Venerology and
Allergology, Charitéplatz 1, 10117 Berlin, Germany
3 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany
Couperosis is a hereditary skin disorder which is characterized by the
weakness of the connective tissues. Topically applied vitamin A1 allows a
causal therapy due to its new collagen forming properties. Many products on
the market containing the active formed as an emulsion. NLC are a well-known
dermal delivery system to improve the penetration of actives by forming an
occlusive film, also called the “invisible patch” [1]. An ex vivo penetration study
was performed to compare these delivery systems with the aim to identify the
delivery system better suited for achieving higher concentrations in deeper skin
layers.
Vitamin A1 loaded NLC suspension (6.0 % retinol 50C, 6.0 % carnauba wax,
2.0 % Miranol Ultra 32, 86.0 % distilled water w/w) was produced by rotorstator homogenization (30 s, 8,000 rpm) (Ultra-Turrax T25, Jahnke und
Kunkel, Germany) followed by hot high pressure homogenization (HPH) (2
cycles, 800 bar, 85°C) (Micron LAB 40, APV, Germany). Vitamin A 1 nanoemulsion (NE) with same active agent and lipid content (6.0 % retinol 50C, 6.0%
MCT, 2.0 % Miranol Ultra 32, 86.0 % distilled water w/w) was prepared as
reference by HPH (1 cycle, 500 bar, 85°C). The particle size distributions were
analysed and compared by laser diffraction analysis (LD, Mastersizer 2000,
Malvern Instruments, UK). An ex vivo tape stripping test on pig ear skin was
performed with tesa Film No. 5529 (Beiersdorf, Hamburg, Germany) after 20
and 60 minutes penetration time.
Before starting the penetration study, the nanoemulsion showed LD diameter
95 % of 0.227, 99 % of 0.274 and 100 % of 0.345 µm. NLC suspension
possessed similar particle size distribution with LD diameter 95 % of 0.210, 99
% of 0.250 and 100 % of 0.305 µm. The relative concentrations of the
penetrated vitamin A1 from NE and NLC suspensions in respective layers of
the stratum corneum (SC) were compared among each other (Table 1). After
20 minutes in the depth of 3 % of SC a high amount of 46 % of the active
agent could be found from NE whereas just 3 % of active agent reached the
same depth applied as NLC. Also in a depth of 12 % of the SC a higher
relative concentration of vitamin A1 could be found from NE (4.7 %) than NLC
(0.5 %).
SC
NE
NLC
depth
[%]
rel.
c(A1)
[%]
46.0
4.7
0.2
rel.
c(A1)
[%]
3.0
0.5
0.0
3
12
81
after
60 min.
after
20 min.
Table 1: The relative concentration of vitamin A1 at different depths of SC as a
function of delivery system and penetration time.
SC
NE
NLC
depth
[%]
rel.
c(A1)
[%]
2.5
0.8
0.5
rel.
c(A1)
[%]
12.0
2.4
1.8
22
36
50
However, after 60 minutes penetration time, the concentrations of vitamin A1 in
the layers of the SC show an obvious change of the penetration profiles for the
benefit of NLC. In the depth of 22 % of the SC just 2.5 % of active could be
detected from NE whereas 12 % (fivefold higher) reached the same depth with
NLC. Considering the SC in the depth of 36 and 50 %, more than a three times
higher concentration of active could be detected from NLC (2.4 and 1.8 %)
compared to NE (0.8 and 0.5 %).
The observed profiles can be explained by a faster release from the
nanoemulsions (liquid lipid droplets) compared to the NLC (solid particle
matrix). The occlusive effect of NLC takes some time to develop, which finally
leads to higher penetration into the stratum corneum. In summary, NE provide
faster initial release, NLC act as prolonged delivery systems with better
penetration.
Reference:
1. Müller, R.M. et al.: Euro Cosmet. 2013, 6: 20-22.
202
Nanoparticles based on poly(D,L-lactic-co-glycolic acid) (PLGA) and stabilised
with poly(vinyl alcohol) (PVA) are widely used in pharmaceutical research [1,2],
but little is known about the glass transition temperature of the resulting
polymer matrix and the possible influences on this. The glass transition
temperature Tg is a characteristic of amorphous polymers like PLGA. Within
this temperature range the material changes from being hard and brittle to a
more soft and reactive manner. For Resomer® RG 502 H, the PLGA polymer
used in this study, depending on the measurement conditions Tg is between
38°C and 45°C. This temperature is easily achieved during particle preparation
by homogenisation or subsequent purification of the nanoparticles.
The aim of this study is to examine the reasons for the differences of glass
transition of pure PLGA (38.6 ± 1.6°C), freeze dried PLGA nanoparticles
(35.6 ± 3.4°C), and PLGA nanoparticles in suspension (32.2 ± 0.9°C). For
examination of the glass transition temperature a differential scanning
calorimetry (DSC) method was established.
In order to exclude the influence of the ingredients a physical mixture of PLGA,
PVA, and mannitol (used in the freeze drying process for stabilisation) as well
as a solid solution of these substances were tested. Neither the presence of
the stabiliser nor an influence of different preparation methods (emulsion
diffusion method, solvent displacement) could be observed. But when
preparing the nanoparticles with emulsion diffusion evaporation method using
a rotary evaporator a shift of Tg to lower temperatures was observed. This was
probably due to residual organic solvent in the nanoparticle formulation.
In all yet mentioned cases PVA was used in one concentration, expecting that
other concentrations of the polymer might have an influence on Tg, which
couldn´t be confirmed.
Residual moisture content might explain the differences between the values for
glass transition of pure PLGA, freeze dried PLGA nanoparticles, and PLGA
nanoparticles in suspension, respectively. Blasi et al. [3] confirmed the
existence of different kinds of water whereat non-freezable water is closely
associated to the polymer chains and lowers the Tg. This observation could be
confirmed, with the result that for freeze dried nanoparticles measured under
moisture displacing conditions a glass transition temperature of 47.0 ± 0.7°C
could be achieved.
References:
1. Vandervoort, J., Ludwig, A.: Int. J. Pharm. 2002, 238(1-2): 77-92.
2. Mundargi, R.C. et al.: J. Control. Release 2008, 125(3): 193-209.
3. Blasi, P. et al.: J. Control. Release 2005, 108(1): 1-9.
PT.14
Strategies to improve colloidal stability of lysozyme-loaded
nanoparticles
Thoma, F.; Langer, K.
Institute of Pharmaceutical Technology and Biopharmacy, Westfälische WilhelmsUniversität Münster, Corrensstraße 48, 48149 Münster, Germany
Proteins become more and more important for the treatment of several
diseases. Due to the high advantages of nanoparticles used as drug carrier, a
focus of current research is on the entrapment of therapeutic proteins into
nanoparticles.
The aim of our work was the entrapment of model compound lysozyme, a
small protein of 14 kDa, into a nanoparticle system based on poly(lactic-coglycolic acid) (PLGA) by solvent displacement method [1].
During nanoparticle preparation the characteristics of lysozyme caused
problems in nanoparticle stability. Lysozyme exhibits a comparatively high
isoelectic point (IEP), which results in a positively charged model protein under
the conditions of PLGA nanoparticle preparation. This led to a high protein
adsorption on PLGA nanoparticles. Hence, before purification lysozyme-loaded
PLGA nanoparticles showed a positive zetapotential, that changed to negativ
values during particle purification. This was due to a removal of adsorptive
bound lysozyme during the purification procedure. Passing the point of neutral
surface charge induced stability problems of the colloidal system. Additionally,
the adsorptive binding of the positively charged lysozyme to a negatively
charged PLGA-matrix is responsible for ionic interactions which results in
strong particle agglomeration and sedimentation.
Within the study we developed different strategies for stabilization of this drug
delivery system during preparation and purification, like different steric
stabilisators, buffers, and coatings.
Reference:
1. Niu et al.: Drug Dev. Ind. Pharm. 2009, 35(11): 1375-1383.
PT.15
Nanocapsules - Is the preparation of core-shell structured
nanosystems that simple?
Wessels, L.1; Tacke, S.2; Mulac, D.1; Langer, K.1
Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster ,
Corrensstr. 48, 48149 Muenster
2 Institute of Medical Physics and Biophysics, University of Muenster, Robert-Koch-Str. 31,
48149 Muenster
1
Nanocapsules seem to have many advantages as a drug delivery system in
comparison to nanospheres. The polymeric shell shields the active pharmaceutical ingredient (API) from degradation as well as it protects tissue from
hazardous effects of the API. Another advantage over nanospheres is the high
drug encapsulation efficiency due to optimized solubility of the API in the core
material in contrast to low polymer content [1].
There are many publications linked with the keyword nanocapsule, which
describe different methods for nanocapsule preparation [1] but often without
any differentiation between spheres and capsules and without a confident
proof of a core-shell structured particle. Furthermore, some groups use the
terms nanocapsules and nanospheres interchangeably.
We investigated solvent displacement method (nanoprecipitation method) to
gain an oily core nanocapsule formulation [2] and double emulsification
method to build nanocapsules with an aqueous core [3]. The resulting particle
systems were analyzed by dynamic light scattering (DLS), scanning electron
microscopy (SEM), and cryo- scanning transmission electron microscopy
(cryo-STEM) for size, size distribution, and morphology. Nanocapsules could
be identified in the samples by the analytical methods used. However in most
cases we observed that the multitude of particles was in shape of nanospheres
instead of capsules. Therefore we conclude that a quantitative formation of
nanocapsules by the methods described in literature is difficult to obtain.
The authors want to acknowledge Ulrike Keller (Institute of Medical Physics and
Biophysics, University of Muenster, Robert-Koch-Str. 31, 48149 Muenster) for her skilful
supportive SEM measurements.
References:
1. Mora-Huertas, C.E.; Fessi, H.; Elaissari, A.: Int. J. Pharm. 2010, 385 (1-2): 113-142.
2. Rübe, A. et al.: J. Control Release 2005, 107 (2): 244-252.
3. Zhu, Y. et al.: J. Surfactants Deterg 2005, 8 (4): 353-358.
PT.16
Mucoadhesive polymyxin B-dexamethasone acetate nanocrystals for ocular delivery
Romero, G.B.1; Keck, C.M.2; Müller, R.H.1; Bou-Chacra, N.A.3
1 FU
2 Fachhochschule/University of Applied Sciences Kaiserslautern, Applied Pharmacy,
Campus Pirmasens, Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany
3 University of Sao Paulo, Faculdade de Ciências Farmacêuticas, Departamento de
Farmácia, Av. Lineu Prestes 580 - Bloco 15, Caixa-Postal: 6608, 05508-900 Sao Paulo,
Brazil
The conventional ophthalmic preparations have limited residence time in the
ocular region as a result of the protective mechanisms of the eye. In addition,
the cornea acts as a barrier contributing to the reduced concentration of the
drug in the vision organ. As a consequence, ophthalmic products show poor
bioavailability. These limitations require patient adherence to treatment
regimen with multiple administrations per day and substantial loss of the
instilled material. These drawbacks can be overcome by nanonization of the
drug powder, resulting in drug nanocrystals with increased saturation solubility,
dissolution velocity and additionally, increased mucoadhesion, which is
specially desired for this application site [1]. In this study, the production of
positively charged nanocrystals of dexamethasone acetate in combination with
polymyxin B was evaluated.
The production method was a super small scale bead milling performed in a 2
mL glass vial with 0.05 mm yttria stabilized zirconium oxide beads as the
grinding media. The coarse suspension was composed of 5% (w/w) dexamethasone acetate, 1% (w/w) stabilizer and optionally 1% polymyxin B. Stirring
was performed on a magnetic stirring plate RCT basic (IKA-Werke GmbH &
Co. KG, Germany) at 1,200 rpm during 24 hours. Subsequently, the 5%
concentrated suspension was diluted to the final desired dexamethasone
acetate and polymyxin B concentration. Particle size and polydispersity index
(PI) were assessed by photon correlation spectroscopy (PCS) using a
Zetasizer Nano ZS (Malvern Instruments, UK) and light microscopy (LM).
The conjunctiva and the cornea are protected by the tear film by secreting
mucin, electrolytes and fluid. Mucin, the mucus layer that coats the corneal
surface, is negatively charged. Therefore, the ideal carrier system for the eye
would be a cationic particle with high adhesion to the mucosa. The electrostatic interaction between the opposite charges of the mucin and the cationic
nanocrystals would allow adequate concentration of drug at the site of action.
This strategy can dramatically improve the drug ocular bioavailability.
Polymyxin B as the only stabilizer resulted in positively charged nanocrystals
with zeta potential of + 11 mV (in original medium) after 24 hours milling, but
the stabilization provided only by polymyxin B was not sufficient to avoid
agglomeration, which could be detected by PCS and light microscopy. Dilution
of this concentrate with traditional nonionic stabilizers at a concentration of
0.1% allowed the disintegration of the agglomerates and revealed nanocrystals
of 491 nm and 687 nm for poloxamer 188 and Tween® 80, respectively.
However, after dilution, the zeta potential reduced to around 0 mV for both
stabilizers, which is not desired. In contrast, when the nanosuspensions were
first produced using a cationic stabilizer such as cetylpyridinium chloride or
benzalkonium chloride and subsequently diluted with polymyxin B solution, the
final zeta potential had a positive value of around +30 mV (in original medium).
Also, for this case, the particle size remained in the sub-micron range, being
130 nm and 176 nm for the nanosuspensions stabilized with cetylpyridinium
chloride and benzalkonium chloride, respectively. This is an elegant strategy to
stabilize such particles, since these two molecules are preservatives, which
are added to a multi-dose topical ophthalmic preparation to prevent the growth
of, or to destroy the microorganisms introduced inadvertently during the
treatment interval.
Positively charged nanocrystals with increased mucoadhesion have been
successfully produced. These formulations have the potential to overcome the
drawbacks of the conventional ophthalmic preparations providing an innovative
ocular drug delivery system by increasing their therapeutic efficacy and safety.
Polymyxin B-dexamethasone acetate nanocrystals can be used to treat
superficial infections such as conjunctivitis, keratoconjunctivitis and bacterial
blepharitis with improved performance and patient compliance compared to
standard formulations.
Acknowledgements: the authors would like to thank PhamaSol GmbH and the Brazilian
ministry of education through the CAPES/DAAD doctoral program (Grant no. 12416/12-6)
for R&D and financial support.
References:
1. Shegokar, R., Müller, R.H.: Int. J. Pharm. 2010, 399: 129-139.
203
PT.17
Tetrahydrocannabinol-loaded NLC for nasal drug delivery
against break-through pain in cancer patients
Hommoss, G.; Keck, C.M.; Zhai, X.; Müller, R.H.
Institute of Pharmacy - Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany
Tetrahydrocannabinol (THC) is a lipophilic molecule, binding non-specifically to
a variety of receptors in the brain and body with analgesic medical property.
For treating permanent pain in cancer patients, regularly fixed doses of drug
are used. But administration of additional dose for treating the high peaks pain
would be a hyper medication. Fast onset of action and easy administration is
highly desired to cover these peaks. Nasal drug delivery systems as spray or
aerosols based on nanoparticles are ideal due to their fast drug release and
can be designed to provide adhesion to the nasal mucosa.
Nanostructured lipid carriers (NLC) loaded with THC were produced for the
development of a nasal delivery system. Due to the lipophilic property of THC,
it is highly suitable to be incorporate into a lipid matrix. Therefore the solubility/miscibility of various lipids with oily THC was investigated. Compritol ATO
888 and cetyl palmitate were identified as optimal lipids. THC-loaded NLC
formulated with 1.0 % lipid blend (0.125 % THC and 0.875 % lipid), 0.05 %
surfactant and water up to 100 % were produced by hot high pressure
homogenization using a Micron LAB 40 homogenizer (APV Deutschland,
Germany). The produced THC-loaded NLC were characterized for particle size
by photon correlation spectroscopy (PCS, Zetasizer Nano ZS) and laser
diffraction (LD, Mastersizer 2000, both from Malvern Instruments, UK). Zeta
potential was measured in conductivity adjusted water (50 µS/cm) by using
again Zetasizer Nano ZS.
First a screening was performed producing unloaded NLC to save costly THC
material. Screened was for optimal composition and production parameters.
With about 300 nm, cetyl palmitate yielded distinctly smaller NLC than
Compritiol ATO 888 and it was chosen as optimal solid lipid. Cetylpyridinium
chloride proved being superior to benzalkonium chloride, yielding NLC being
about 100 nm smaller. Final production parameters were only 1 homogenization cycle at 800 bar, being industrially friendly.
Loading with THC decreased the particle size of the NLC. A small particle size
of about 220 nm was obtained, no large particles or agglomerations were
observed. Cetylpyridinium chloride as cationic surfactant reversed the negative
charge of the NLC; the zeta potential was close to +50 mV. First this high zeta
potential provides sufficient physical stability in long-term storage and during
the spraying process in application to the nose. In addition it increases
adhesion to the negatively charged mucosa. Spray-ability test was performed
using a commercial spray nasal bottle and the PARI BOY inhalator. The
particle size of the THC-loaded NLC changed little after spraying with both
delivery systems. The particle size was 280 nm after spraying from the nasal
spray bottle and 247 nm after nebulizing from the PARI BOY (n=3), indicating
good physical stability of produced THC-loaded NLC. Presently the formulation
is in a long-term stability study at different temperatures.
PT.18
Characterisation of PEGylated nanoparticles comparing the
nanoparticle bulk to the particle surface using UV-vis spectroscopy, SEC, 1H NMR spectroscopy and X-ray photoelectron
spectroscopy
Spek, S.1; Haeuser, M.1; Schaefer, M.2; Langer, K.1
1 Institute
of Pharmaceutical Technology and Biopharmacy, University of Muenster,
Corrensstraße 48, 48149 Muenster, Germany
2 nanoAnalytics, Heisenbergstraße 11, 48149 Muenster, Germany
Nanoparticles prepared of poly(lactic-co-glycolic acid) (PLGA) were assembled
using an emulsion-evaporation method. Different mixtures of PLGA and
PEGylated PLGA polymer were used for particle preparation and led to
nanoparticles containing up to 15 wt.% PEG. For all polymeric mixtures the
preparation technique required the use of polyvinyl alcohol (PVA) as a
stabilising agent. Overall, 4 nanoparticle species of different PEGylation
degree, each of about 200 nm in diameter, were characterised with respect to
residual PVA and PEG content. This work focusses on the quantitative
determination of all utilised polymeric compounds using different analytical
204
approaches, comparing the polymer content of the particle bulk to the particle
surface.
For polymer quantification of the nanoparticle bulk, PVA was determined using
a historically well-established UV-vis spectroscopic method [1] as well as size
exclusion chromatography (SEC), and 1H nuclear magnetic resonance (NMR)
spectroscopy. The need for different PVA quantification methods was due to
the fact that the photometric quantification of a coloured PVA complex using
Lugol’s solution and boric acid cannot be analysed as soon as PEG was
present in the nanoparticle sample. This was due to the fact that PEG itself
forms a water insoluble complex with the chemicals used. In this work, all three
quantification methods for PVA were compared to each other under standardised conditions. Additionally, PEG determination of the nanoparticle bulk was
carried out using quantitative 1H-NMR spectroscopy. The amount of PLGA in
particle bulk was calculated indirectly by subtraction of PVA and PEG from the
overall solids content.
Furthermore, all prepared nanoparticle samples were investigated by X-ray
photoelectron spectroscopy (XPS), giving reliable information about the
relative polymer composition of the nanoparticle surface. In conclusion, the
comparison of nanoparticle bulk to nanoparticle surface showed an increase of
hydrophilic polymers at the surface with increasing PEGylation degree.
Furthermore, interesting information about the arrangement of a PEG corona
can be extracted from the data comparing the results of the nanoparticle
surface and the nanoparticle bulk.
Reference:
1. Pritchard, J.G., Akintola, D.A.: Talanta 1972, 19: 877-888.
PT.19
“Nano-Curry” for improved health
Rostamizadeh, K.1,2; Gerst, M.2,3; Scholz, P.2,3; Arntjen, A.2; Keck, C.M.3,4
1 Zanjan
Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical
Sciences, Zanjan, Iran
2 Applied Pharmacy, University of Applied Sciences Kaiserslautern – Campus Pirmasens,
Pirmasens, Germany
3 Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Institute of
Pharmacy, Freie Universität Berlin, Berlin, Germany
4 PharmaSol GmbH, Berlin, Germany
Introduction: Antioxidants are important to improve health as it has been
shown in vivo and in vitro. Nowadays, isolated antioxidants are not recommended anymore, because they can also show pro-oxidative effects (e.g.
isolated vitamin E). Thus natural antioxidants in natural combinations are of
great interest. Curry is composed of several spices, possessing a high
antioxidant capacity (AOC). However, at least some compounds of curry are
poorly soluble, e.g. curcumin. Therefore, to further increase the antioxidant
capacity, the solubility of curry should be increased. Thus, the production of
nanocrystals which increase solubility might be a good strategy to increase the
AOC of curry powder. Therefore the aim of this study was to produce
nanosized curry powder and to compare its AOC to the original powder.
Materials and Methods: Curry was purchased from the local supermarket and
was homogenized by high pressure homogenization (HPH). The AOC was
measured by a modified DPPH method [1]. Particle size was analyzed by
photon correlation spectroscopy (PCS), laser diffractometry (LD), and light
microscopy.
Results: LD measurements indicated that the original powder possessed a
large particle size of about 210µm (d(v) 50%). Light microscopy revealed that
the original sample contained large crystals and needles. During the homogenization process, coarse curry microparticles were disintegrated into nanoparticles. Subsequently, the mean particle size decreased with an increase in
homogenization cycles. The lowest mean particle size of the curry nanopowder
obtained after 10 homogenization cycles at 1500 bar was 333 nm (PCS).
Further homogenization to up to 20 cycles lead to a slight increase in particle
size, probably due to heat increase. The AOC of original curry powder and the
curry nanoparticles were determined via the DPPH method. The results
revealed that nanosized curry possessed a much higher AOC than the original
powder. In addition to the high value of AOC, the decrease in absorption was
faster for the homogenized curry, when compared to the original curry powder.
Conclusion: Nanosized curry could be obtained by high pressure homogenization. The decrease in particle size led to an increase in the AOC. Thus the
production of “Nano-Curry” might be a successful strategy to further improve
the health benefit of curry powder for either oral or dermal application. Further
studies will focus on the development of an optimized production method to
obtain optimized “Nano-Curry”.
Acknowledgments: The authors are grateful for the financial support of this project by
Germany academic exchange Service (DAAD).
References:
1. Pisoschi, A.M.; Cheregi, M.C.; Danet, A.F.: Molecules 2009, 14: 480-493.
PT.20
Gene Delivery and Knockdown using Novel Lipopolyplexes
Pinnapireddy, S.R.; Bakowsky, U.
Institute for Pharmaceutical Technology and Biopharmacy, Philipps University, 35037 Marburg, Germany.
This study was aimed at formulating nucleic acid carriers which are composed
of a Liposomal shell encapsulating a Polymer and Nucleic acid complex
together termed Lipopolyplex. These are intended for transfection and for
knockdown or down-regulation of a particular gene. In this current study we
investigated the in-vitro efficacy of DOPE (1, 2-dioleoyl-sn-glycero-3phosphoethanolamine), DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine)
and Cholesterol based liposomes which were used to encapsulate a PEI
(Polyethylenimine) based polymer and Nucleic acid complex. The physical
characteristics of the Lipopolyplexes were analysed using Dynamic Light
Scattering, Laser Doppler Micro-electrophoresis and Scanning Electron
Microscopy. Complex stability analysis was done using Complex Stability
Assay and Heparin Competition Assay. Transfection efficiency was done using
Luciferase reporter gene assay, GFP expression and RNAi. Cytotoxicity was
evaluated using MTT assay. With a size of 237 ± 5 nm and Zeta potential of 6
± 2 mV, the complexes were well within the suitable range for use in Transfection. Luciferase expressions in the range higher than 1000000 RLU/mg protein
were achieved. Comparison against other transfection agents showed a better
cytotoxicity profile which was relatively lower. Acknowledging the results of the
study, it can be concluded that Lipopolyplexes are by far a better alternative to
the conventional transfection reagents bearing in mind their cytotoxicity
profiles.
The decline of DAM followed 1st order kinetics while formation of DMG as well
as one unknown degradation product could by described by zero order
kinetics. The degradation rates were distinctive temperature dependent. Based
on an acceptable DAM-content of 90% (w/w) RSDL® proofed to be stable for
several years at the recommended storage conditions from 15°C to 30°C.
Calculated shelf life was 38.3a at 15°C and 7.6a at 30°C. For the degradation
product DMG a valid threshold value of 0.1% (w/w) was considered. Time span
to meet 0.1% was 22.6a at 15°C and 5.0a at 30°C. In both cases higher
temperatures led to a remarkable shortening of shelf life. An additional
unknown degradation product arose during our experiments. For unknown
degradation products ICH Q3B(R2) gives threshold values (percentage of AI)
of 0.05% (reporting threshold), 0.1% (identification threshold) and 0.15%
(qualification threshold) [4]. If these values established for drugs were applied
analogously for the medical device RSDL®, even the highest value of 0.15%
was reached after 1.0a at 15°C and 0.5a at 30°C.
The quality of RSDL® is warranted for a period of 4 years. According to our
calculations in that timespan the content of the AI as one crucial parameter of
quality will not fall under an acceptable limit if RSDL® is stored correctly. For
specified or unknown degradation products an obvious limitation of shelf live
could be shown even if stored at the recommended conditions. Thus a short
retest-period is necessary due to a risk orientated quality monitoring of drugs
and medical devices being stockpiled or used in missions abroad.
References:
1. Schwartz, M.D. et al.:Curr Pharm Biotechnol. 2012, 13 (10): 1971-1979.
2. http://www.rsdecon.com/pages/aboutUS.htm
3. Grimm, W. in: Drug Stability, Principles and Practices (Taylor & Francis) 2000.
4. ICH Harmonised Tripartite Guideline Q3B(R2), Impurities in New Drug Products, 2006.
PT.22
Are polymeric nanoparticles able to cross the gastrointestinal
barrier?
Söbbing, J.; Grünebaum, J.; Mulac, D.; Langer, K.
Institute of Pharmaceutical Technology and Biopharmacy, University of Münster,
Corrensstraße 48, 48149 Münster
PT.21
Stability testing of medical devices –
estimation of shelf life for Reactive Skin Decontamination Lotion
(RSDL®)
Bogan, R.; Klaubert, B.; Zimmermann, T.
Central Institute of the Bundeswehr Medical Service Munich, Ingolstädter Landstr. 102,
85748 Garching-Hochbrück, Germany
For regulatory reasons stability studies are mandatory for drugs. As medical
devices do not pass identical procedures during the authorization and
certification process, stability data are not always strictly collected. German
Armed Forces stockpile medical devices over a long period and use them in all
climatic zones. Therefore stability is an essential matter of concern. In this
work we describe the estimation of shelf life for the Reactive Skin Decontamination Lotion (RSDL®), a class IIa medical device with CE certification for the
European market, which is currently procured.
RSDL® contains polyethylene glycol monomethylether and water as solvent
system and, in a proprietary formulation, 2,3-butandione monoxime (=
diacetylmonooxime, DAM) as active ingredient (AI) for the decontamination of
chemical warfare agents and toxic industrial compounds on the skin [1,2].
RSDL® was exposed to temperatures of 40°C, 53°C and 70°C up to 4 months.
We analysed the contents of DAM, of the putative degradation product
dimethylglyoxime (DMG) and of unknown degradation products by means of
reversed phase high pressure liquid chromatography (RP-HPLC) with diode
array detection (DAD). Based on the gathered concentration-time curves the
kinetics of degradation was determined and the temperature dependent rate
constants were calculated according Arrhenius equation [3]. These rate
constants were used to calculate the time span until defined threshold values
for DAM, DMG and unknown degradation products were exceeded. These
data were basis for estimation of shelf life at different storage conditions.
Oral delivery of active pharmaceutical ingredients (API) is the most popular
and most patient-friendly form of drug administration. However, there are
several challenges, which have to be overcome. In case, APIs of the Biopharmaceutical Classification System (BCS) classes II or IV, the poor solubility and
the poor permeability are the biggest problems.1 Therefore drug carrier
systems like nanoparticles (NP) are discussed to increase the bioavailability of
these drugs due to an enhanced delivery of pharmaceuticals over the
gastrointestinal (GI) barrier. In literature many groups described that nanoparticles are able to deliver insulin over the GI barrier.2 However, in most of these
studies only secondary effects e.g. the blood glucose response after insulin
administration were measured. The real transport of NP through the barrier
was not detected until now.
The aim of this study was to develop an improved drug delivery system for an
oral application. In detail, 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin
(mTHPP), a second-generation photosensitizer, was embedded into polymeric
NP to enhance the bioavailability in comparison to the free drug.
NP based on polymers like poly(DL-lactide-co-glycolide) (PLGA) or
poly(butyl methacrylate-co-(2-dimethylaminoethyl)
methacrylate-co-methyl
methacrylate) (Eudragit® E) were formed using the emulsion-diffusion-method.3
Different stabilizing agents, such as chitosan hydrochloride or didodecyldimethylammonium bromide (DMAB) were used for particle preparation in order to
influence the transcellular pathway. Furthermore surface modification of these
particles with ligands should enable specific transport mechanisms.
The photosensitizer mTHPP was successfully embedded into the different NP
formulations. Furthermore a surface modification of the NP could be achieved.
Dynamic light scattering analysis showed an average diameter ranging from
200 to 300 nm and a polydispersity index < 0.1. All formulations showed no
cytotoxicity concerning Caco–2 cells and could be tested in the GI barrier
model. The model was validated regarding barrier integrity, tight junction
formation and permeability of control compounds. Barrier integrity was
controlled continuously with an online measurement of the transepithelial
electrical resistance. Previously described NP formulations were investigated
over a period of 24 hours, where no permeation of particles could be detected.
The obtained results are in contrast to the literature, which is why we see the
205
need for further investigations to differentiate between particle permeation and
carrier-mediated drug transport.
PT.23
Nanoparticles for gene delivery
Look, J.1; Wilhelm, N.2; Gorjup, E.2; von Briesen, H.2; Noske, N.3; Rodriguez,
J.R.4; Prosper, F.4; Serra, M.5; Carrondo, M.5; Alves, P.5; Langer, K.1
1 Institute
of Pharmaceutical Technology and Biopharmacy, University of Münster,
Corrensstraße 48, 48149 Münster, Germany
2 Fraunhofer Institute for Biomedical Engineering, Ensheimer Str. 48, 66386 St. Ingbert,
Germany
3 apceth GmbH & Co. KG, Max-Lebsche-Platz 30, 81377 München, Germany
4 Clinica Universidad de Navarra, Av. Pio XII 36, 31008 Pamplona, Spain
5 Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras,
Portugal
Gene therapy is a promising tool for treating many serious diseases like cancer
or genetic disorders. The difficulty is to develop a safe and effective DNA
vector, which is able to transport the DNA to the nucleus without prior
degradation and with a controlled DNA release at the target location. Until now
only viral gene transfer vectors work efficiently but are still plagued with safety
concerns. Aim of this study is the development of a nanoparticular gene
transfer system as a safer alternative to the risky use of viral gene transfer
vectors. The obtained nanoparticles should be biocompatible, biodegradable,
cell specific by ligand modification and should enable receptor mediated
uptake and a controlled DNA vector release.
Therefor DNA vector loaded nanoparticles based on the protein human serum
albumin were prepared by a well-established desolvation method [1]. Different
stabilisation methods were tested to check the influence on DNA vector
release. To enable receptor mediated uptake the nanoparticles were surfacemodified. Different cell penetrating peptides were coupled to the primary amino
groups on the nanoparticle surface using a bifunctional PEG-based crosslinker.
Nanoparticles with monodisperse size distribution and an effective DNA
incorporation were obtained. Preservation of plasmid functionality during
particle preparation was shown by plasmid extraction from the final nanoparticle suspensions followed by Lipofectamine transfection. In vitro release studies
showed a stable entrapment of the DNA vector under storage conditions. The
surface modification worked successfully which could be revealed by peptide
quantification using C18-RP-HPLC analytics. Cell uptake and binding studies
proved a higher cell uptake for the surface-modified compared to unmodified
nanoparticles. Transfection studies showed gene expression for the surfacemodified nanoparticles.
Acknowledgements:
This work was financially supported by the BMBF (13N115391, 13N115402, 13N115413).
tides as found in gelatine preparations are an interesting example of such
materials, as they promote cell adhesion, are non-immunogenic, economic and
comparably easy to process. For the required cross-linking we utilized a
recently established oligomeric cross-linker and are able to engineer a variety
of hydrogels with different physic-chemical properties [2, 3]. Hydrogels
fabricated from Collagel® (type B, Gelita AG, Germany) and different
anhydride-containing cross-linkers were investigated as controlled release
systems for Polyethylenimine (PEI)-complexed nanoparticles for target gene
knockdown.
To investigate the distribution of oligonucleotides in the hydrogels, a model
plasmid DNA (pDNA) was covalently linked to a fluorophore using the mirusbio
Lable IT(R) kit. The pDNA was then complexed by branched PEI F25 for half an
hour4, diluted to an appropriate volume to rehydrate the lyophilized, preformed
hydrogel and finally cryo-sectioned and analysed using fluorescence microscopy.
In order to determine the release of functionally active nanoparticles a model
system was established. Hydrogels were loaded with PEI-based complexes
containing siRNAs against luciferase and stably luciferase-expressing SKOV3-Luc were cultured on the hydrogels. The biological activity was determined
by the luciferase knockdown of the released siRNA-containing nanoparticles.
Our results show that complexed oligonucleotides can by loaded to hydrogels
with varying modes of deposition dependent on pore structure and surface
characteristics. We could verify that immortal as well as primary cells grow and
spread on various gel formulations. Cell number, distribution and penetration
depend on physical and chemical properties of the gels. Cells, cultured in
osteogenic medium, formed extracellular matrix.
To evaluate the hydrogel’s performance for bone regeneration, human adipose
tissue-derived stem cells (hASC) were proliferated and differentiated along the
osteogenic lineage on hydrogels. hASC proliferation was observed via CLSM
imaging. Calcium as a late marker of osteogenic differentiation was determined
using a colorimetric method with cresolphthaleine as complexing reagent.
Moreover, we evaluated the knockdown of luciferase activity in stable
luciferase transfected SKOV cells via RNAi as a proof of concept.
These results are promising towards the development of a local siRNA release
platform for osteogenic regeneration. In addition, the gels appeared to be well
osteocompatible as they allowed for adhesion and differentiation of adult stem
cells.
Funded by Sächsisches Staatsministerium für Wissenschaft und Kunst (SMWK) and
DFG-Transregio 67 A1
References:
1. Schneider et al.: Tissue Eng Part A 2014, 20: 335-345.
2. Loth, T. et al.: React. Func. Polym. 2013, 73: 1480–1492.
3. Loth, T. et al.: Biomacromolecules 2014, 15: 2104–2118.
4. Ewe, A. et al.: Acta Biomaterialia 2014, 10, 2663-2673.
Reference:
1. Steinhauser, I. et al.: J. Drug Target. 2009, 17 (8): 627-637.
PT.25
PT.24
Influence of cross-linker type and content on oligonucleotide
deposition and delivery from gelatine-based hydrogels
Schwabe, K.1; Ewe, A.2; Kascholke, C. 1; Aigner, A.2; Hacker, M.C.1; SchulzSiegmund, M.1
Universität Leipzig, Institute of Pharmacy, Pharmaceutical Technology, Eilenburger Str
15a., 04317 Leipzig, Germany
2 Universität Leipzig, Faculty of Medicine, Rudolf-Boehm-Institute of Pharmacology and
Toxicology | Clinical Pharmacology, Härtelstraße 16-18, 04107 Leipzig, Germany
1
With the objective to improve upon current treatment of severe bone defects,
that are typically addressed with implants containing unphysiologically large
amounts of Bone Morphogenetic Protein 2 (BMP-2), we strive to suppress
BMP-2 antagonist locally by the controlled delivery of specific siRNA from
implant materials and thus increase the osteogenic potential of low doses of
BMP-2 [1]. In this context, we focus on hydrogels as one possible cell carrier
and delivery system due to their similarity to natural extracellular environments
and stabilizing effects on embedded nanoparticles and therapeutic proteins.
Through the use of natural polymers, hydrogel formulations with good
biocompatibility and optimized degradation kinetics can be obtained. Polypep-
206
Oral drug delivery of therapeutic gases- carbon monoxide
release for gastrointestinal diseases
Steiger, C.; Lühmann, T.; Meinel, L.
Purpose: Carbon monoxide (CO) has therapeutic effects in various gastrointestinal diseases [1] yet clinical use today is challenged by inappropriate
delivery modes [2]. Consequently, we developed a tablet referred to as oral
carbon monoxide release system (OCORS) (Figure 1) providing precise,
controlled and targeted CO delivery for the treatment of gastrointestinal injury
and inflammation, respectively.
Methods: OCORS is an oral tablet based on sulfite induced CO release from
the CO releasing molecule 2 (CORM-2) [2]. OCORS performance was detailed
as a function of the presence of buffer within the tablet core and characteristics
of a water-insoluble cellulose acetate coating, forming a semipermeable shell
around the tablet core. Amperometric detection was deployed for recording CO
release profiles throughout 10 hours.
Results: OCORS was tuned for environmental pH insensitivity by appropriate
buffer systems blended within the tablet core. OCORS delivered CO for up to
10 hours, contrasting CORM-2 suspensions delivering the gas within 1.5
hours. Zero order CO release was observed within approximately 30 to 240
minutes in different release media and was tuned by the thickness of the
semipermeable shell.
Conclusion: OCORS is a readily available tablet for oral use. The controlled
release system reliably delivered CO independent of environmental pH, such
that the therapeutic gas can be safely generated at gastric, intestinal or colonic
sites. In vivo experiments of OCORS are required to demonstrate the
pharmacokinetics and clinical potential of this oral delivery platform for
therapeutic gases.
Injectibility measurements indicate a very good syringeability caused by the
low viscosity of the ISFO. They show a dependence of the injection rate on the
injection force. The higher the injection rate the higher is the required force.
For the application into the chicken slightly higher forces are needed compared
to the beaker due to the backpressure of the surrounding tissue. By the use of
25G cannulas, very low injection forces of less than 9 N are required for all
ISFO and injection rates, providing the possibility of a further reduction in
needle size.
The decrease in conductivity of buffered salt solutions by addition of NMP can
be used for determining the extraction kinetics out of the applied ISFO. The
small amount of the biocompatible solvent NMP is released within 6-10 h,
leading to the solidification. This observation is almost independent from the
LMOG concentration. The formation of a bulk gel structure causes dissolved
molecules to be highly mobile due to the low micro viscosity and to diffuse
easily to the interface.
Figure 1: Schematic drawing of the oral carbon monoxide release system
(OCORS). Left part of the cartoon: Water permeates the semi-permeable
cellulose actate shell, dissolving the swelling coating layer around the sodium
sulfite crystals which readily dissolve. Right part of the cartoon: The Na2SO3
interacts with CORM-2, thereby causing CO release.
References:
1. Motterlini, R.; Otterbein, L.E.: Nature Reviews Drug Discovery, 2010, 9: 728–743.
2. Steiger, C.; Luhmann, T.; Meinel, L.: Journal of controlled release : official journal of the
Controlled Release Society, (2014).
PT.26
In Situ Forming Oleogels: In Vitro Investigation of Application,
Solidification and Degradation
Windorf, M.; Mäder, K.
Martin-Luther-University Halle-Wittenberg, Department of Pharmaceutical Technology and
Biopharmaceutics, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
Introduction
Parenteral depot systems provide a sustained drug release over several days,
weeks or even months. However, the often used polymers consisting of lactic
acid and glycolic acid monomers show a complex and hardly predictable drug
release kinetic and furthermore, the accumulation of acidic degradation
products often leads to local irritation or influences the stability of the active
ingredient [1]. In contrast, liquid and solid lipids more and more turn out to be a
promising alternative as matrix forming materials [2].
In situ forming oleogels (ISFO) are composed of a solvent or oil, a low
molecular weight organogelator (LMOG) and a small amount of a biocompatible organic solvent [3]. After application, the solvent diffuses into the surrounding tissue and the LMOG self-assembles into aggregates, which build a solid
network by inter-molecular physical interactions with incorporated oil droplets.
Their relatively simply production, the gentle manufacturing conditions for
temperature resp. shear sensitive drugs as well as the ease and painless
administration are just some advantages.
In this study, the application of the LMOG 12-Hydroxystearic acid (12-HSA) to
produce injectable ISFO based on peanut oil is investigated. The solidification
of the formulation by extraction of the solvent N-Methyl-2-pyrrolidone (NMP)
and the degradation behaviour of the implant in the presence of lipase are
presented.
Methods
The injection force was measured at different injection rates and with different
cannula sizes into a beaker or chicken wings. Conductivity measurements
were used to determine the extraction velocity of NMP after injection of the
ISFO in phosphate buffered saline leading to the solidification of the implant.
To determine the degradation rate, ISFO were injected in phosphate buffered
saline containing different amounts of Lipoprotein lipase. Every 48 hours the
surrounding medium was removed. The time-dependent degradation of the
implants was determined by weighing after drying and calculating the mass
loss.
The degradation rate can be controlled by the concentration of the LMOG and
ranges from a few days to months. Lowering the LMOG concentration of the
ISFO and increasing the lipase activity in the release medium leads to
enhanced implant degradation. The implants are degraded layer-by-layer from
the surface and mass erosion and rupture does not occur. Therefore, a
continuous release of suspended drugs is possible.
The authors would like to thank the BMBF (ProNet-T³; Th-03) for the financial support.
References:
1. Fredenberg, S. et al.: Int. J. Pharm. 2011, 415(1-2): 34-52.
2. Kreye, F. et al.: Expert Opin. Drug Deliv. 2008, 5(3): 291-307.
3. Vintiloiu, A. et al.: J. Control. Release 2008, 125(3): 179-192.
PT.27
smartLipids® - the next generation of lipid nanoparticles by
optimized design of particle matrix
Müller, R.H.1; Ruick, R.1; Keck, C.M.2
1 FU
2 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany
Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were
invented as drug carrier systems about 25 years and 15 years ago, respectively [1]. Meanwhile they have established themselves as delivery systems being
investigated and developed by many research groups world-wide. Identical to
the development of liposomes, entering the cosmetic market before appearance of pharmaceutical products, NLC meanwhile are applied in cosmetic
products world-wide on the market (e.g., La Prairie, Amore Pacific etc.). The
SLN and NLC posses a particle matrix typically made from a single solid lipid
(SLN) or a lipid blend (solid lipid plus oil, NLC). To improve the drug loading
and to ensure a firm drug incorporation during shelf life, the design of the
particle matrix was optimized by “more complex nanostructuring” to yield
smarter lipidic nanoparticles (smartLipids®).
Many lipids used in SLN and NLC can undergo polymorphic transitions - also
known to be influenced e.g., by the stabilizers used [2] – directly after
preparation or on storage. This influences the physical stability as well as drug
loading properties, i.e., loading capacity and potential resulting drug expulsion
(typically more pronounced in SLN, less in NLC). The aim was to develop a
new lipid matrix primarily exhibiting an α-modification, which is less densely
packed than lower energy β´- and β-modifications and therefore providing a
higher drug loading. The aim was also to hinder or delay polymorphic
transitions towards higher energy modifications during storage. This should
avoid drug expulsion and - from the production side - reduce the dependency
on certain stabilizers and processing parameters applied.
The developed smartLipids® ideally consist of a multiple mixture (up to 10 or
more) of different solid lipids containing mono-, di-, and triglycerides with
different chain length (ranging from C10 to C22 or longer) optionally having
additional liquid lipids (oils) in the blend. This leads to a polydisperse and less
structured matrix with imperfections and hinders the transition to more stable
polymorphs (β´- and β-modification), thus increasing the drug loading. A direct
comparison to classic SLN formulations containing Dynasan 118 (Tristearin)
was made.
The lipid matrix consisted either of Dynasan 118 (= SLN) or a smartLipids®
blend. Final formulations contained 15 % lipid matrix, different stabilizers i.e.,
anionic, cationic or nonionic stabilizers (sodium dodecyl sulfate, Lanette E,
polyvinyl alcohol, Tween 20, cetylpyridinium chloride or Plantacare 818) and
207
Milli-Q water up to 100 %. The formulations were produced by hot high
pressure homogenization (3 cycles at 500 bar) and were characterized by
photon correlation spectroscopy (PCS) (Zetasizer Nano ZS), laser diffraction
(LD) (Mastersizer 2000, both Malvern Instruments, UK), light microscopy
(Orthoplan, Zeiss) and wide angle X-ray diffraction (WAXD) (Philips PW 1830,
Philips, Netherlands).
The polymorphic modifications were determined by WAXD where one single
peak located at 2Ө = 21.4° (corresponding 4.15 Å) is indicating an αmodification and the peak at 2Ө = 19.4° (corresponding 4.6 Å) was chosen to
identify the presence or absence of the β-modification, because no superposition occurs at this angle. Within the various Dynasan SLN formulations only 1
sample showed a complete α-modification, 2 showed a full β-Modification and
3 a mixture of both depending on the stabilizer. In contrast within the
smartLipids® formulations, 3 samples showed a complete α-modification and
the other 3 an α-modification with only small amounts of β-modification. The
independency from the stabilizer type on the polymorphic state could be shown
for smartLipids® formulations in contrast to conventional SLN formulations.
Samples which remained stable showed a z-average mainly ranging from 100
nm to 400 nm (polydispersity index mainly between 0.15 and 0.30).
By combining multiple lipids a lipid particle matrix could be designed which
predominantly forms an α-modification and exhibits polydisperse and less
ordered structure. In contrast to the highly ordered β-modification this enables
higher drug encapsulation and firmer drug inclusion. The dependency on the
applied stabilizer of smartLipids® formulations regarding polymorphic transitions is reduced. Therefore smartLipids® represent a “smarter version” of the
NLC facilitating drug incorporation, especially when formulating drugs with a
priori low solubility in lipids (e.g., dexamethasone acetate) and reducing the
problem of drug expulsion.
the NLC. Hence, the structure of the vehicle seems to modify the film formation
of NLC. Reasons for this are manifold, e.g. inhomogeneous distribution of the
NLC within the vehicle and/or agglomeration of the particles during the
application and/or due to drying of the vehicle. The influence of the vehicle
structure on film formation and skin hydration of NLC was even more
pronounced when polyacrylate gel was used as vehicle. The gel alone led to a
strong hydration effect 1h after application, after 4h and 24h the skin hydration
was less than the control, indicating a strong drying effect of the hydrogel.
Addition of NLC to the gel decreased the strong increase in hydration and the
hydration effect after 4h and 24h was also lower than for the gel alone. An
explanation for these results is that NLC in hydrogel led to a partial film
formation directly after application, i.e. as observed for the NLC in crema
basalis. However over time the water of the gel evaporates, the gel structure
collapses and the NLC can form a tight film with highly occlusive properties.
Conclusions: The type of vehicle used for incorporation of NLC and the
concentration of NLC strongly influences the film formation of NLC upon
dermal application und thus the occlusive properties of NLC. Corneometer
measurements are suitable to identify changes in skin hydration. However, in
case of NLC containing formulations the data represent a superposition of
effects caused by the film formation of NLC (decrease in measured hydration)
and effective skin hydration (increase in measured hydration). Therefore,
further studies will focus on the evaluation of additional skin parameters and
different bases to identify optimized formulations for the dermal application of
NLC with tailor-made dermal properties.
References:
1. Müller, R.H. et al.: Current Drug Discovery Technologies 2011, 8: 207-227.
2. Müller, R.H. et al.: H&PC Today 2014, 9(2): 18-25.
3. Müller, R.H. et al.: EURO COSMETICS 2013, 6: 20-22.
References:
1. Müller, R.H.; Shegokar, R.; Keck, C.M.: Curr. Drug Discovery Technol. 2011, 8(3): 207227.
2. Bunjes, H.; Koch, M.J.; Westesen, K.: Molecular Organisation on Interfaces (Springer
Berlin Heidelberg) 2002.
PT.28
Skin hydration of NLC due to formation of invisible patch:
Influence of vehicle
Arntjen, A.1; Maus, A.1; Keck, C.M.1,2
1 Applied
Pharmacy, University of Applied Sciences Kaiserslautern – Campus Pirmasens,
Pirmasens, Germany
2 Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Institute of
Pharmacy, Freie Universität Berlin, Berlin, Germany
Introduction: Nanostructured lipid carriers (NLC) consist of a solid lipid matrix
being composed of a blend of liquid and solid lipid and possess a size typically
in the range between 80-500nm [1]. After dermal application they form an
“invisible patch” which causes occlusion and thus an increase in skin hydration
[2]. For dermal application NLC need to be incorporated into a vehicle, i.e.
ointments, creams or gel bases. As different types of vehicle possess different
properties, the hydration effect of NLC, i.e. the ability of NLC to form the
“invisible patch”, might be affected by the type of vehicle. Therefore, the aim of
this study was to evaluate the influence of different vehicles, i.e. water, crema
basalis (Basiscreme) and polyacrylate gel on the skin hydration effect of NLC.
Material and methods: Fresh pig ears were used as ex-vivo model. The NLC
were admixed in 5% and 10% concentration in each base. As controls only the
bases were topically applied. Additionally untreated pig ear skin was measured
(n=3). Skin hydration was analysed using a Corneometer® CM 825 (Courage &
Khazaka Electronic GmbH, Köln, Germany) following the instructions of the
manuals. Measurements were performed prior to application and 1h, 4h and
24 h after application of the formulations, respectively.
Results: The application of pure water showed no effect in comparison to the
untreated control. Water containing 5% or 10% of NLC showed lower skin
hydration values in comparison to the controls. The results confirm the
formation of the invisible patch upon the addition of NLC, i.e. less skin
hydration is measured due to the formation of the lipidic NLC film [3]. However,
the standard deviations for 5% NLC are much higher than for the 10% NLC
formulations, indicating a less tight film formation for 5% NLC. The application
of crema basalis and crema basalis with NLC increased the skin hydration.
The hydration effect was more pronounced and longer lasting with NLC 5%
and was most pronounced with 10% NLC. In comparison to the NLC in water
the skin hydration was detectable for the NLC in crema basalis, indicating that
the addition of NLC to a semi solid cream prevents complete film formation of
208
PT.29
Multi-Targeting Modifiers of Nano-Carriers for individual Therapy
and Diagnosis of Cancer
Krebs, L.1; Peters, T.1; Langguth, P.1; Goerigk, G.2; Schweins, R.3; Nawroth, T.1
Gutenberg University, Inst. Pharmacy and Biochemistry, Pharmaceutical Technology
Department, Staudingerweg 5, D-55099 Mainz
2 HZB, Institute of Soft Matter and Functional Materials , BESSY Synchrotron, ASAXS, D14109 Berlin, Germany
3 Institut Laue Langevin ILL, Group DS/LSS, 71 Avenue des Martyrs, F-38042 Grenoble
CEDEX 9, France
1
The MultiTarget project shall improve therapy and diagnosis of severe
diseases, e.g. cancer, by individual targeting of drug-loaded nanopharmaceuticals towards cancer cells. Specific ligands, which are recognized
by the diseased cells, are bound to the nanoparticle surface, and thus capable
of directing the drug carriers
[1-3]. In the current concept a multiple ligand set is coupled by a fast assembly
technique (click link) in the very last step of the formulation. In the clinical
application the ligands set (2-5 different) will be selected according to the
biopsy analysis of the patient tissue e.g. from tumor.
We synthesize multi-targeting modifiers of metal drug loaded nanoparticles
which consist of four structure domains (fig.1). The components are varied and
optimized in a case specific manner. The nanoparticles, e.g. biodegradable
polymer (PLGA), lipid particles as well as the anchor domain are hydrophobic.
The linker binds the ligand in two steps: adsorption and a covalent bond
formation as terminal step. The hydrophilic spacer is essential for keeping the
distance from the nanoparticle surface. The structure of the modified nanoparticles is analyzed by dynamic light scattering DLS, neutron small angle
scattering SANS and metal specific X-ray scattering ASAXS, while the effect of
the drug is proven in cell culture tests [4]. The multi-targeting modification is
applied to lanthanide loaded polymer nanoparticles (PLGA) for radiation
therapy [5] and liposomes as a fast available carrier.
multi-targeting nanoparticle modifiers for individual medicine
Table 1: Comparison between the fluorescence signals in the heart of
two rats two hours after the application of a fluorescence-labeled contrast agent. The microscopic images show only a fluorescence signal in
the infarcted tissue, which is also infiltrated with macrophages.
Animal
Rat with
infarction
x 10
10
8
6
4
2
Rat
without
infarction
x 10-6
2.5
2
1.5
1
0.5
infarction
References:
1. Ferrara, T.A. et al.: Current Opinion in Molecular Therapeutics 2009, 11, 37-42.
2. Alemdaroglu, F.E. et al.: Adv. Materials 2008, 20, 899-902.
3. Jahn, M.R. et al.: J. Pharm. Pharmacol. 2011, 63, 1522-1530.
4. Buch, K. et al.: Radiation Oncology 2012, 7, 1-6.
5. EU-Patent 11 007 401.0; PCT 13 07 12, 2012 “A particulate system for use in
diminishing cell growth / inducing cell killing”, keyword "Lanthanide", Gutenberg
University Mainz, inventors: Buch, K., Nawroth, T., Langguth, P., Schmidberger, H..
PT.30
Imaging of cardiac infarction by labeling the oil phase of a fluorcontaining MRI contrast agent
Keller, T.1,2,3; Dietrich, T.3; Bourayou, R.4; Wittstock, K.3; Meyborg, H.3; Licha,
K.5; Schnackenburg, B.6; Fleck, E.3; Bunjes, H.1
1 Technische
Universität Braunschweig, Institute of Pharmaceutical Technology,
Mendelssohnstraße 1, 38106 Braunschweig, Germany
2 B.Braun Melsungen AG, Carl Braun Straße 1, 34212 Melsungen, Germany
3 Deutsches Herzzentrum Berlin,Kardiologisches Forschungslabor, Seestraße 13, 13353
Berlin, Germany
4 www.bourayou.de, Weichselplatz 8, 12045 Berlin, Germany
5 Mivenion GmbH, Robert-Koch-Platz 4, 10115 Berlin, Germany
6 Philips Healthcare, Philipsstraße 14, 20099 Hamburg, Germany
Inflammatory events in the circulatory system can be visualized by Magnetic
Resonance Imaging (MRI) using emulsion-based contrast agents containing
fluorinated substances as part of the oil phase. In the bloodstream, the oil
droplets are phagocytized by cells of the immune system before they enrich
especially in inflamed tissues [1]. The aim of the present study was to label the
oil phase of such a contrast agent with a Near-Infrared-Fluorescence-dye
(NIRF-dye) to allow fluorescence imaging and microscopy in order to follow the
in vivo fate of the oil droplets with high sensitivity and resolution [2].
An oil-in-water-emulsion containing perfluorohexyloctane (F6H8) as part of the
oil phase was prepared as MRI contrast agent. The NIRF-dye “PM-ITCC”,
which was covalently coupled to heptadecafluoroundecylamine to increase its
affinity to F6H8, was used as fluorescence label for the oil droplets. The
labeled emulsion was incubated with human plasma to ensure that the NIRFdye was not washed out by contact with plasma components. The contrast
agent formulation was tested by intravenous injection to rats with a myocardial
infarction as inflammatory model (three rats with an infarction vs. one rat
without infarction). Two hours after injection, the rats were sacrificed and the
hearts of the animals were characterized with a fluorescence imager ex vivo. In
addition, cryosections of the hearts were inspected by brightfield and fluorescence microscopy using hematoxylin-eosin (H.E.)- and CD68-stainings
(macrophages).
The ex vivo NIRF images of the infarcted hearts and the corresponding
microscopic images displayed a significantly higher fluorescence signal than
the non-infarcted heart (see individual values of the scale for each fluorescence image in Table 1). Furthermore, the infarcted tissues were heavily
infiltrated with macrophages (see red-stained cells in the CD68-image in Table
1).
The fluorescence-labeled formulation can thus be used to assess the
distribution of the F6H8-containing contrast agent with high sensitivity and
resolution.
Macrophages
References:
1. Flögel, U. et al.: Circulation 2008, 118(2): 140-148.
2. Klohs, J. et al.: Mol Imaging 2006, 5(3): 180-187.
PT.31
Proficiency test on ofloxacin eye drops
Maul, K.J.1; Diergardt, T.2; Feldmann, D.3; Wätzig, H.1
1 Technische
Universität Braunschweig – Institute of Medicinal and Pharmaceutical
Chemistry, Beethovenstraße 55, 38106, Germany
2 Physikalisch-Technische Bundesanstalt – Braunschweig, Bundesallee 100, 38116,
Germany
3 Bausch+Lomb, Brunsbütteler Damm 165-173, 13581, Germany
Proficiency testing is a good performance-monitoring procedure and an
important part in quality assurance, which should also include validation of
analytical methods, the use of certified reference material and a routine
internal quality control [1].
This proficiency test is the second of a series in the East African Community
(EAC). It is a part of a project called “Establishment of a regional Quality
Infrastructure for the pharmaceutical sector in the EAC” initiated by the
Physikalisch-Technische Bundesanstalt. It was conducted in April 2014 with 15
participants from 5 different countries. Ofloxacin eye drops were distributed as
test material. These were analyzed according to the USP monograph for the
amount of active ingredient and the pH-value. The acceptance criteria for the
assay are 90.0-110.0% and the pH-value should be between 6.0 and 6.8.
Furthermore, the system suitability test was performed. The interlaboratory
standard deviation for the pH-value is 0.15 (RSD%=2.41 %) and for the assay
using the submitted results 0,622 mg/ml (RSD%=22,1 %). For the evaluation
the mean of all laboratories was employed as assigned value. Moreover the zscore for each laboratory was calculated (Figure 1). All laboratories measured
a pH-value within the specification. The results for the assay have to be
recalculated to eliminate calculation errors.
Assay (submitted results)
2
z-score
Acknowledgments: We are grateful for the funding by the German ministry of science and
education BMBF, grant 05KS7UMA.
Microscopic image (100 x)
H.E.
CD68
-6
The authors thank Dr. J. Schmitt, Dr. D. Röthlein, Dr. V. Krüger and W. Schlemermeyer
(all B. Braun) for supporting the project.
0
-2
5 10 3
-4
1
4
9 11 15 6
8
7
2 12 13 14
lab. #
pH-value
3
z-score
Fig.1: Case- and person specific therapy and diagnosis by cell specific nanopharmaceuticals : The multi-targeting modifiers for drug nanoparticles consist
of four domains: 1) hydrophobic anchor, 2) hydrophilic spacer, 3) linker (two
parts) and 4) receptor ligand (protein or bioorganic ligand), coupled by fast
assembly.
Microscopic image (400 x)
Black/white
Fluorescence
NIRF
1
-1
-3
5
2
1 10 9 13 12 15 4
7 14 6
8 11 3
lab. #
209
Figure 1: Z-score charts. The z-scores of each participant are
shown for assay and pH. A z-score between -2 and 2 (white area)
is very good. Z-scores between -3 to -2 and 2 to 3 (black area) are
acceptable.
References:
1. Thompson, M. et al.: Pure Appl. Chem. 2006, 78(1): 145-196.
PT.32
Accessing the Nanoparticle Corona in Pulmonary Surfactant
Raesch, S.1,2; Tenzer, S.3; Storck, W.3; Ruge, C.1; Schäfer, U.F.1; Lehr, C.-M.1,2
1 Department
of Biopharmaceutics and Pharmaceutical Technology, Saarland University,
66123 Saarbruecken, Germany
2 Department of Drug Delivery, Helmhotz Institute for Pharmaceutical Research Saarland,
66123 Saarbruecken, Germany
3 Institute for Immunology, University Medical Center of Mainz, Langenbeckstrasse 1,
55101 Mainz, Germany
Nanoparticles (NP) are of high interest in medical and pharmaceutical
research, especially regarding their toxicological profile, but also potential use
as carrier systems. Understanding the outstanding properties of NP´s can not
only help assess the possible harm which can arise from them, but it also
opens new perspectives for targeted and controlled drug delivery. NP, that
come in contact with a biological fluid, are opsonized by biomolecules such as
proteins, which build a “corona”.
It has generally been accepted that an evolution of such a biomolecule layer
occurs, and that this time-dependent layer of adherent biomolecules typifies
the actual biological identity of the NP. Considering the many different NP with
varying surface modifications which are produced worldwide, differences in
resulting corona seem plausible and were identified in the coronas on NP in
plasma [2, 3].
The lung is an attractive pharmaceutical target, as the air-blood barrier is a
less than 2 µm thin layer with an enormous alveolar surface area larger than
100m2. The enormous amount of potentially polluted air, which passes the
lung, makes an effective maintenance system essential. In the alveolar region
cells are only covered by a thin pulmonary surfactant (PS) layer and clearance
is mainly carried out by alveolar macrophages. PS, secreted by type II alveolar
cells, allows gas diffusion and its surface tension lowering effect is essential for
stability of the alveoli during breathing cycle. The surfactant layer consists of
approximately 90% lipids (mainly phospholipids, especially DPPC) and 10%
proteins with about half of them being surfactant specific proteins (SP). Albeit
the PS is very thin (~200 nm), it is the first biological barrier an airborne NP
comes in touch with. Before NP are either taken up by the alveolar cells or
ingested by macrophages, they are coated by the PS building a lipoprotein“corona”.
So far, it remains to be elucidated whether the fate of inhaled NP depends on
the coating obtained from the surfactant layer, though there is evidence for the
influence of the SP on macrophage uptake [1]. With knowledge about the
relationship between the surfaces of NP, the subsequent built coronas, and the
recognition of these patterns by cells, an approach for longer residence time of
NP or targeted uptake by alveolar or macrophage cells could be exploited.
Although understanding the surfactant-NP interaction is fundamental for the
fate of NP in the lung, there is, so far, no reproducible method for the analysis
of the NP-corona.
The unique composition, structure, and properties of the lipid-rich PS require
different and more advanced analytical methods for the assessment of the NPcorona in the deep lung.
Hence, we used a native pulmonary surfactant preparation, isolated from
porcine lungs, for the development of a method, to access the lipid-proteincorona. Magnetic separation of NP was found to be most suitable in comparison to various commonly used techniques. We present data for three model
particles with diverse surface properties and the influence on the adsorbed
proteins. Furthermore, future possibilities for the analytical determination of all
biomolecules present in the nanoparticle corona in pulmonary surfactant are
discussed.
210
Chiara de Rossi, Jesús Perez-Gil
References:
1. Ruge, C.A. et al.: PLoS ONE 2012, 7(7): e40775.
2. Monopoli, M.P. et al.: J. Am. Chem. Soc. 2011, 133(8): 2525–2534.
3. Tenzer, S. et al.: Nat. Nanotechnol. 2013, 8(Oct): 772–781.
PT.33
Development of a Bioequivalent Taste Masked Cetirizine HCl
10mg Zydis® Dosage Form using Cyclodextrin
Grother, L.; Warren, N.; Cusack, A.
Catalent Pharma Solutions, Frankland Road, Blagrove, Swindon, SN5 8RU, United
Kingdom
Purpose
Cetirizine HCl is a bitter drug indicated for the treatment of allergies. Due to the
nature of allergic episodes, medication is often required at unpredictable times.
Consequently dose forms such as orally disintegrating tablets (ODT) are
frequently preferred by patients due to the greatly enhanced convenience they
provide. In order for the drug to be delivered using an ODT such as the Zydis®
dosage form, effective taste masking is required. The aim of this study was to
investigate if cyclodextrin could be used to taste mask cetirizine HCl 10mg
when formulated in a freeze dried ODT (Zydis®). A chemically stable product
with bioequivalence to the reference product was a prerequisite for success.
Methods
Prototype freeze dried formulations containing cyclodextrin were manufactured
using the Zydis® formulation and process1. Dissolution testing was performed
on the prototype formulations using USP apparatus II (50rpm) with 900ml
purified water. Assay and stability testing was conducted by HPLC.
A taste trial was conducted by a panel of 30 volunteers. The probe bioequivalence study was a single dose, open label, randomized, two period, two
treatment crossover study in which 10 healthy adult subjects received two
separate single dose administrations of cetirizine HCl 10mg, following an
overnight fast of at least 10 hours. Subjects received Cetirizine HCl 10mg
Zydis® ODT and Zirtek® 10mg reference product in a randomized fashion.
Results
The cetirizine and cyclodextrin were successfully incorporated into the Zydis®
dosage form resulting in elegant ODTs with rapid disintegration properties (<2
seconds). Stability and dissolution testing results were comparable or
favorable to the Zirtek® control. The results of the 30 subject taste panel were
positive, demonstrating that taste masking had been achieved by the use of
the cyclodextrin (Figure 1). The probe bioequivalence study in 10 subjects
showed that the Zydis® product was bioequivalent to the Zirtek® comparator.
Figure 1. Taste trial questionnaire results
Conclusion
This study confirmed that cyclodextrin can be successfully incorporated into
the Zydis® ODT. The formulation is chemically stable, taste masked and is
bioequivalent to the Zirtek® reference product. The work confirms the feasibility
of developing a commercially viable ODT formulation of cetirizine HCl 10mg.
Reference:
1. Seager, H.: J.Pharm.Pharmacol. 1998, 50: 375-382.
PT.34
Expression and localization of various tight junction-associated
proteins in porcine hair follicles and their contribution to transdermal barrier function
Mathes, C.1; Brandner, J.2*; Hansen, S.3; Schäfer, U.F.1; Lehr, C.-M.1,3*
1 Biopharmaceutics
and Pharmaceutical Technology, Saarland University, Saarbrücken,
Saarland, Germany, [email protected]
2 Department of Dermatology and Venerology, University Hospital Hamburg-Eppendorf,
Hamburg, Germany
3 Department of Drug Delivery, Helmholtz-Institute for Pharmaceutical Research Saarland
(HIPS), Helmholtz-Center for Infection Research (HZI), Saarbrücken, Germany
*Correspondence author
Barrier properties of hair follicles gained increasing interest during the last few
years, especially with respect to non-invasive antigen delivery via the
transfollicular route [1].Tight junctions (TJs), which are barrier-forming
paracellular junctions composed of various TJ transmembrane proteins (e.g.
claudin-1 to -24, occludin, tricellulin, and junctional adhesion molecules
(JAMs)), as well as intracellular scaffold proteins (e.g. ZO-1, -2, -3) [2], have
been shown to be localized and expressed in the human hair follicle as was
demonstrated several years ago by Brandner et al [3]. In simple epithelia and
endothelia their role as a barrier has been well characterized; in stratified
epithelia only first barrier properties have been elucidated. Due to difficulty in
obtaining human tissue for these types of experiments, we were interested in
comparing human and porcine TJ-associated proteins in the hair follicle in
order to determine whether the pig ear is suitable for further barrier property
investigation. Thus, we performed immunohistochemical staining on paraffin
sections (5 µm) of full thickness porcine ear skin using antibodies specific for
claudin 1, claudin 4, occludin and ZO-1.
Expression and localization of claudin 1, occludin and ZO-1 in pig were in
accordance with data reported by Brandner et al. in human [3]. Claudin 1 and 4
revealed a broader distribution in porcine trichocytes than in human tissue,
however, after restaining human tissue the same distribution was seen
concluding that this effect was most likely caused by higher sensitivity of the
new batch of antibody used. Claudin 1 showed the broadest distribution
throughout the entire hair follicle, as can also be seen in the epidermis. Colocalizations of at least two proteins were also seen in areas previously
reported in the human hair follicle. In conclusion, no difference was seen in the
expression and localization of the four TJ-associated proteins making the pig
ear model suitable for future investigations.
References:
1. Mittal, A. et al.: Vaccine, 2013, 31(34): 3442-3451.
2. Aijaz, S.; Balda, M.S.; Matter, K.: Int. Rev. Cytol. 2006, 248: 261-298.
3. Brandner, J.M. et al.: Arch. Dermatol. Res. 2003, 295(5): 211-221.
211
AUTHORS INDEX
Abdel-Aziz, H. ................................................................. 98, 151, 188
Abdelmohsen, U.R. ...................................................................... 182
Abe, I. ........................................................................................... 110
Abts, H.F. ........................................................................................ 75
Achenbach, J. ............................................................................... 167
Adler, M. ....................................................................................... 103
Admas, T.H. .................................................................... 72, 176, 178
Ahmad, K. ..................................................................................... 138
Aigner, A. ...................................................... 107, 130, 133, 134, 206
Akakawa, T. .................................................................................. 110
Alasel, M. ...................................................................................... 141
Alban, S. ............................................................................... 115, 186
Albishri, H.M. ................................................................ 144, 162, 168
Al-Gousous, J. .............................................................................. 198
Alhazmi, H.A. ................................................................ 144, 162, 168
Allegretta, G. ................................................................................. 165
Alves, P. ....................................................................................... 206
Andrews, K.T. ................................................................................. 58
Ansari, N. ........................................................................................ 75
Apel, A.K. ...................................................................................... 143
Appelt, A.K. ................................................................................... 170
Argyrou, A. .................................................................................... 157
Arisawa, M. ................................................................................... 109
Arntjen, A. ............................................................................. 204, 208
Asfaw, H. ...................................................................................... 158
Asseburg, H. ................................................................................. 185
Avery, V.M. ..................................................................................... 58
Awwad, K. ....................................................................................... 26
Ayata, K. ....................................................................................... 122
Bacher, A. ............................................................................. 153, 162
Bähre, H. ...................................................................................... 140
Bakowsky, U. ........................................................................ 160, 205
Baleerio, R. ..................................................................................... 64
Balk, A. ......................................................................................... 195
Ball, A.-K. ...................................................................................... 124
Ballell, L. ....................................................................................... 157
Barandun, L.J. .............................................................................. 161
Barho, M.T. ........................................................................... 113, 161
Bartuschat, A.L. ............................................................................ 169
Basavarajappa, D. ........................................................................ 138
Bassett, D.J.P. ................................................................................ 27
Basu, D. ................................................................................ 131, 155
Baudiß, K. ..................................................................................... 122
Bauer, M.R. .................................................................................... 45
Baumeister, S. .............................................................................. 159
Baumgärtel, A. .............................................................................. 188
Bautista, O. ................................................................................... 180
Bechthold, A. .................................................................................. 55
Becker, C. ............................................................................. 129, 131
Beese, K. .............................................................................. 136, 172
Begley, D. ..................................................................................... 154
Behrendt, C.T. ...................................................................... 153, 162
Bendas, G. .................................................................... 128, 129, 130
Bender, A. ....................................................................................... 49
Bensinger, D. ................................................................................ 175
Berger, T. ...................................................................................... 159
Bermudez, M. ......................................................................... 70, 179
Bernat, V. ........................................................................ 72, 176, 178
Bernhardt, G. ........................................................................ 171, 180
Bernhardt, P. ................................................................................ 106
Berressem, D. ....................................................... 123, 184, 185, 192
Betz, M. ........................................................................................ 161
Bhandari, D. .................................................................................... 60
Biel, M. .......................................................................................... 156
212
Biela, A. ........................................................................................ 142
Bischoff, I. ..................................................................................... 123
Blank, S. ....................................................................................... 194
Blaßhofer, F. ................................................................................. 134
Blättermann, S. ............................................................................. 180
Blöcher, R. .................................................................................... 172
Blohm, A. ...................................................................................... 156
Bock, A. .......................................................................................... 99
Bock, S. ........................................................................................ 159
Boddy, A.V. ................................................................................... 195
Bodem, J....................................................................................... 159
Boeckler, F.M. ................................................................................ 45
Bogan, R. ...................................................................................... 205
Bolte, K. ........................................................................................ 191
Bonnet, D. ....................................................................................... 47
Bonus, M......................................................................................... 53
Boomgaren, M. ............................................................................. 159
Boos, J. ......................................................................................... 195
Borchiellini, M. .............................................................................. 185
Borek, C. ....................................................................................... 128
Borghardt, J.M. ............................................................................. 196
Botermann, L. ............................................................................... 151
Böttcher, M.M. .............................................................................. 160
Bou-Chacra, N.A. ......................................................................... 203
Bourayou, R. ................................................................................. 209
Bracher, F. ............................................................................ 123, 156
Brandner, J. .......................................................................... 187, 211
Braun, A. ............................................................................... 157, 167
Brewster, M.E. ................................................................................ 90
Brezesinski, G. ............................................................................... 77
Briel, D. ......................................................................................... 178
Britz, H. ......................................................................................... 150
Brockmeyer, J. .............................................................................. 198
Brönstrup, M. ................................................................................ 135
Brosig, H. .............................................................................. 148, 170
Brötz, E. ........................................................................................ 186
Brox, R. ................................................................... 72, 176, 177, 178
Brücher, K. .................................................................................... 162
Brück, S. ....................................................................................... 152
Brüggerhoff, A. ............................................................................. 139
Brüßler, J. ..................................................................................... 160
Büchold, C. ................................................................................... 141
Bührmann, M. ............................................................................... 155
Büllesbach, K. ............................................................................... 180
Bunjes, H. ..................................................................................... 209
Burghaus, R. ................................................................................... 44
Busch, D. ...................................................................................... 152
Buschauer, A. ......................................................... 69, 139, 171, 180
Butz, L........................................................................................... 139
Caffrey, C.R. ................................................................................. 183
Calderon, M. ................................................................................. 199
Camacho, C. ................................................................................... 52
Cardinaux, J.-R. ............................................................................ 176
Carlino, L. ..................................................................................... 136
Carlomagno, T. ............................................................................... 51
Carrasco-Gomez, R. ..................................................................... 174
Carrier, L. ...................................................................................... 176
Carrondo, M. ................................................................................. 206
Cheng-Chang, C. .......................................................................... 156
Cheung, S.-Y. ............................................................................... 125
Chhatwal, G.S. ............................................................................. 184
Chirinda, B. ................................................................................... 165
Chung, C....................................................................................... 157
Cieslik, M.B. .................................................................................... 45
Ciglia, E. ............................................................................... 142, 143
Cinatl jr., J. .................................................................................... 134
Clement, B. ................................................................................... 173
Codutti, L. ....................................................................................... 51
Collins, C. ..................................................................................... 142
Cools, F. ....................................................................................... 120
Cristofoletti, R. ................................................................................ 93
Culmsee, C. ...................................... 29, 32, 113, 161, 189, 190, 191
Cusack, A. .................................................................................... 210
Dahse, H.-M. ................................................................................ 159
Dai, B. ........................................................................................... 123
Danhof, M. .................................................................................... 120
Darras, F.H. .................................................................................. 163
Dassinger, N. ........................................................................ 141, 145
De Min, A. ..................................................................................... 177
de Sousa Amadeu, N. .................................................................. 142
de Sousa, L.R.F............................................................................ 182
Debaene, F. .................................................................................. 161
Decher, N. ...................................................................................... 32
Decker, H. ............................................................................. 194, 196
Decker, M. ...................................................................... 71, 163, 187
Degenhardt, I. ....................................................................... 113, 159
Denzer, I. ...................................................................................... 189
Diederich, F. ................................................................................. 161
Diederich, W. ........................................................................ 159, 166
Diedrich, A. ..................................................................................... 64
Diedrich, D. ................................................................................... 143
Diehl, O. ........................................................................................ 125
Diemert, S. .................................................................................... 191
Diergardt, T. .......................................................................... 149, 209
Diesel, B. ...................................................................................... 123
Diethelm, S. .................................................................................. 106
Dietrich, T. .................................................................................... 209
Diken, M. ........................................................................................ 77
Dingemanse, J. ............................................................................. 147
Dings, C. ....................................................................................... 147
Dircks, M.G. .................................................................................. 149
Dirksen, U. .................................................................................... 128
Dirsch, V.M. .................................................................................... 54
Dittmar, F. ..................................................................................... 140
Dodel, R. ....................................................................................... 190
Doi, T. ........................................................................................... 111
Dolga, A.M. ..................................................................... 32, 190, 191
Dömling, A. ..................................................................................... 52
Dörje, F. ........................................................................................ 149
Dos Santos Capelo, R. ................................................................. 139
Drescher, S. .................................................................................. 163
Duffy, S. .......................................................................................... 58
Ebeling, S. .................................................................................... 186
Ebert, R. ....................................................................................... 167
Ecke, M. ........................................................................................ 155
Eckert, G.P. .................................................. 123, 184, 185, 191, 192
Efferth, T. ................................................................................ 95, 182
Ehrig, K. ........................................................................................ 186
Ehrlich, S.M. ................................................................................. 132
Eickhoff, C. ................................................................................... 151
Einsle, O. ........................................................................................ 85
Eisenreich, W. .............................................................................. 153
El Deeb, S. ................................................................... 144, 162, 168
Elewa, M. ...................................................................................... 163
Elgaher, W.A.M. ........................................................................... 168
El-Hady, D.A. ................................................................ 144, 162, 168
Elsässer, K. .......................................................................... 113, 189
Empting, M. .......................................................................... 144, 165
Endreas, W. .................................................................................. 160
Engel, F. ....................................................................................... 134
Engel, J. ................................................................ 129, 131, 155, 165
Engels, B. ............................................................................. 128, 170
Erdelmeier, C.A.J. ........................................................................ 183
Erker, T. ........................................................................................ 173
Eschenhagen, T. .......................................................................... 176
Esparza, I........................................................................................ 77
Ewe, A. ......................................................................................... 206
Exner, T.E. ...................................................................................... 51
Fabian, J. ...................................................................................... 161
Fan, A. .......................................................................................... 140
Fang, Z. ........................................................................................ 129
Faust, A. ....................................................................................... 114
Feeder, N. ....................................................................................... 50
Fehler, S.K. ................................................................................... 169
Feizabad, M.S. ............................................................................. 164
Feldmann, D. ........................................................................ 149, 209
Fenical, W. .................................................................................... 106
Ferreirόs, N. .................................................................................. 124
Fersht, A.R...................................................................................... 45
Fettel, J. ........................................................................................ 126
Fischer, B...................................................................................... 188
Fischer, K...................................................................................... 125
Fischer, M. ............................................................................ 153, 162
Flaßhoff, M.................................................................................... 131
Fleck, E. ........................................................................................ 209
Fleckenstein, K. .............................................................................. 88
Fleming, I. ....................................................................................... 26
Flesch, D............................................................................... 167, 174
Flinspach, K. ................................................................................. 143
Förster, F. ....................................................................................... 56
Fox, D. .......................................................................................... 154
Franco, R. ..................................................................................... 181
Frank, J. ................................................................................ 163, 184
Franke, N. ..................................................................................... 123
Franke, R. ..................................................................................... 135
Fransson, I. ................................................................................... 171
Fredriksson, K. ............................................................................... 51
Freeman, B.A. ................................................................................ 26
Freigang, M................................................................................... 184
Friedland, K. ........................................................... 87, 147, 149, 189
Friedland-Leuner, K. ............................................................. 184, 189
Friedrich, C. .................................................................................. 165
Friess, W....................................................................................... 199
Fritz, D. ........................................................................................... 77
Fröhlich, T. .................................................................................... 132
Fröhling, S. ..................................................................................... 83
Frömel, T. ....................................................................................... 26
Frötschl, R. ................................................................................... 134
Fruth, M. ....................................................................................... 144
Fuchs, S........................................................................................ 183
Fujii, H............................................................................................. 40
Fujii, N............................................................................................. 41
Fukuda, H. .................................................................................... 109
Funari, S. ........................................................................................ 77
Fürst, R. ................................................................................ 123, 183
Fürtig, M........................................................................................ 149
Gabler, M. ............................................................................. 167, 174
Gajer, M. ......................................................................................... 85
Gallacher, G.J. .............................................................................. 120
Ganjam, G.K. ........................................................................ 190, 191
Garscha, U.................................................................................... 124
Geisslinger, G. ........................................................................ 26, 124
George, S. .................................................................................... 139
Gerber, U. ..................................................................................... 128
Gerbier, R. .................................................................................... 168
Gerhardt, S. .................................................................................... 85
Germershaus, O. .......................................................................... 157
Gerst, M. ....................................................................................... 204
Gerstmeier, J. ............................................................................... 124
Gerth, K. ....................................................................................... 184
Getlik, M........................................................................................ 165
Gieselmann, V. ............................................................................. 117
213
Gillard, M. ..................................................................................... 179
Glaser, J. ...................................................................................... 183
Gmeiner, P. .................................................................................. 169
Gnanapragassam, V.S. ................................................................ 123
Goerigk, G. ................................................................................... 208
Gohlke, H. ....................................................................... 53, 142, 143
Gollos, S. ........................................................................................ 62
Gomeza, J. ................................................................................... 179
Göring, S. ..................................................................................... 175
Gorjup, E. ..................................................................................... 206
Gorzelanny, C. .............................................................................. 129
Gossmann, R. ............................................................................... 198
Gotta, V. ....................................................................................... 120
Grabow, N. ..................................................................................... 36
Grathwol, C. .................................................................................. 170
Gräwert, T. ............................................................................ 153, 162
Grebenstein, N. ............................................................................ 184
Grevelding, C.G. ........................................................................... 156
Griese, N. ..................................................................................... 151
Griesinger, C. ................................................................................. 51
Grimm, C. ..................................................................................... 156
Groh, M. ........................................................................................ 168
Groll, M. ........................................................................................ 153
Grother, L. .................................................................................... 210
Grün, J. ......................................................................................... 182
Grünebaum, J. .............................................................................. 205
Grünweller, A. ............................................................... 107, 130, 133
Grütter, C. ..................................................................................... 165
Guenther, S. ................................................................................... 60
Guetschow, M. .............................................................................. 180
Günther, S. ............................................................................. 68, 174
Guo, Z. .......................................................................................... 176
Guthoff, R. ...................................................................................... 63
Gutmann, M. ................................................................................. 167
Haas, H. .......................................................................................... 77
Haase, C. ...................................................................................... 164
Hachenthal, N. .............................................................................. 123
Hack, C. .......................................................................................... 88
Häcker, G. .................................................................................... 186
Hacker, M.C. ................................................................................. 206
Häckh, M. ....................................................................................... 68
Haefeli, W.E. ................................................................................. 147
Haeuser, M. .................................................................................. 204
Häfner, A.-K. ........................................................................... 26, 124
Hagl, S. ................................................................................. 184, 191
Hähn, S. ........................................................................................ 153
Hahne, M. ....................................................................................... 34
Hamacher, A. .................................................................................. 58
Hammami, M. ............................................................................... 132
Hammamy, M.Z. ........................................................................... 164
Hanefeld, A. .................................................................................... 64
Hanh, B.D. ...................................................................................... 63
Hanke, T. ...................................................................................... 125
Hansen, F.K. ................................................................... 58, 142, 143
Hansen, S. .................................................................................... 211
Harder, M. ....................................................................................... 28
Hardick, J. ..................................................................................... 155
Hardt, S. ....................................................................................... 192
Harenberg, J. ................................................................................ 152
Harloff, M. ..................................................................................... 133
Harmer, N.J. ................................................................................. 154
Hartmann, R.K. ............................................................. 107, 130, 133
Hartmann, R.W. ...................................................... 18, 144, 165, 168
Hasenfuss, G. ............................................................................... 176
Hassan, T.H. ................................................................................. 199
Haupenthal, J. ...................................................................... 144, 168
Hausch, F. .................................................................................... 189
Häussinger, D. ................................................................................ 53
Havemeyer, A. .............................................................................. 173
214
Hawarden, A. ................................................................................ 156
Heide, H. ......................................................................................... 26
Heilmann, J. .................................................................................. 187
Hein, M. ........................................................................................ 154
Heine, A. ....................................................... 132, 141, 142, 161, 166
Heinrich, M.R. ............................................................................... 169
Held, J............................................................................. 58, 153, 162
Hellmann, N. ......................................................................... 194, 196
Hellwig, M. .................................................................................... 134
Helmstädter, A. ............................................................................. 182
Hempel, G............................................... 86, 128, 154, 161, 173, 195
Hengl, T. ......................................................................................... 75
Hennen, S. .................................................................................... 179
Henrich, A. .................................................................................... 136
Hentschel, U. ................................................................................ 182
Hergenröther, A. ........................................................................... 152
Herrmann, J. ................................................................................... 56
Herz, T. ......................................................................................... 173
Herzog, J. ..................................................................................... 132
Heuckmann, J.M. .......................................................................... 165
Heusler, E. .................................................................................... 170
Hibert, M. ........................................................................................ 47
Hilgenfeld, R. ................................................................................ 164
Hillebrecht, A. ............................................................................... 159
Hils, M. .......................................................................................... 141
Hintersteininger, M. ...................................................................... 173
Hinz, S. ......................................................................................... 181
Hirao, T. ........................................................................................ 109
Hirayama, S. ................................................................................... 40
Höbel, S. ....................................................................................... 134
Hoeglinger, G.U. ........................................................................... 191
Hofmann, B. .................................................... 26, 124, 125, 126, 127
Hohmann, C.................................................................................... 31
Holak, T. ......................................................................................... 52
Holländer, A. ................................................................................. 158
Höllein, L. ...................................................................................... 153
Höllerhage, M. .............................................................................. 191
Holloway, S. .................................................................................. 159
Holzgrabe, U. ................................ 153, 154, 157, 166, 183, 190, 195
Homann, J. ................................................................................... 124
Hommoss, G. ................................................................................ 204
Hönzke, S. .................................................................................... 122
Hoppstädter, J. ............................................................................. 123
Horstkorte, R................................................................................. 123
Hoser, S. ................................................................................. 98, 188
Hoß, S.G. ...................................................................................... 128
Hou, Z. ............................................................................................ 41
Hsieh, L.T. .................................................................................... 183
Huang, G. ..................................................................................... 187
Huber, S.M.................................................................................... 139
Hübner, H. .................................................................................... 169
Hummel, M. .................................................................................. 198
Hüsecken, K. ................................................................................ 144
Hüttel, S. ......................................................................................... 56
Hüwel, D. ...................................................................................... 156
Idzko, M. ....................................................................................... 122
Ihling, C......................................................................................... 116
Ilan, N. .......................................................................................... 128
Ilko, D............................................................................................ 157
Illarionov, B. .......................................................................... 153, 162
Imhof, A. ....................................................................................... 136
Imming, P...................................................................... 157, 158, 163
Imran, I.......................................................................................... 192
Irsheid, L. ...................................................................................... 128
Isaak, J. .......................................................................................... 26
Ishida, Y. ....................................................................................... 111
Ishimura, K.................................................................................... 109
Itami, K.......................................................................................... 114
Iwai, T. ............................................................................................ 40
Jaehde, U. ........................................................................ 62, 88, 134
Jakob, F. ....................................................................................... 167
Jakobs, H. ..................................................................................... 173
Jakubzig, B. .................................................................................. 129
Janiak, C. ...................................................................................... 142
Janke, J. ......................................................................................... 64
Jantscheff, P. ................................................................................ 129
Jenner, D. ..................................................................................... 154
Jensen, A.A. ................................................................................. 190
Jiménez-Ruiz, A. .......................................................................... 154
Jin, N..................................................................................... 200, 201
Jivishov, E. ................................................................................... 185
Jivishova, S. ................................................................................. 185
Jockers, R. .................................................................................... 168
Joerger, A.C. .................................................................................. 45
Joerger, M. ..................................................................................... 42
John, C. ........................................................................................ 173
Johnson, R. .................................................................... 81, 194, 196
Jones, G. ................................................................................ 35, 170
Jose, J. ......................................................................................... 134
Juli, C. ........................................................................................... 154
Jung, A. .......................................................................................... 78
Jung, M. .......................................................................... 85, 136, 172
Junker, A. ..................................................................................... 114
Kaever, V. ............................................................................. 140, 160
Kahnt, A.S. ................................................................... 124, 125, 139
Kaiser, A. ...................................................................................... 102
Kaitsiotou, H. ................................................................................ 131
Kalayda, G.V. ......................................................................... 62, 134
Kamal, A. ...................................................................................... 144
Kanitz, M. ...................................................................................... 159
Kapitzke, C. .................................................................................. 143
Karaman, B. .................................................................................... 85
Kascholke, C. ............................................................................... 206
Kassack, M.U. ................................................................................ 58
Katryniok, C. ................................................................................. 138
Kaul, A. ......................................................................................... 176
Kaule, S. ......................................................................................... 36
Kauzor, D. ..................................................................... 148, 149, 170
Kaysser, L. .................................................................................... 106
Keck, C.M. .................................... 200, 201, 202, 203, 204, 207, 208
Kees, F. ........................................................................................ 148
Kelber, O. ................................................................. 97, 98, 151, 188
Keller, M. ...................................................................................... 156
Keller, T. ....................................................................................... 209
Kempin, W. ..................................................................................... 36
Kersten, E. .................................................................................... 194
Kersting, D. ................................................................................... 102
Kesselring, J. ................................................................................ 170
Keßler, V. ...................................................................................... 150
Keusgen, M. ......... 134, 141, 143, 145, 148, 158, 164, 183, 185, 186
Khoshakhlagh, P. ........................................................... 81, 194, 196
Khoury, K. ....................................................................................... 52
Kiefer, W. ...................................................................................... 159
Kiemer, A.K. ................................................................................. 123
Kietz, A. ........................................................................................ 134
Kim, N.H. ........................................................................................ 27
Kindgen, S. ............................................................................. 81, 195
Klaubert, B. ................................................................................... 205
Klebe, G. ............................................... 132, 141, 142, 159, 161, 166
Klein, J. ........................................................................... 30, 123, 192
Klein, S. ........................................................................................ 194
Klemm, M. .................................................................................... 157
Klitsche, F. .................................................................................... 172
Kloft, C. ........................................... 20, 136, 148, 149, 151, 170, 196
Knolle, P. ........................................................................................ 64
Ko, Y.D. .......................................................................................... 88
Koch, E. ........................................................................................ 183
Koch, K.A. ............................................................................. 123, 192
Koeberle, A. .................................................................................... 25
Koeberle, S. .................................................................................... 25
Koes, D. .......................................................................................... 52
Kolb, P. ......................................................................................... 141
Kölbel, K. ...................................................................................... 116
Koling, S. ........................................................................................ 86
Kölln, C. .......................................................................................... 34
Konerding, M. ............................................................................... 194
Konietzka, J. ................................................................................. 192
König, B. ....................................................................................... 171
Kontny, N.E................................................................................... 195
Konzuch, S. .................................................................................. 162
Kornhuber, J. .................................................................................. 87
Kortum, F. ..................................................................................... 156
Kostenis, E............................................................................ 179, 180
Kostewicz, E.S. ............................................................................... 92
Kotz, S. ......................................................................................... 134
Kouretova, J.................................................................................. 164
Kowarz, E. .................................................................................... 137
Kraft, K. ................................................................................. 151, 187
Kramer, C. ...................................................................................... 46
Kranz, L.M. ..................................................................................... 77
Kraus, A.L. ............................................................................ 161, 189
Krause, A. ..................................................................................... 147
Krauth-Siegel, R.L. ....................................................................... 154
Krebs, F. ....................................................................................... 165
Krebs, L. ................................................................................. 81, 208
Kreiter, S. ........................................................................................ 77
Kretschmer, S.B.M. .............................................................. 125, 126
Krischke, M. .................................................................................. 195
Krueger, K..................................................................................... 151
Krüger, M. ..................................................................................... 102
Küchler, S. ............................................................................ 122, 199
Kühn, A. ........................................................................................ 137
Kühnreich, R. ................................................................................ 166
Kullmann, M. ........................................................................... 62, 134
Kunfermann, A. ............................................................................. 153
Kunz, C. ........................................................................................ 196
Kuroda, N........................................................................................ 40
Kurz, T. ........................................................... 58, 142, 143, 153, 162
Ladwein, K.I. ........................................................................... 85, 136
Läer, S. ........................................................................................... 89
Laino, V......................................................................................... 190
Lamers, C. ............................................................................ 172, 174
Lang, M. ........................................................................................ 178
Lange-Grünweller, K. .................................................... 107, 130, 133
Langer, B. ..................................................................................... 102
Langer, K. ..................................... 173, 198, 202, 203, 204, 205, 206
Langguth, P. ............................. 80, 81, 119, 194, 195, 196, 198, 208
Lappe, S. ...................................................................................... 202
Lategahn, J. .................................................................................. 131
Laufer, S. ........................................................................................ 25
Laufs, U. ....................................................................................... 176
Laven, A.......................................................................................... 89
Lechtape, B................................................................................... 128
Lee, W. ......................................................................................... 170
Lehmann, C. ................................................................................. 124
Lehnen, D. .................................................................................... 122
Lehr, C.-M. ...................................................................... 64, 210, 211
Lehr, T. ........................................................................... 43, 147, 150
Leigh, M. ......................................................................................... 91
Lemcke, T. .................................................................................... 171
Lendlein, A. ..................................................................................... 63
Li, L. .............................................................................................. 167
Li, S.-M. ................................................................................ 140, 146
Li, W.............................................................................................. 117
Licha, K. ........................................................................................ 209
Lieb, S........................................................................................... 180
Liebhold, M. .................................................................................. 146
215
Liebl, J. ........................................................................... 57, 131, 132
Lienau, C. ............................................................................. 153, 162
Lietsche, J. ................................................................................... 192
Lill, A.P. ........................................................................................ 127
Lill, N. .............................................................................................. 26
Lingelbach, K. ............................................................................... 159
Link, A. .................................................................................. 136, 172
Lippert, J. ........................................................................................ 44
Listing, M. ..................................................................................... 125
Löbler, M. ........................................................................................ 63
Loesgen, S. .................................................................................. 106
Look, J. ......................................................................................... 206
Lorenz, K. ..................................................................................... 176
Lorimer, D. .................................................................................... 154
Löscher, D. ................................................................................... 137
Lucas, X. ................................................................................. 68, 174
Lüdeke, S. .............................................................................. 68, 143
Lühmann, T. ........................................................... 35, 167, 170, 206
Lühn, S. ........................................................................................ 115
Lum, L.G. ........................................................................................ 27
Lunter, D.J. ................................................................................... 118
Lutz, S. ......................................................................................... 176
Luzhetskyy, A. .............................................................................. 186
Mäder, K. ...................................................................... 163, 199, 207
Mäder, P. ...................................................................................... 156
Madisch, A. ................................................................................... 151
Maginn, S.J. .................................................................................... 50
Maier, T.J. ............................................................................... 26, 125
Maison, W. .................................................................................... 172
Maiwald, A. ................................................................................... 132
Mandl, M. ........................................................................................ 57
Marek, L. ......................................................................................... 58
Markert, C. .................................................................................... 147
Marschalek, R. ................................................................ 84, 137, 138
Martin, J. ....................................................................................... 188
Massing, U. ................................................................................... 129
Mathes, C. .................................................................................... 211
Matsuda, T. ................................................................................... 109
Matsuda, Y. .................................................................................. 110
Maul, K.J. .............................................................................. 149, 209
Maus, A. ....................................................................................... 208
Mayer-Wrangowski, S. ................................................................. 144
Mazur, A. ........................................................................................ 51
Mederos y Schnitzler, M. .............................................................. 101
Meinel, L. ................................................ 35, 157, 167, 170, 195, 206
Meinhardt, K. ................................................................................ 194
Meinke, M.C. ................................................................................ 202
Meister, S. ...................................................................................... 58
Memmel, E. ............................................................................ 35, 167
Meng, M. ......................................................................................... 77
Merfort, I. .............................................................................. 186, 187
Merk, D. ................................................................................ 167, 174
Merk, H. ........................................................................................ 131
Merkel, O.M. ................................................................................... 27
Merkens, J. ................................................................................... 138
Merten, N. ..................................................................................... 179
Metz, H. ........................................................................................ 199
Metzger, E. ................................................................................... 136
Metzger, S. ............................................................................. 62, 134
Meyborg, H. .................................................................................. 209
Michaelis, M. ................................................................................. 134
Michler, V. ..................................................................................... 165
Mielke, M.G. ......................................................................... 143, 148
Mikula, M. ..................................................................................... 173
Mikulits, W. ................................................................................... 132
Milanos, L. .................................................................................... 177
Milne, T. .......................................................................................... 82
Mishenzon, N. ............................................................................... 188
Miyata, N. ....................................................................................... 39
216
Mohr, K. ........................................................................ 165, 177, 179
Mohsen, A.Y. ................................................................................ 190
Möller, H.M. .................................................................................... 51
Möllmann, U.................................................................................. 157
Moore, B.S. ................................................................................... 106
Mordmüller, B. ................................................................ 58, 153, 162
Morhenn, K. .................................................................................. 176
Mori, T........................................................................................... 110
Morissette, P. ................................................................................ 120
Mozafari, M. .................................................................. 144, 162, 168
Mueller, M. .................................................................................... 185
Muenster, U. ................................................................................... 76
Mulac, D........................................................................ 198, 203, 205
Müller, C. ...................................................................................... 150
Müller, C.E. ........................................................... 117, 178, 179, 181
Müller, G. ...................................................................................... 140
Müller, J. ....................................................................................... 187
Müller, M. ........................................................................................ 68
Müller, R. ........................................................................ 56, 131, 184
Müller, R.H. ........................................... 200, 201, 202, 203, 204, 207
Münsterberg, M. ........................................................................... 171
Murase, H. .................................................................................... 111
Muromoto, R. ................................................................................ 109
Myler, P......................................................................................... 154
Nachbar, M. .................................................................. 144, 162, 168
Nadithe, V. ...................................................................................... 27
Nagase, H. ...................................................................................... 40
Nagel, N. ......................................................................................... 73
Nagel, S. ......................................................................................... 36
Naggi, A.M. ................................................................................... 128
Nakagawa, H. ......................................................................... 39, 111
Nam, S.-J. ..................................................................................... 106
Narkhede, Y. ................................................................................. 164
Nawroth, T. ............................................................. 81, 194, 196, 208
Neffe, A.T........................................................................................ 63
Negri, M. ....................................................................................... 144
Neitemeier, S. ....................................................................... 190, 191
Neochoritis, C. ................................................................................ 52
Netter, M. ........................................................................................ 32
Neumann, D.................................................................................. 176
Neumann, S. ................................................................. 148, 185, 186
Neumann-Haefelin, T. .................................................................... 31
Nguyen, M.A. .................................................................................. 80
Nieber, K. ................................................................................ 98, 188
Nishiyama, S................................................................................. 112
North, N.J........................................................................................ 85
Norville, I.H. .................................................................................. 154
Noske, N. ...................................................................................... 206
Obreque-Balboa, J. ...................................................................... 171
Obst, K. ......................................................................................... 199
Oertel, W.E. .................................................................................. 191
Oetjen, E. ...................................................................................... 176
Ohno, H. ......................................................................................... 41
Ohta, E.......................................................................................... 112
Oishi, S. .......................................................................................... 41
Okamura, H. ................................................................................... 48
Okpanyi, S.N............................................................. 97, 98, 151, 188
Olbrich, C. ....................................................................................... 74
Oli, S. ............................................................................................ 182
Oltmann-Norden, I. ....................................................................... 169
Onila, I. ........................................................................................... 51
Oppermann, S. ..................................................... 113, 161, 189, 190
Ortmann, R. .................................................................................. 159
Ostrowki, J. ................................................................................... 173
Oswald, S. .................................................................................... 152
Ott, I. ............................................................................................. 135
Pahl, A. ......................................................................................... 176
Pannek, M....................................................................................... 85
Parnham, M.J. .............................................................................. 124
Parra-Guillen, Z. ........................................................................... 136
Pasternack, R. .............................................................................. 141
Pauly, A. ......................................................................................... 87
Pawlowska, D. ................................................................................ 77
Pein, M. .......................................................................................... 37
Penning, M. .................................................................................. 198
Peters, J.-U. .................................................................................... 94
Peters, L. ...................................................................................... 179
Peters, T. ...................................................................................... 208
Petersen, S. .................................................................................... 36
Pfankuchen, D. ............................................................................. 130
Pinnapireddy, S.R......................................................................... 205
Pischetsrieder, M. ................................................................. 184, 189
Plank, C. ....................................................................................... 185
Platzer, C. ..................................................................................... 128
Plesnila, N. ..................................................................................... 32
Pohland, M. .......................................................................... 185, 191
Popa, A.-L. ...................................................................................... 77
Potratz, J. ..................................................................................... 128
Prante, O. ..................................................................................... 169
Preidl, J.J. ..................................................................................... 123
Prinz, E.-M. ................................................................................... 119
Prochnicka, A. .............................................................................. 135
Proschak, E. ........................................................... 96, 125, 167, 172
Prosper, F. .................................................................................... 206
Prothiwa, M. .................................................................................. 156
Pyo, S.M. ...................................................................................... 202
Quentin, T. .................................................................................... 176
Rademann, J. ............................................................................... 123
Radke, C. ...................................................................................... 161
Rådmark, O. ................................................................................. 138
Radziwill, R. .................................................................................... 31
Raesch, S. .................................................................................... 210
Rauh, D. ....................................................... 129, 131, 144, 155, 165
Raynor, A. ..................................................................................... 129
Rechlin,C. ..................................................................................... 166
Redweik, S. .......................................................................... 144, 168
Reichl, S. ........................................................................................ 34
Reinehr, R. ..................................................................................... 53
Reitz, E. ........................................................................................ 198
Reske, T. ........................................................................................ 36
Reuter, K.C. .................................................................................... 77
Richter, A. ..................................................................................... 157
Richter, F. ....................................................................................... 78
Richter, M. .............................................................................. 32, 190
Richters, A. ................................................................................... 165
Riederer, U. .................................................................................. 153
Riegel, K. ........................................................................................ 75
Rietscher, R. ................................................................................... 64
Rinne, A. ....................................................................................... 100
Roberto, S. ................................................................................... 134
Rödl, C.B. ..................................................................... 125, 126, 127
Rodriguez, J.R. ............................................................................. 206
Roessler, C. .................................................................................... 85
Rohde, M. ..................................................................................... 184
Rohe, A. ........................................................................................ 128
Romero, G.B. ........................................................................ 200, 203
Römpp, A. ....................................................................................... 60
Roos, D. ........................................................................................ 187
Roos, J. .......................................................................................... 26
Rösch, P. ...................................................................................... 154
Ross, T. ........................................................................................ 129
Rostamizadeh, K. ......................................................................... 204
Rothweiler, F. ............................................................................... 134
Rox, K. .......................................................................................... 184
Royer, H.D. ................................................................................... 130
Ruberg, E.-M. ............................................................................... 104
Ruberg, K. ...................................................................................... 88
Rudolph, C. ................................................................................... 105
Rudolph, I. .................................................................................... 157
Ruff, A. ............................................................................................ 92
Ruge, C......................................................................................... 210
Ruhe, D......................................................................................... 149
Ruick, R. ....................................................................................... 207
Rumpf, T. ........................................................................................ 85
Ruth, P. ................................................................................... 16, 139
Rüther, A....................................................................................... 143
Rutherford, T.J. ............................................................................... 45
Saal, C. ........................................................................................... 65
Saarberg, W.................................................................................. 183
Sadek, M.S. .................................................................................. 168
Sahin, U. ......................................................................................... 77
Sahner, J.H. .................................................................................. 168
Saitoh, T. ...................................................................................... 112
Samadi, S. .................................................................................... 183
Sanglier-Cianférani, S. ................................................................. 161
Sannajust, F.................................................................................. 120
Sarin, N. ........................................................................................ 134
Sarkar-Tyson, M. .......................................................................... 154
Sasaki, S......................................................................................... 48
Sato, H. ......................................................................................... 111
Sauer, K. ....................................................................................... 123
Saul, M.J. ...................................................................................... 124
Schader, T. ................................................................................... 125
Schaefer, C. .................................................................................. 128
Schaefer, L. .................................................................................. 183
Schaefer, M. ......................................................................... 156, 204
Schäfer, E.-M. ............................................................................... 159
Schäfer, J........................................................................................ 89
Schäfer, U.F.......................................................................... 210, 211
Schäfer-Korting, M. ....................................................................... 122
Schäftlein, A.......................................................................... 147, 150
Schäke, F...................................................................................... 178
Scheer,F. ...................................................................................... 166
Scheffler, K. .................................................................................... 61
Schepmann, D. ............................................................................. 114
Scherließ, R. ................................................................................... 64
Scherzberg, M.-C. ........................................................................ 138
Schichtel, J. .................................................................................. 119
Schiede, M.l. ................................................................................... 85
Schiedel, C.A. ............................................................................... 181
Schiewe, J. ................................................................................... 196
Schiffmann, R. .............................................................................. 156
Schilcher, P................................................................................... 136
Schiller, S........................................................................................ 64
Schirmeister, T. .................................................... 128, 159, 170, 182
Schlager, H. .................................................................................. 147
Schleithoff, C. ............................................................................... 128
Schlesinger, M. ..................................................................... 128, 129
Schlinzig, K. .................................................................................... 75
Schlitzer, M. .................................................. 113, 156, 159, 161, 189
Schlossarek, S. ............................................................................. 176
Schmetter, R. .................................................................................. 58
Schmidt, B. ................................................................................... 175
Schmidt, C. ................................................................................... 135
Schmidt, C.Q. ................................................................................. 28
Schmidt, I. ..................................................................................... 154
Schmidt, M. ................................................................................... 128
Schmiedel, K................................................................................. 147
Schmitt, M. .................................................................................... 136
Schmitz, B....................................................................................... 53
Schmueser, L. ...................................................................... 194, 196
Schnackenburg, B. ....................................................................... 209
Schneider, G. ................................................................................ 174
Schneider, I................................................................................... 150
Schneider, K. .................................................................................. 37
Schneider, L.S. ............................................................................. 132
Schneider, M................................................................................... 64
217
Schneider, T. ........................................................................ 170, 186
Schober, Y. ..................................................................................... 60
Schoenfeld, A. .............................................................................. 115
Scholl, F. ....................................................................................... 138
Scholz, B. ..................................................................................... 138
Scholz, P. ............................................................................. 201, 204
Schrader, F.C. ...................................................................... 113, 189
Schrage, R. ................................................................................... 179
Schröder, M. ................................................................................... 64
Schröder, R. ......................................................................... 179, 180
Schroeder, S. ................................................................................ 176
Schröpf, S. .................................................................................... 149
Schubert-Zsilavecz, M. ................................. 125, 167, 172, 174, 191
Schüle, R. ..................................................................................... 136
Schulte, F.W. ................................................................................ 133
Schulz, M. ..................................................................................... 151
Schulz-Fincke, J. .......................................................................... 136
Schulz-Siegmund, M. ................................................................... 206
Schurigt, U. ........................................................................... 154, 183
Schutkowski, M. ...................................................................... 85, 128
Schwab, J. ...................................................................................... 24
Schwabe, K. ................................................................................. 206
Schwan, G. ................................................................................... 178
Schwarz, R. .................................................................................. 116
Schweimer, K. .............................................................................. 154
Schweins, R. ........................................................................... 81, 208
Seeber, F. ..................................................................................... 159
Seibel, J. ......................................................................... 35, 128, 167
Seibt, F.B. ..................................................................................... 181
Seidler, S. ..................................................................................... 133
Seidlitz, A........................................................................................ 36
Seifert, R. ...................................................................... 140, 160, 176
Sergeev, G. .................................................................................. 135
Serno, P. ......................................................................................... 79
Serra, M. ....................................................................................... 206
Seyfried, S. ................................................................................... 140
Shibasaki, M. .................................................................................. 19
Shimizu, T. ...................................................................................... 25
Shindou, H. ..................................................................................... 25
Shopova, T. .................................................................................. 158
Shuto, S. ....................................................................................... 109
Siebenand, S. ............................................................................... 182
Siegmund, H.-U. ............................................................................. 44
Siegmund, W. ............................................................................... 152
Simmet, T. ...................................................................................... 28
Simon, K. ...................................................................................... 179
Sinclair, D.A. ................................................................................... 85
Singh, O. ....................................................................................... 157
Sinz, A. ......................................................................................... 116
Sippl, W. ......................................................................... 85, 128, 136
Skinner-Adams, T.S. ...................................................................... 58
Školová, B. ................................................................................... 122
Skopp, S. ........................................................................................ 67
Smith, S. ....................................................................................... 155
Snitko, M. ...................................................................................... 159
Söbbing, J. .................................................................................... 205
Sochorová, M. .............................................................................. 122
Soler, M.M. ................................................................................... 199
Sommerfeld, A. ............................................................................... 53
Sorg, B.L. ...................................................................................... 126
Sosio, M. ....................................................................................... 143
Sotriffer, C.A. ........................................................ 154, 163, 164, 170
Spek, S. ........................................................................................ 204
Spengler, B. .................................................................................... 60
Staab, A. ....................................................................................... 196
Stachs, O. ....................................................................................... 63
Stacy, R. ....................................................................................... 154
Stark, H. ................................................................ 124, 125, 126, 127
Stasch, J.-P. ................................................................................. 160
218
Stauber, R..................................................................................... 128
Staufenbiel, S. ...................................................................... 200, 201
Steegborn, C. ................................................................................. 85
Stegen, B. ..................................................................................... 139
Steiger, C. ..................................................................................... 206
Stein, J. ......................................................................................... 188
Steinbrink, S.D. ............................................................................... 26
Steiner, I.S. ................................................................................... 159
Steinhilber, D. ................. 26, 124, 125, 126, 127, 138, 139, 172, 188
Steinicke, F. .................................................................................. 169
Steinmetz, M. ................................................................................ 176
Steinmetzer, T. ............................................................. 132, 160, 164
Stelzer, E. ....................................................................................... 75
Stenzel, K. ...................................................................................... 58
Steri, R. ................................................................................. 125, 167
Sternberg, K.................................................................................... 63
Steuber, H..................................................................................... 159
Stieler, M....................................................................................... 141
Stölting, D.P. ................................................................................. 130
Storck, W. ..................................................................................... 210
Storr, M. ........................................................................................ 151
Strödke, B. .................................................................................... 123
Strohmeier, J. ............................................................................... 152
Studenik, C.R. .............................................................................. 173
Suess, B. ...................................................................................... 138
Sumanadasa, S.D.M. ..................................................................... 58
Sun, Q........................................................................................... 171
Suzuki, B....................................................................................... 183
Suzuki, T ......................................................................................... 39
Suzuki, Y......................................................................................... 41
Swyter, S. ............................................................................. 136, 172
Syntschewsk, V. ........................................................................... 142
Szekely, N....................................................................... 81, 194, 196
Tacke, S........................................................................................ 203
Tadros, S.A.A. .............................................................................. 168
Takakura, Y. ................................................................................. 109
Takats, Z. ........................................................................................ 22
Tanaka, N. .................................................................................... 162
Taniguchi, Y. ................................................................................... 48
Tänzler, D. .................................................................................... 116
Tasar, A. ......................................................................................... 88
Telukunta, K.K. ............................................................................. 174
Tenzer, S. ..................................................................................... 210
Terpolilli, N. ..................................................................................... 32
Teufel, R. ...................................................................................... 106
Thakur, A. ....................................................................................... 27
Thoma, F. ..................................................................................... 202
Thomas, M. ........................................................................... 133, 187
Thomas, R.K. ................................................................................ 165
Thommes, M. .......................................................................... 33, 198
Thuermann, P.A. .......................................................................... 152
Tillmanns, A. ................................................................................. 128
Tocchetti, A. .................................................................................. 143
Tokimizu, Y. .................................................................................... 41
Tomioka, K...................................................................................... 38
Toth, P. ......................................................................................... 166
Tschammer, N. ....................................................... 72, 176, 177, 178
Tschöp, M. .................................................................................... 140
Tuereli, A.E. .................................................................................. 119
Ulrich-Merzenich, G. ..................................................................... 151
Ulrich-Rückert, S. ................................................................. 138, 188
Umeda, T. ..................................................................................... 162
Umezawa, K. ................................................................................ 112
Urban, N. ...................................................................................... 156
Usman, M. .................................................................................... 154
van Ammel, K. .............................................................................. 120
van der Graaf, P.H. ....................................................................... 120
Vasylyeva, V. ................................................................................ 142
Vávrová, K. ................................................................................... 122
Vermeiren, C. ............................................................................... 179
Verstraelen, J. ................................................................................ 34
Vieira, R.P. ................................................................................... 122
Vielmuth, C. .................................................................................. 178
Vierfuß, S. ..................................................................................... 115
Vinson, B.R. .................................................................................. 151
Visser, S.A.G. ............................................................................... 120
Vlodavsky, I. ................................................................................. 128
Vogt, D. ................................................................................. 125, 126
Voigt, K. ........................................................................................ 157
Völler, S. ....................................................................................... 195
Vollmar, A.M. ............................................................ 56, 57, 131, 132
Volmer, D.A. ................................................................................... 59
Volz, A.-K. ..................................................................................... 147
von Briesen, H. ............................................................................. 206
von Hammerstein, F. .................................................................... 159
von Löwenich, F. .......................................................................... 186
von Schwarzenberg, K. ................................................................ 132
Vornicescu, D. .............................................................. 134, 141, 145
Wachtel, H. ............................................................................. 78, 195
Wada, N. ......................................................................................... 40
Wagner, E. ...................................................................................... 21
Wagner, S. ............................................................................ 114, 132
Wahl, O. ........................................................................................ 153
Wahl-Schott, C. ............................................................................ 156
Wakimoto, T. ................................................................................ 110
Walden, P. ...................................................................................... 64
Wallmeyer, L. ................................................................................ 122
Waltering, I. .................................................................................... 86
Wardecki, T. ......................................................................... 186, 187
Warren, N. .................................................................................... 210
Wätzig, H. ............................................. 144, 149, 162, 168, 169, 209
Weaver, C. .................................................................................... 180
Weber, B. ...................................................................................... 196
Weber, J. .............................................................................. 141, 172
Weckenbrock, W.V. ...................................................................... 134
Wegener, J. .................................................................................. 180
Wegscheid-Gerlach, C. ................................................ 113, 161, 189
Wehle, S. ...................................................................................... 163
Weickert, A. .......................................................................... 128, 170
Weidel, E. ..................................................................................... 165
Weidner, T. ................................................................................... 159
Weigandt, M. .................................................................................. 64
Weigert, A. .................................................................................... 138
Weimer, D. .................................................................................... 142
Weirauch, U. ................................................................. 107, 130, 133
Weischer, M.-L. .............................................................................. 97
Weiser, C.

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