IDrugs The Investigational Drugs Journal

Transcription

Vol 1 No 1 May 1998
IDrugs
The Investigational Drugs Journal
Editor Ian Tarr
Deputy Editors Barbara Chan, Tara Dyson
London Office
IDrugs
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Simon Campbell (UK)
Daniel TW Chu (USA)
T Colatsky (USA)
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IDrugs
IDrugs (ISSN 1369-7056) is published
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Copyright © 1998 by Current Drugs
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IDrugs
Contents
1
Editorial
42
42
Cancer gene therapy
D Banerjee
45
Cell-based gene therapy and late-breaking news
E Hutchinson
47
Highlights of selected symposia
AF Chambers
Meeting Reports
3
American Chemical Society - 215th National
Meeting
3
PET and Radionuclides
P Robins
4
Adenosine receptors as targets for drug discovery
JE Gomez-Galeno
6
Protein farnesyltransferase and adenosine receptors
P Robins
American Association for Cancer Research 89th Annual Meeting
49
Medicinal Chemistry in Eastern England Ninth Symposium
P Norman
55
8
Protein prenylation
D Leonard
10
ATP site-directed kinase inhibitors
WA Denny
63
13
Anticoagulation strategies
P Robins
67
17
Amyloid aggregation inhibitors
MG Zagorski
19
Medicinal Chemistry Awards Symposium
P Robins
21
Cytochrome P450 and drug design
C Wu
22
Angiogenesis
MS Wolfe
26
Aromatics, heterocycles and porphyrins
KG Pinney
28
Total syntheses of alkaloids, antibiotics, anticancers
and antivirals
D Pleynet
30
Glucosidase and fucosidase inhibitors & Advances in
nucleosides and nucleotides
ZJ Witczak
32
Ernest Guenther award in the chemistry of natural
products
KG Pinney
35
Combinatorial carbohydrate chemistry - where are we
now?
M Panigot
T Wright
Selected state of the art topics - organic chemistry
poster session
E Juaristi
40
Interview with the Chair of the ACS MEDI division,
Annette Doherty
P Robins
Prostate Cancer - CHI Conference
PB Fisher
American Society for Clinical Pharmacology
& Therapeutics - 99th Annual Meeting
D Morgan
Reviews
73
Neurokinin antagonists as potential therapies for
inflammation and rheumatoid arthritis
Andreas von Sprecher et al
Is there a clinical potential for the use of neurokinin
antagonists in inflammatory diseases?
92
38
Japanese Pharmacological Society - 71st
Annual Meeting
Plant-derived anticancer agents currently in clinical
use or in clinical trials
Hui-Kang Wang
Throughout history, plant products and their modified
analogs have been rich sources of clinically useful drugs,
including anticancer agents. Cancer chemotherapeutic
drugs, including vinblastine, vincristine, Navelbine,
etoposide, Teniposide, Taxol and, most recently, Taxotere,
topotecan, and irinotecan are reviewed.
103 Mucosal immunity elicited by DNA vaccines
Taff Jones
Are there prospects for the clinical use of DNA vaccines?
This review charts the progress towards the challenge of
eliciting mucosal immunity through the delivery of DNA
vaccines.
109 Involvement of 5-HT receptors in learning and memory
François Roman & Evelyne Marchetti
141 Tranilast (Kissei)
M Konneh
Research has suggested that the 5-HT system plays a
modulatory role in cognitive processes, particularly in
learning and memory. However, few data are currently
available for the role of the 5-ht5, 5-ht6 and 5-HT7 receptors.
This article presents an overview of their brain locations
and possible involvement in these highly cognitive
processes.
Tranilast, a known anti-allergic agent, is now being
developed as a prophylactic agent for restenosis. Clinical
trials have so far demonstrated a reduced rate of
restenosis in patients.
O
OH
O
O
N
H
Drug Evaluations
O
122 GS-4104 (Gilead)
A Billich
147 Iralukast (Novartis)
A Bramley
A neuraminidase inhibitor currently in phase III clinical
trials for influenza virus infection. Current therapies for this
indication are suboptimal and novel therapies, especially in
elderly patients and infants, are required.
This LTD4 / LTE4 antagonist is currently in phase II trials for
the treatment of asthma. Animal studies have shown it to be
more potent than other LT antagonists.
O
O
S
O
O
OH
O
O
O
OH
O
N
H
O
OH
F
F
F
NH 2
152 Epristeride (SmithKline Beecham)
SS Hedge
129 Cystemustine (INSERM)
B Palmer
A non-competitive steroid 5-α-reductase inhibitor in phase
II trials for the treatment of benign prostatic hypertrophy.
Analysts estimate that epristeride will be launched in
Japan between 1999 and 2000 and is predicted to attract
over 10 billion Yen in sales.
This chloroethyl-nitrosourea is being developed as an
anticancer drug. It is currently in phase II trials for several
cancers.
O
O
O
S
O
Cl
N
H
N
H
N
N
O
H
136 KA-672 (Schwabe)
H Mucke
An α1 and D2 antagonist currently in phase II clinical
trials for the treatment of Alzheimer’s disease.
O
O
N
O
N
H
Cl
H
O
O
158
O
H
HO
Patent News
Peter Steele
Highlights of the patent literature over the past month from
Schering-Plough, IDUN, NIH, SmithKline Beecham,
Rhone-Poulenc Rorer, Sanofi, Merck, Eisai, Nutrimax, Eli
Lilly, Meiji Seika Kaisha, Affymetrix, Shionogi, Columbia
University, Johns Hopkins University, Harvard College,
Millennium, Hokuriku Seiyaku, Kissei, Takeda, Du Pont
Merck, Demeter, Guilford, Pherin, Hoechst Marion Roussel
and ImmuLogic.
Editorial
1
Editorial
Ian Tarr
Welcome to the first issue of a new journal, IDrugs – the
Investigational Drugs journal. Each month, IDrugs will
bring to its readers' attention, in a lively manner, the most
noteworthy information reported on investigational drugs
in the preceding weeks. In doing so, we will place particular
emphasis on the following:
Although there are now several excellent general magazines
and review publications covering various aspects of
pharmaceutical R&D, few assess the scientific aspects of
drug R&D in a commercial context. In contrast, articles in
IDrugs will emphasize the latter.
•
•
•
Neurokinin antagonists in the clinic?
Rapid publication of meeting reports
Timely reviews and analysis of R&D programs
Expert evaluations of the literature on new drugs
Much of the most valuable information on both the
discovery and subsequent development of investigational
drugs is first disclosed at scientific meetings – long before
full papers appear in the primary literature. For this reason,
IDrugs will provide unparalleled coverage of such meetings
via its team of scientific reporters and editors.
For example, the article by von Sprecher et al in this issue
reviews the basic pharmacology of neurokinin antagonists
in the context of drugs in active development and their
prospective indications. The aim is to assess whether there is
sufficient evidence to suggest a place for these novel
compounds in the clinic. Reviews in future issues will place
an even greater emphasis on the commercial implications of
drug R&D, adding, for example, comments and forecasts
from market analysts.
Meetings in this issue – focus on the ACS
Drug evaluations: a core review service
In this first issue of IDrugs, we cover the following meetings
in depth:
•
•
•
•
the 215th National Meeting of the American Chemical
Society,
the 71st Annual Japanese Pharmacological Society
meeting,
the 89th Annual Meeting of the American Association
for Cancer Research,
the 99th Annual Meeting of the American Society for
Clinical Pharmacology and Therapeutics.
All these meetings took place less than 6 weeks prior to
publication of this issue and we will strive to maintain this
rapid publication process for meeting reports in future
issues of IDrugs.
In many cases, we will have several reports from the same
conference, reflecting the multidisciplinary nature of the
larger meetings and the number of parallel sessions in their
programs. For example, the most recent ACS meeting is
covered in this issue via no less than 14 separate reports; we
also carry an interview with Dr Annette Doherty, who
organised the program for the ACS’ Medicinal Chemistry
Division. The interview provides a valuable insight into the
original selection of symposium topics and reflects on which
sessions proved to be most successful and the probable
reasons for their success or otherwise).
Here at Current Drugs Ltd, we have systematically collected
all published references to investigational drugs over a
period of several years. The resulting drug bibliographies
are a core component of our Investigational Drugs database
(IDdb). As part of this service, we periodically commission
experts to review each of these bibliographies, so providing
users of the IDdb with a guide to the literature cited in the
database. This whole literature collation, organization and
evaluation process results in a rich seam of value-added
information on investigational drugs – one that will be
mined regularly by IDrugs.
The choice of which drug evaluations to include in IDrugs
will be governed by the strength of commercial or scientific
interest in the drug (or drug class) concerned. In this first
issue, we have selected six drugs currently in clinical trials
for a wide range of indications: this choice is intended to
give our readers a general impression of the scope and
quality of the literature evaluations that will be included in
future issues of the journal.
We hope that this unique blend of timely meeting reports,
reviews and drug-literature evaluations will make IDrugs an
indispensable read for anyone with an interest in
investigational drugs. We also hope that you enjoy it.
Meeting Report 215th ACS
3
American Chemical Society 215th National Meeting
PET and radionucleotides
29 March - 1 April 1998, Dallas, Texas, USA
Peter Robins
Address
Middlesex House
London
W1P 6LB
UK
Email: [email protected]
IDrugs 1998 1(1):3
© Current Drugs Ltd ISSN 1369-7056
Over 10,000 scientists attended over the five days of the 215th
National Meeting of the American Chemical Society (ACS). The
ACS is the largest scientific society in the world with more than
155,000 members and 29 March - 2 April was duly designated
’American Chemical Society Week’ by the Mayor, Ronald Kirk.
This is the first of a series of ACS reports beginning with the
medicinal chemistry program followed by reports from the organic
chemistry symposia.
Diagnosis with PET
Of the presentations on the first day, particularly striking were
several that considered the growing use of diagnostic PET.
When normal cells start to become cancerous, they develop
an enormous appetite for glucose. Therefore, after injection
18
with an [ F]glucose analog, a whole body PET scan can then
be used to determine the presence of a tumor and moreover,
whether it is malignant, or if it has metastasized. Michael
Phelps (UCLA School of Medicine, USA) described how
such studies may be used in patients with lung, breast, head
and neck, colorectal, ovarian and prostate cancers. These
techniques may also identify the early metabolic alterations
associated with neurodegenerative diseases, such as
Alzheimer’s disease (AD), Parkinson’s disease (PD) and
Huntington’s disease (HD). Further studies of HD, and the
genetic form of AD have indicated that PET is able to detect
metabolic abnormalities associated with these diseases prior
to the appearance of symptoms. Serial scans may be used to
monitor the progression of the disease through the brain. A
18
similar approach is taken with PD, except [ F] L-DOPA is
used as the radiotracer.
On January 1, 1998, the US Federal Government began
reimbursing physicians who use PET to diagnose and
identify the stage of lung cancer. The government has also
agreed to perform expedited reviews of 12 other clinical
indications for PET. In addition, the Food, Drug and
Cosmetic Act was amended this year to direct the US FDA
to take account of the special diagnostic capability of PET,
and whilst this takes place, the US Pharmacopoeia (USP)
will be used as the legal standard of FDA approval. The USP
has approved the above mentioned radiotracers for numerous diagnostic indications in cancer, heart disease and
neurological diseases such as AD, PD, cerebrovascular
ischemia and epilepsy. Dr Phelps stated that advances in
radiotracer chemistry are now allowing PET to take its place
in every day medical practice.
Radionuclides and bone cancer
Researchers from Brookhaven National Laboratory described
advances in the use of radionuclides for a variety of medical
uses, but particularly bone cancer. This debilitating condition
causes excruciating pain to more than 200,000 Americans each
year, and is characterized by painful lesions which are often
spread throughout the bony tissue. Conventional treatments are
far from satisfactory, causing decreases in quality of life, or
being of only limited effectiveness. Suresh Srivastava described
how Brookhaven, in conjunction with SUNY at Stony Brook,
has developed a new agent, tin-117m DTPA, which appears to
provide pain relief with virtually no side-effects, including the
bone marrow damage seen with other nuclear medicines.
In a phase I/II clinical trial, tin-117m DTPA completely or
substantially relieved the pain in 75% of 47 enrolled patients,
with relief lasting up to one year. Bone marrow toxicity was
also much less than comparable therapies. Two multi-center,
double-blind, randomized clinical trials are currently in
progress in 100 prostate cancer patients and 75 patients with
osteolytic bone metastases (including breast and lung). This
therapy may also hold promise for the slowing of metastatic
cancer and prolonging patient survival time, and even for
treating primary bone cancer and rheumatoid arthritis.
Ritalin
For many years, synthetic drugs have routinely been
marketed as racemates. However, it has only recently become
clear that individual enantiomers may possess quite distinct
pharmacokinetic and side-effect profiles. The failure to take
this into account has occasionally been catastrophic, as in the
case of thalidomide. Advances in chemical technology have
facilitated the routine synthesis of pure enantiomers, permitting
the refinement of the previously ’dirty’ profiles of a number of
drugs, such as methadone and local anesthetics. The latest
such candidate is Ritalin (methylphenidate), widely prescribed in the US (an estimated 2 million recipients, with sales of
$350 million) for the treatment of attention deficit disorder.
Yu-Shin Ding (Brook-haven National Laboratory, NY, USA)
described how evidence suggests that Ritalin may only be
beneficial in its d-threo form, and that furthermore, the presence
of the l-threo isomer may in fact compromise its effectiveness.
PET scans following individual administration of the enantio13
13
mers of [ C]Ritalin revealed that d-threo-[ C]Ritalin binds
exclusively to dopamine receptors, whilst the binding of its
enantiomer was mostly non-specific. The high specificity of d13
threo-[ C]Ritalin for dopaminergic neurons suggests that it
might be used as a radiotracer to probe the neuronal loss that
accompanies neurodegenerative diseases, echoing the work of
Dr Ding’s colleague, Dr Srivastava.
4
IDrugs 1998 Vol 1 No 1
Adenosine receptors as targets for drug discovery
Jorge E Gomez-Galeno
receptor antagonist. This xanthine derivative was licensed to
Biogen from CV Therapeutics and has entered phase II
clinical trials as a diuretic for the treatment of the edema
associated with congestive heart failure (CHF).
Address
Metabasis Therapeutics
4575 Eastgate Mall
San Diego
CA 92121-1911
USA
IDrugs 1998 1(1):4-5
This symposium was held on March 30, 1998 as part of the 215th
American Chemical Society National Meeting. It was presided by
PR McGuirk of Pfizer and was particularly aimed at medicinal
chemists. There were more than 200 attendees at the symposium.
Introduction
The initial lecture by Professor RA Olsson (University of
South Florida, USA), covered the subject of adenosine
receptors (A1, A2A, A2B and A3) and their implication for drug
development. Activation of these cell surface, G-protein
coupled receptors causes cell-specific physiological responses
with potential utility for the treatment of a variety of disease
states. Intervention at the adenosine receptor level can be
achieved in several ways: (i) through direct action on the
receptor by either agonists or antagonists; (ii) by enhancement
of tissue adenosine concentration by compounds which
interfere with its metabolism; (iii) by treatment with adenosine
precursors, such as ribose or AICAR (5-amino-4 carboxamido1-ribosylimidazole; Figure 1). Benefits and drawbacks to each
of the approaches were reviewed.
BG-9719
Russell C Peter of Biogen (Cambridge, MA, USA) presented
some of the pharmacology of BG-9719 (1,3-dipropyl-8-[2-(5,6
epoxynorbornyl)]xanthine (Figure 1), previously known as
CVT-124, a highly potent and selective adenosine A1
Typical diuretics act mainly by inhibiting reabsorption in the
kidney distal tubule but inhibition of reabsorption at the
proximal tubule appears to be more clinically relevant in CHF.
The distribution of adenosine A1 receptors means that BG9719 is expected to inhibit reabsorption of sodium and
chloride at the proximal and distal tubules, causing increased
diuresis without an increase of potassium excretion.
In saline loaded rats, oral dosing of BG-9719 at 1 mg/kg
increased urine output to the same extent as furosemide at
30 mg/kg. There was an increase in potassium excretion
which was statistically significant but was not believed to be
clinically relevant. In humans, phase I clinical trials showed
that sodium excretion was increased by a factor of about 2.3
in iv drug-treated patients versus a placebo-treated group.
Potassium excretion was also increased, but not to a
clinically relevant level. These effects peaked about 1 h after
infusion. The effect on sodium excretion for BG-9719 (0.3
mg/kg, iv) was similar to the effect observed with
furosemide (20 mg/kg, po). The compound has entered
phase II clinical trials.
A3-selective antagonists
The therapeutic potential of, and challenges associated with,
intervention at the A3 adenosine receptor were discussed by
Marlene Jacobson (Merck Research Labs, PA, USA). There
are unique differences in tissue distribution and affinity to
xanthine antagonists between the rat and human adenosine
A3 receptors. This has presented a challenge for the
development of animals model relevant to clinical
situations. Levels of A3 adenosine receptors are increased in
individuals with airway inflammation. It has been proposed
Figure 1. Drugs targeting adenosine receptors
F
O
NH
NH2
N
NH2
N
O
O
H3C
N
H
HO
OH
AICAR
(Gensia)
O
N
N
N
N
O
HO
N
H
N
H
O
H3C
H
H3C
HO
BG-9719
(University of Florida)
GP-3269
(Gensia)
OH
that selective A3 antagonists may be useful as anti-asthmatic,
anti-inflammatory, and anticancer agents.
Dr Jacobson described two approaches for the study of the
role of the adenosine A3 receptor. The first, library screening
and lead optimization, led to the identification of L-249313
(6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]triazolo[5,1-a][2,7]naphthyridine) which binds to the human
A3 receptor with Ki = 5.4 nM, and shows high selectivity over
A1 and A2A receptors. L-249313 increases cAMP levels and to
decreases GTP-γS binding, which is not due to covalent
modification.
Jacobson’s second approach entailed development of an A3
knockout mouse, in collaboration with S Tilley (University
of North Carolina, USA). This model simulates the effect of
chronic antagonism at the A3 receptor. Amongst the most
relevant preliminary observations were the lack of
histological differences with wild-type mice, the absence of
an effect on fertility and growth (which was interesting,
given the high density of A3 receptor in testis) and the
absence of any effect on baseline mean arterial pressure and
heart rate. There was no apparent effect on the density of A1
125
and A2A receptors. As expected, [ I]ABA did not bind to
bone marrow mast cell membranes derived from the
transgenic mice. There was no observed increase of
hexosaminidase release induced by adenosine or 2-chloroIB-MECA (a selective A3 agonist) in mast cells derived from
the transgenic mice. This last result was relevant because it
indicates that the A3 receptor, which has also been found in
human lung eosinophils, plays an important role in mast cell
degranulation, a key step in allergic reactions and asthma.
Adenosine regulating agents
The use of adenosine regulating agents (ARAs) to modulate
pathways responsible for the production or metabolism of
adenosine were described by Mark Erion (Metabasis
Therapeutics, San Diego, CA, USA). The approach is based
on the observation that adenosine is produced primarily at
sites undergoing ATP breakdown; ARAs are therefore
expected to enhance endogenous adenosine production in a
site- and event-specific manner. Two enzymes were targeted
as ARAs: adenosine kinase (AK) and AMP deaminase
(AMPDA).
GP-3269 (Gensia Sicor; 4-N-(4-fluorophenylamino)-5phenyl-7-(5-deoxy-1-β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine; Figure 1) inhibits AK with IC50 = 5 nM. It also inhibits
maximal electroshock-induced seizures in rats with ED50 = 5
mg/kg after oral administration. This inhibition occurs in
the absence of hemodynamic effects, and its antagonism by
theophylline indicates that adenosine is the key mediator of
the antiseizure activity. GP-3269 has been tested in phase I
clinical trials. In vitro studies reveal that inhibition of AK
5
results in decreased neutrophil adhesion and TNFα
production. This suggests a further potential use of AK
inhibitors for the treatment of inflammation.
Coformycin
monophosphate-based
transition
state
inhibitors of AMPDA were designed and synthesized which
were expected to have high binding affinity and enzyme
specificity. The high affinity would allow for removal of the
phosphate group from the molecule, and therefore enhance
the cell penetration of these compounds. Highly potent
AMPDA inhibitors were described (Ki = 2 nM) that also
showed high selectivity versus adenosine deaminase. In rat
hepatocytes subjected to anoxia, there is a decrease of about
30% in ATP levels. Treatment with AMPDA inhibitors
increased the levels of ADP and ATP, and decreased IMP
levels. AMPDA inhibitors were also shown to have good
event specificity in these cells.
Purinergic ligands
The history and therapeutic potential of purinoceptors were
reviewed by Michael Williams (Abbott Laboratories, IL,
USA). In addition to a discussion on P1 (adenosine) receptors
(A1, A2A, A2B and A3) and AK, he also reviewed the therapeutic potential and challenges associated with the eleven P2
(ATP/UTP) purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7).
It was commented that 5’-deoxy-5’-iodotubercidin, an
adenosine kinase inhibitor, had an analgesic effect in the hot
plate and carrageenan hyperalgesia models of pain in rats,
without a significant effect on their heart rate or blood
pressure.
P2 purinoceptors offer potential for the treatment of a variety
of conditions, such as cystic fibrosis (P2Y2) and pain (P2X3).
Many of these potential applications have been recently
reviewed (Trends Pharmacol Sci (1997) 18(3):83; Curr Opin
Neurobiol (1997) 7(3):346). The challenges associated with P2
purinoceptor intervention include the lack of selective
pharmacological tools and of reliable binding assays. For
example, the pharmacology of P2 purinoceptors is defined
by the functional effect of agonists. Also, the known
antagonists are relatively weak and have poor selectivity
and high molecular weight.
Recently, the P2X7 purinoceptor was found to have a role in
apoptosis (Neuropharmacol (1997) 36(9):1149). It has also been
implicated in the killing of bacteria by human macrophages,
and in the mediation of host immunity to tuberculosis.
These results open up a wide range of potential applications
to drugs that interact with this receptor.
Conclusion
This symposium highlighted the importance of purinergic
receptors as viable, novel therapeutic targets.
6
Protein farnesyltransferase and adenosine receptors
Peter Robins
Address
Middlesex House
London
W1P 6LB
UK
IDrugs 1998 1(1):6-7
Many exciting advances in the treatment of cancer were presented at
the symposium entitled ’Protein prenylation’ which was chaired by
Professor Richard A Gibbs (Wayne State University, Detroit, MI,
USA). Prenylation, or the covalent attachment of isoprenoid lipids, is
now recognized as an important component in the post-translational
localization and functionalization of many cellular proteins. The
majority of prenylated proteins are modified by one of two specific
enzymes: protein farnesyl-transferase (FTase) and geranylgeranyltransferase type I (GGTase-I). The finding that farnesylation of
oncogenic forms of Ras proteins is required for expression of their
transforming activity has led to FTase becoming an important target
for anticancer drug design.
Farnesyltransferase inhibitors
About one quarter of all human cancers are caused by
genetic malfunctions in the Ras biochemical pathway,
including up to 90% of pancreatic cancers, half of all colon
cancers, and a quarter of all lung cancers. DM Leonard
(Parke-Davis, MI, USA) explained how, as such, this is one
of the most common types of mutation responsible for
cancer, and how mutation of as little as one amino acid (eg,
Gln61 to Leu61) may cause oncogenicity. She then discussed
a series of histidine-N-(benzylglycinamides), including PD169451 (Figure 1), which have been developed as FTase
inhibitors, and presented structure-activity relationship
studies of this class. Parke-Davis uses three assays to assess
activity against FTase: an enzyme scintillation proximity
assay using rat affinity FTase purified from SF9 cells; a
cellular mobility shift assay in NIH 3T3 cells which express
H-ras (used if activity is detected in the above); and a twolayer soft agar system in which NIH 3T3 cells are
continuously exposed to drug for several days. One
compound, PD-161956, exhibited IC50 values of 0.062, 0.1 and
4.4 mM, respectively, in these assays, and served as a
template for further SAR studies, which are ongoing.
RJ Doll (Schering-Plough, NJ, USA) presented a series of
novel trihalobenzocycloheptapyridine heterocycles with
which his company claims to have overcome the problems
of in-cell potency and oral bioavailability that have beset
FTase inhibitors in the past. Again, several assays have been
employed, including an in-cell H-ras FTase inhibition assay,
and an anchorage-independent colony forming assay. In
addition, pharmacokinetic studies have been carried out in
mouse and cynomologus monkey, following both iv and po
administration. Early leads showed only mediocre potency,
but were selective for FTase over GGTase. The compounds
that resulted from modification of these leads, including
Sch-54419, Sch-56620 and Sch-44342 (Figure 1), suffered from
poor pharmacokinetics, but provided useful clues for further
SAR because of their characteristic metabolism. One of the
compounds that has resulted, Sch-66336, entered the clinic in
the third quarter of 1997. It is not clear which cancers the
drug will be developed for if results from phase I studies are
satisfactory, although it seems likely that these will be
pancreatic in origin.
Structure-activity studies of early FTase inhibitors were
presented by SL Graham (Merck Research Laboratories, PA,
USA). These compounds, which included L-744832 (Figure
1), all contained a thiol group which, because of the
potential for immune-mediated toxicities, led to the
abandonment of the series at the preclinical stage. SAR
studies, some of which have been reported in J Med Chem
(1996) 39:1345, led to a non-thiol series of cyanobenzylimidazole derivatives of piperazinones. An illustrated
compound produced 100% inhibition of tumor growth at a
dose of 80 mg/kg in h-ras positive mice, and 79% at a dose
of 160 mg/kg in k-ras positive mice. These experiments in
transgenic animals support the premise that FTase inhibitors
will find utility in clinical oncology. Dr Gibbs, the
symposium chair later commented: "The field of protein
prenylation is a tremendously exciting one, not only because
of its intrinsic scientific interest, but also because of the
potential of prenylation inhibitors as anticancer drugs".
Adenosine receptors
Earlier in the day, CR McGuirk chaired a symposium on the
use of adenosine receptors as drug targets. Perhaps the most
useful talk for the novice was, somewhat perversely,
reserved until last. M Williams (Abbott, IL, USA) began with
an overview of currently known members of the class, then
gave a short history of the molecular biology of the group,
and, perhaps most usefully, clarified the IUPHAR position
on the naming of its members. There are currently four P1
receptors (A1, A2A, A2B and A3) and eleven P2 receptors which
form the focus of an intensive research interest. In contrast
to the preceding lectures which focused on adenosine
receptors, Dr Williams gave an overview of the latter class of
purinoreceptors
by
describing
the
pharmacology,
therapeutic potential and clinical status of modulators of
each member receptor.
Of the other presentations, that by Marlene Jacobson was
particularly interesting as it dealt with the A3 receptor. This
is mysteriously different from other adenosine receptors in
that it shows unique species differences between rodents
and humans, both in the pattern of distribution of
expression and sensitivity to classical xanthine antagonists.
Pharmacological characterization was hampered until recently
by a lack of selective modulators, although many claims have
been made in the literature in this regard. Merck are interested
in the pulmonary applications of A3 modulators, since, in
humans, the receptor is most strongly expressed in the lung.
7
Figure 1. Farnesyl transferase inibitors
O
O
H
N
H
N
O
H3C
CH3
N
N
N
O
H
O
N
N
N
N
H
O
Cl
PD-169451
(Parke-Davis)
Sch-54419
(Schering-Plough)
O
N
N
HS
O
O
O
H2N
H
H3C
O
S
CH3
O
Sch-44342
(Schering-Plough)
The company has used a classical SAR approach, but has
also created an A3-knockout mouse in order to study the loss
of function phenotype. A compound has emerged from
these studies, L-249313, which binds to cloned human A3
receptors with Ki = 5.4 ± 0.34 nM (compared to 32.7 ± 12.9
mM for rat).
Buckminsterfullerenes
Elsewhere in the conference, an interesting presentation by
JM Alford (TDA Research, CO, USA) described a new
process for the manufacture of buckminsterfullerenes that
has decimated their market cost. Commercial, much less
medical applications of buckminsterfullerene and its
derivatives have been slow to present themselves, hampered
partly by the limited availability of the raw materials.
CH3
O
N
Cl
CH3
H
N
H
N
CH3
L-744832
(Merck & Co)
However, the new method employs a continuous
combustion process, in contrast to the labor-intensive and
wasteful carbon-arc process that is currently used. TDA
Research is currently capable of synthesizing up to one
pound of the material per day and claims that it could easily
be carried out on a multi-ton scale.
The most promising clinical application of the fullerenes
relies on their ability to act as free-radical traps; as
antioxidants, they are much more potent that vitamin E.
There is preclinical work underway at Washington
University Medical School in St Louis for amyotrophic
lateral sclerosis, and Lou Gehrig’s disease, but clinical trials
are at least a year away.
8
Protein prenylation session
Daniele Leonard
Address
Parke-Davis Pharmaceutical Research
2800 Plymouth Road
Ann Arbor
MI 48105
USA
IDrugs 1998 1(1):8-9
This session, which took place on Monday, 30 March 30 1998, was
organized by Richard A Gibbs and Kim D Janda, and presided by
Richard A Gibbs (Wayne State University, Detroit, MI, USA).
The first two speakers discussed the mechanism of protein
prenylation in yeast and mammalian species, while the next three
lectures were about inhibitors of one of the prenylation enzyme,
farnesyl transferase. As more research is carried out in identifying
farnesyl transferase inhibitors, with companies starting clinical
studies, it was of interest to have a session on this topic. The
meeting was well attended, with about 300 people in the audience.
Prenylation
Prenylation is a post-translational modification which
allows the attachment of a lipophilic isoprenoid group onto
a protein. There are two types of prenylation, namely,
farnesylation and geranylgeranylation. The enzymes for
each of the respective prenylations have been isolated and
characterized: farnesyl transferase (FTase) and geranylgeranyl transferase (GGTase-I). Their substrates are proteins
with the C-terminal motif CAAX (C is cysteine, A is any
aliphatic amino acid, and X is either Ser or Met for proteins
undergoing farnesylation, or Leu or Phe, for proteins
undergoing geranylgeranylation).
The Ras proteins undergo farnesylation at the cysteine
residue in the presence of FTase that uses farnesyl
pyrophosphate as a donor, forming a thioether linkage. The
three terminal amino acids (AAX) are then proteolytically
cleaved, followed by methyl esterification at the new Cterminal cysteine residue. The Ras protein then associates
with the cell membrane, which enables signaling events
leading to cell replication and transformations. Oncogenic
versions of these proteins are also farnesylated and
subsequently localized to the cell membrane, leading to
uncontrolled growth. The ras oncogenes are implicated in a
wide variety of human cancers. Since the farnesylation of
the protein is the necessary step which allows for the
membrane localization, inhibition of this modification via
the inhibition of FTase could prove to be useful, and may
have potential as antitumor therapy.
Mechanistic studies of protein
farnesyltransferase
Professor C Dale Poulter (University of Utah, USA) was the
first speaker in this session. His talk focused on the
mechanism of prenylation by the yeast FTase enzyme, with
regards to substrate binding for a fluorescent labeled
peptide, difluorofluorescein-Arg-Thr-Arg-Cys-Val-Ile-Ala.
Farnesylation can occur via a 1,4 condensation mechanism,
involving the nucleophilic attack of the protein onto carbon1 on farnesyl pyrophosphate to give the farnesylated
protein. Fluorescence anisotropy was used to determine the
kinetic constants. For the substrate difluorofluorescein-ArgThr-Arg-Cys-Val-Ile-Ala, the Kcat was determined to be 0.6/s
and the KM 2 µM. The Kd value was 2.5 µM (at pH 7) and
independent of pH (Kd = 2.0 µM at pH 5.75, and 1.9 µM at
pH 9). It was suggested that the binding of the protein to the
yeast enzyme is independent of the cysteine side chain being
either the thiol or the thiolate. In the yeast enzyme, it was
also shown that the residues Asp-307, Cys-309 and His-363
2+
are involved in the binding of Zn .
The mechanism of protein prenylation of human FTase was
discussed by Professor Patrick J Casey (Duke University
Medical Center, USA). Interestingly, for the human enzyme,
the Kd value is pH-dependent, unlike the yeast enzyme. The
human enzyme was shown to preferentially bind the
thiolate form of the cysteine residue of the protein. The
crystal structure of FTase was also discussed. A structure of
FTase and farnesyl pyrophosphate (FPP) (3.5 Å) was
presented. The binding pocket of the FPP moiety is sizespecific, causing the C-1 of the farnesyl moiety to interact
2+
with Zn .
Protein farnesyltransferase inhibitors
In vivo studies of L-744832 (Merck & Co; Figure 1), in four
new transgenic mice models, ie, H-ras/p53 knockout mice, Hras/myc overexpression mice, N-ras mice and K-ras mice,
were discussed by Samuel L Graham (Merck Research
Laboratories, West Point, PA, USA). Although this
compound was active in vivo, it did present liabilities: the
presence of the thiol group, the prodrug efficacy, and the
pharmacokinetic and pharmacodynamic profiles in rodent
were not optimal. A new class of FTase inhibitors was then
developed, and the results from SAR studies were
presented. IL-744832 inhibited tumor growth inhibition by
100% at a concentration of 80 mg/kg/day, in H-ras (Rat 1
cells), and by 79% at 160 mg/kg/day against the K-ras tumor
cell line (oral administration).
The SAR studies around PD-169451 (Figure 1) and the
results of in vivo evaluation in a panel of human tumor cell
lines were presented by Daniele M Leonard (Parke- Davis
Pharmaceutical Research, Ann Arbor, MI, USA). PD-169451
inhibited FTase with an IC50 value of 0.004 µM, and was
effective at inhibiting H-ras processing at the concen-tration
of 0.05 µM. Further evaluation of this compound in a panel
of human tumor xenografts showed that it inhibited tumor
growth for the following tumor cell lines: MCF-7 (breast): >
120% (200 mg/kg); HT-29 (colon): 108% (125 mg/kg); H460
(lung): 100% (125 mg/kg) and A549 (lung): 140% (125
mg/kg).
Ronald J Doll (Schering-Plough Research Institute,
Kenilworth, NJ, USA) reported on a tricyclic FTase inhibitor,
9
Figure 1. Protein farnesyltransferase inhibitors
O
HS
O
O
CH3
O
H2N
O
H
H3C
CH3
N
H
N
H
N
H
N
H
N
H
O
CH3
O
N
O
H3C
N
H
O
H3C
S
O
CH3
O
L-744832
(Merck & Co)
PD-169451
(Parke-Davis)
N
C
Br
Cl
CH3
N
H
N
Br
O
F
N
F
N
N
N
N
NH2
F
O
O
SCH-66336
(Schering-Plough)
trihalobenzocycloheptapyridine, Sch-66336 (Figure 1). This
compound was developed from structure-activity relationships studies of a original tricyclic FTase inhibitors which
had moderate potencies against FTase. Sch-66336 was active
against FTase with an IC50 value of 1.9 nM, and inhibited Hras processing with an IC50 value of 10 nM. In an anchorageindependent soft agar assay it was active at 72 nM for H-ras
and was 7-fold less active for K-ras (500 nM). The
pharmacokinetic profile of this compound was reported: it is
76% and 50% bioavailable in mouse and in monkey,
respectively; its half-life (iv) is 1.4 and 3 h in mouse and
monkey, respectively. Sch-66336 inhibited tumor growth by
Compound A
(Merck & Co)
100%, in both EJ (bladder) and NIH 3T3–CVLS cell lines
(nude mice xenograft sc qid, 40 mg/kg po), and is not
cytotoxic. It is currently in phase I clinical trials.
Conclusion
The prenylation session was interesting and demonstrated
the high level of activity in this field, from academia to
industry. A better understanding of the enzyme and its
interaction with the various classes of inhibitors will help to
elucidate how these compound are useful as antitumor
agents.
10
ATP site-directed kinase inhibitors
William A Denny
Address
Auckland Cancer Society Research Centre
Faculty of Medicine and Health Science
The University of Auckland
New Zealand
IDrugs 1998 1(1):10-12
This symposium was organised by Dr Michael South
(Monsanto/Searle Discovery Research, USA), and Dr Alex
Bridges (Parke-Davis, MI USA), as a half-day symposium within
the Medicinal Chemistry Division of the ACS. It was intended to
provide an update to the chemical community of recent developments in signal transduction enzymes, especially the progress
towards clinical evaluation of a number of classes of ATP sitedirected inhibitors. This is a rapidly expanding field, as judged by
both the near capacity attendance and the number of additional
posters on this subject at the meeting.
Michael South opened the symposium with the comment that there
were a large number of kinase enzymes now known. These show
considerable homology at the ATP site, and it was thus originally
expected that inhibitors targeting this site were unlikely to be
selective. However, the discovery of potent and selective ATP site
inhibitors has been a major step forward in the development of
clinical agents.
ras/ERK pathways
Melanie Cobb (University of Texas Southwestern Medical
Center, Dallas, USA) began the meeting with an overview of
recent structural and mechanistic findings among kinases in
the ras/ERK pathways. ERK1 and 2 are terminal enzymes in
three-kinase cascades, activated by MAP/ERK kinases 1 and
2, and controlling gene transcription and cytoskeletal changes
in cells. Phosphorylation at both tyrosine and threonine
residues is required for full activation. A comparison of crystal
structures of ERK2 in both its low-activity (non-phosphorylated) and high-activity (twice-phosphorylated) states
showed that phosphorylation induces major conformational
changes in the activation loop. These changes drive head-totail homodimerization by interaction between these loop
regions.
Crystal structures
The literature on crystal structures of tyrosine kinase
enzymes was reviewed by Ravi Kurumbail (Searle, IL, USA).
About ten different structures have now been reported,
including both apo-enzymes and complexes with ATP
and/or inhibitors. These are typically bilobal structures,
with a wide variability in interdomain angles. The Nterminal domains are comprised largely of β-sheets, and the
C-terminal domains of α-helices, with the ATP site situated
at the base of the N-terminal domain. ATP binds quite
similarly to the different enzymes, with the adenosine
accepting hydrogen (H)-bonds, the sugar donating H-bonds,
and the triphosphate bonding to a conserved lysine-rich
loop via metal interactions. Unlike the situation in many
other ATP-accepting enzymes, the adenosine is deeply
buried. A comparison of several enzymes complexed with
diverse classes of inhibitors (isoquinolinesulfonamides,
staurosporines, benzylindoles and isoflavones) showed that,
in all cases, the inhibitors form both H-bonds and lipophilic
interactions at the buried adenosine site, but have little
common effects at the sugar and triphosphate sites. The high
selectivity shown by ATP site binding inhibitors is thus
surprising from a structural point of view, and depends
critically on small inter-enzyme changes in the adenosine
binding region.
p38 MAP kinase
p38 MAP kinase is part of a stress-induced signal transduction pathway downstream of MKK, leading to the
synthesis of several pro-inflammatory cytokines (eg, IL-1 and
TNF), and Tim Gallagher (SmithKline Beecham, PA, USA)
reviewed the development of aminopyrimidinylimidazoles as
inhibitors of this enzyme. The series of compounds were
originally pursued as anti-inflammatory cyclooxygenase/THF
inhibitors, but radiolabeling studies showed p38 MAP kinase
as the sole molecular target. The 4-pyridyl analogs were very
selective inhibitors, but also inhibited cytochrome P450 due to
high-affinity binding of the 4-pyridyl group to the heme iron.
Analogs where this was replaced by a 2-aminopyrimidine
retained potent (IC50 = 20 nM) and selective p38 MAP kinase
inhibition without P450 suppression, and downregulated TNF
in vivo by approximately 80% at 5 mg/kg/day. Structureactivity studies showed that the 4-fluorophenyl ring was
important for selectivity, fitting into a small hydrophobic
pocket. The synthesis of these compounds has been adapted
to a solid-phase combinatorial approach, to enable rapid
further study of structure-activity relationships.
trkA
A staurosporine-based inhibitor of trkA, a receptor for NGF,
as a potential drug for androgen-independent pros-tate cancer
was discussed by Bob Hudkins (Cephalon, PA, USA). At least
90% of human prostate cancers overexpress one or more of the
trk receptors, and the disease is driven by a neurotrophin/
NGF endocrine loop. Prostate cancers are low growth fraction
tumors, poorly responsive to standard cell cycle-dependent
drugs. Staurosporine analogs are known inhibitors of trks,
and an extensive structure-activity study on more than 500
compounds showed that few changes could be made in the
lactam or indole rings. The modified sugar derivative, CEP751 (Figure 1), was selected for development. This
compound is a potent (IC50 = 3 nM) and selective (1000-fold
over PKC, VEGF, PDGF) inhibitor of trkA. It is active in the
refractory Dunning H rat prostate model in vivo, producing
65% tumor inhibition at 5 mg/kg/day po, and causing
substantial tumor cell apoptosis. Because of low solubility, a
series of prodrugs was evaluated, and a lysine/β-alanine
analog was selected for phase I trials on the basis of high
solubility and adequate stability.
isolated EGFR enzyme, and showed that both N1 and N3
quinazoline nitrogens were required. Small lipophilic
substituents at the 3’-anilino position and electron-donor
substituents at the 6- and/or 7-quinazoline/ pyridopyrimidine positions greatly enhanced activity. These data are
consistent with a model in which the drug binds in the ATP
site, with the 3’-substituted anilino ring fitting into a
751
hydrophobic pocket (partly defined by unique Cys and
742
Met residues) adjacent to the ATP site. The N1 and N3
769
766
quinazoline atoms accept H-bonds from Met and Thr
residues, and the drug 6- and 7-positions point towards the
entrance of the ATP binding pocket. The latter feature has
been exploited by attaching weakly basic side chains at these
positions to improve aqueous solubility, and two such
reversible inhibitors, ZD-1839 (Zeneca; Figure 2) and CP358774 (Pfizer; Figure 2) are currently undergoing clinical
evaluation.
Figure 1. CEP-751
H
N
O
N
N
O
CH3
H
OH
H3C
O
CEP-751
(Cephalon)
Despite this success, there are potential advantages in
irreversible inhibitors, since the compounds must compete
with high levels of ATP, and even the tightest-binding
reversible inhibitors shut down the enzyme for only a few
hours. Irreversible inhibitors should provide longer enzyme
shutdown, overcome only by synthesis of new receptors
(turnover approximately 22 h). Modeling studies showed
that 6-acrylamide analogs positioned the acrylamide directly
773
over a unique cysteine (Cys ) not far from the mouth of the
ATP binding pocket, and kinetic studies showed that these
773
compounds rapidly and selectively alkylated the Cys of
EGFR, but not other kinases. Structure-activity studies
Epidermal growth factor receptor
A joint program between Auckland University and ParkeDavis on the development of 4-anilinoquinazoline/
pyridopyrimidine acrylamides as irreversible inhibitors of
the epidermal growth factor receptor (EGFR) was outlined
by Bill Denny (Cancer Research Centre at the University of
Auckland, New Zealand). This is a transmembrane receptor
tyrosine kinase which is overexpressed in a significant
proportion of tumors. Early work established 4-anilinoquinazolines as spectacularly potent reversible inhibitors of the
Figure 2. Inhibitors of EGFR
O
N
H3C
H3C
O
O
O
N
H3C
O
N
O
N
N
11
HN
O
CH
Cl
HN
H
Cl
F
ZD-1839
(Zeneca)
CP-358774
(OSI)
O
N
O
N
N
HN
H2C
HN
O
PD-169540
(Parke-Davis)
Br
C
12
showed that a 6-acrylamide is superior to a 7-analog for
irreversible inhibition, and that an additional weakly
basic solubilizing substituent at the 7-position improved
in vivo activity. The 7-propyloxymorpholide, PD-169540
(Figure 2), showed good in vivo activity against a range of
human tumor xenografts in mice, with growth delays of
10 to 30 days using oral dosing at 6 to 25 mg/kg/day.
This compound, and a small number of analogs, are in
advanced development.
Conclusion
This was a well-planned program, with the first two talks
providing the audience with an update on the functions
and structures of many signal transduction kinase
enzymes. The three concluding talks then gave disparate
examples of the structure-based design of new classes of
inhibitors directed at the ATP sites of different kinase
targets. This field seems set to grow explosively, and to
provide many new classes of drugs.
13
Anticoagulation strategies
Peter Robins
Address
Middlesex House
London
W1P 6LB
UK
IDrugs 1998 1(1):13-16
A number of individual presentations at this meeting dealt with the
burgeoning field of anticoagulation strategies, and the fourth day
(Wednesday) provided one of the most interesting symposia of the
meeting, entitled ’Anticoagulation Strategies: Thrombin and Factor
Xa Inhibition’. Chair RR Wexler (DuPont Merck, PA, USA)
pointed out that classical anticoagulants, such as warfarin, suffer
from poor oral bioavailability, lack of selectivity and short plasma
half-life. In addition, there are safety issues, for instance, an increased
risk of bleeding, and a large peak-to-trough ratio. Hirudin is a
recently-approved thrombin inhibitor, but is suitable for parenteral
administration only. New therapies are needed and many advances
in this field were presented in the ensuing sessions.
Thrombin inhibitors
3DP-4147
JC Spurlino (3-Dimensional Pharmaceuticals, PA, USA)
began the session with a description of the coagulation
cascade and the role that thrombin plays. Scaffold selection
for the lead compound in the program was achieved using a
DirectedDiversity library. 3-Dimensional then employed a
combination of structure-based design and combinatorial
chemistry to generate a series of thrombin inhibitors. These
include 3DP-4147 which has greater than 90% oral
bioavailability and a duration of action in excess of 6 h in
dogs. In addition, it is highly potent (ED 50 less than 10 nM)
and, being non-peptidic, is easily synthesized. An unnamed
compound from this series has been advanced for clinical
trials.
L-375378
Thrombin is a serine protease which has several
procoagulant actions. For instance, it cleaves fibrinogen to
generate fibrin, activates Factors V, VIII and XIII, and
activates platelets via the thrombin receptor. Therapeutic
indications for antagonists include deep vein thrombosis,
pulmonary embolism, and the prophylaxis of cardiogenic
thromboembolism. PEJ Sanderson (Merck Research Laboratories, PA, USA) explained how peptide based inhibitors
have a relatively long history, but are far from satisfactory.
Early compounds in Merck’s research program, were based
on an efagatran template and included L-370518 and L371912. However, none of these compounds was
significantly orally bioavailable, or particularly selective.
The next generation of compounds, including L-373890,
were based on a pyridinone acetamide template, but the
basic guanidine group that they contained meant that,
although the compounds were potent, poor oral
bioavailability was still a problem.
Replacing this group with aminopyridine led to a
compound with good selectivity. The most potent of these,
L-374087, exhibited Ki values of 0.5 and 3200 nM against
thrombin and trypsin, respectively. However, the
pharmacokinetic data were still inadequate to support
further development, since the company’s aim was to
provide a therapy suitable for once- or twice-daily dosing.
The researchers found that by introducing an electron
withdrawing group in to the central pyridine ring, the
compound could be made more metabolically stable. L375378 (Figure 1), which showed the superior biological
profile of the compounds that resulted, has Ki values of 0.8
and 1800 nM against thrombin and trypsin, respectively, a
plasma t1/2 following iv injection of 185 min, and is 92%
orally bioavailable. This compound has been selected for
development in the clinic.
UK-156406
The structure-activity studies that led to the development of
UK-156406 (Figure 1), a potent, orally-active thrombin
inhibitor that was disclosed towards the end of last year,
were presented by David Fox (Pfizer, CT, USA). As with all
such research programs, Pfizer’s main aim was to produce a
potent, orally bioavailable inhibitor which was highly
selective for thrombin over other serine proteases, such as
plasmin and trypsin. Early benzamides in the program had
moderate selectivity but suffered from a short duration of
action due to biliary elimination. Incorporation of a
tetrahydroiso-quinoline moeity into the structure produced
a series of compounds that were well-tolerated, with good
activity, but these compounds, which included UK-141364
and UK-141366, showed lethal hemodynamic toxicities.
Introduction of a carboxyl group produced UK-142773,
which prolonged thrombin time in rat at 10 mg/kg
intraduodenally and was well-tolerated in mice at 50 mg/kg
iv. Finally, the introduction of unsaturation at the piperidine
moiety resulted in UK-156406, which shows increased
potency and decreased hepatic clearance as a result of
improved lipophilicity. In dog, UK-156406 demonstrates
moderate clearance (11 ml/min/kg), a good volume of
distribution (0.9 l/kg), a plasma half-life of 0.8 h, oral
bioavailability of 45%, and a dose-related prolongation of
clotting times following oral administration. The compound
has progressed to phase I clinical trials, and the full
pharmacokinetic profile in humans will be presented at a
later date.
Factor Xa inhibitors
ZX-807191
Berlex maintained a high profile in the Medicinal Chemistry
section this year as a result of its Factor Xa (FXa) inhibitor
program. At the poster session on Wednesday morning,
some 13 posters relating to structure activity studies had
been displayed, and KJ Shaw from the company presented
an outline of this program. FXa plays a central role in the
coagulation cascade and affects coagulation without
affecting platelet function. When Berlex began its program
14
Figure 1. Thrombin inhibitors
HN
CH3
N
CH3
CH3
O
O
O
N
N
S
NH
NH
NH2
N
O
O
NH2
O
L-375378
(Merck Research Laboratories)
N
H3C
approximately five years ago, only a few low molecular
weight inhibitors were known. One of these was E,E-2,7-bis(4-amidinobenzilidene)-cycloheptan-1-one (BABCH). Berlex
quickly identified the Z,Z isomer of this compound, which
was designated ZK-805412, as a highly-active lead. It was
also selective (Ki = 0.66 and 530 nM against FXa and FIIa,
respectively), but was photochemically unstable, and, being
a bisaryl-amidine, belonged to a family for which there were
little known toxicological data.
With ZK-805412 as a lead, the company used an indolebased template and undertook an extensive series of SAR
studies to optimize selectivity and potency whilst enhancing
photochemical stability (structural alterations at each part of
the molecule were reported in individual posters). The
resulting compound, ZK-807191 (Figure 2), showed Ki
values of 0.10 and 2000 nM against FXa and thrombin,
respectively, and was efficacious in several in vivo models,
eg, the rat model of venous stenosis. The compound is
currently in phase I clinical trials and a detailed presentation
of its pharma-cology will be presented at the forthcoming
1998 FASEB meeting.
SK-549
DuPont Merck is another company which has been
investigating the use of FXa antagonists. FXa was selected as
a target because the thrombin cascade involves signal
amplification with one molecule of FXa activating many
molecules of prothrombin to thrombin. It was therefore
reasoned that inhibition of FXa would be a more economical
way of inhibiting coagulation. Mimi Quan, described how a
lead was generated by screening the company’s IIb/IIIa
library, since a similarity was detected between the binding
site of the two enzymes based on inspection of the Arg-GlyAsp sequence of the former. Early compounds from the FXa
program, including SF-303 and SF-324 were effective
antithrombotics, but possessed an ester side chain at the
chiral center. Replacement of the ester group with a nonhydrolyzable moiety led to SM-084 and SK-549. The latter
shows an ID50 of 0.035 µmol/kg/h (23 mg/kg/h) in the
arteriovenous shunt thrombosis model in anesthetized
rabbits, and a Ki of 0.49 nM against FXa.
OH
UK-156406
(Pfizer)
RPR-130737
WR Ewing (Rhone-Poulenc Rorer (RPR), PA, USA) stated
how FXa is uniquely situated at the junction of the intrinsic
and extrinsic branches of the coagulation cascade, and is the
sole enzyme capable of generating thrombin. These
attractive characteristics led RPR to begin an FXa program
three years ago. The researchers began with a benzamidine
ligand and used SKELGEN (see Toderov NPJ: Comput Aided
Des (1997) 11:175) to generate a lead with an IC50 of 1.1 µM.
However, this compound suffered from slow binding
kinetics. Optimization via the use of a pyrrolidinone linker
led to RPR-120844, and ultimately to RPR-130737 (Figure 2),
which is highly potent (Ki = 2 nM) with more than 1000-fold
selectivity against other serine proteases. It is a competitive
inhibitor of FXa and is effective against the prothrombinase
complex. In addition, it has no effect on heart rate or blood
pressure at therapeutic doses. However, no indication was
given regarding progression of this compound to the clinic.
Poster session
Both the medicinal chemistry poster sessions at this year’s
conference were relatively modest in size, but some
interesting disclosures were nonetheless made.
RWJ-64777
R W Johnson Pharmaceutical Research Institute revealed
details in MEDI 170 of its program to identify novel and
selective inhibitors of p56 Lck tyrosine kinase. This enzyme
is expressed primarily in T-cells and natural killer cells, and
is critical for their proliferative and effector functions. One
of the series of indandiones that the company has
developed, RWJ-64777 (Figure 3), shows an IC50 against Lck
of 32 nM, compared to 460 and 8000 nM for c-Src and PKA,
respectively.
H3 antagonists
In a series of three posters (MEDI 145, 146, 147), Gliatech
described SAR studies for a series of histamine H3 receptor
antagonists. One of these compounds, GT-2227 (Figure 3),
Figure 2. Factor Xa inhibitors
N
NH2
N
N
HN
N
CH3
OH
N
O
O
N
N
O
O
S
O
H2N
NH
O
N
O
F
F
F
OH
N
H3C
HO
F
NH2
F
HN
O
ZK-807191
(Berlex)
SK-549
(DuPont Merck)
NH
S
O
NH2
O
S
O
N
N
HN
OH
RPR-130737
(Rhône-Poulenc Rorer)
Figure 3. Structures from Medicinal Chemistry Poster Session
H
O
H
H
H
O
O
HO
OH
H2N
Br
O
Br
HN
OH
GT-2227
(Gliatech)
RWJ-64777
(R W Johnson Pharmaceutical
Research Institute)
O
CH3
H3C
N
O
H3C
O
N
N
N
O
H3C
O
H3C
CH3
N
N
H
N
N
N
N
CH3
N
N
H3C
N
N
Br
H3C
N
H
N
Br
NH
H3 C
CH3
H3C
CH3
H3C
CH3
CH3
SJ-948
(DuPont Merck)
SC-241
(DuPont Merck)
O
Cl
N
SG-058
(DuPont Merck)
H
15
16
shows Ki values of 13407, 4469 and 3.2 nM against human
cloned H1, H2 and H3 receptors, respectively, and has been
"chosen for additional characterization". It was also
described as "…a possible clinical candidate".
CRH-R1 antagonists
In a series of five posters (MEDI 140-144), DuPont Merck
described SAR studies on a series of triazolopyridines and
pyrimidines which are corticotropin releasing hormone
(CRH-R1) antagonists. Compounds named on the posters
included SC-241, SG-058 and SJ-948, (Figure 3). The last
compound is in late preclinical development, and extensive
in vivo (male beagles) and in vitro data were presented. SJ948 was not in fact the most active of the compounds
presented (Ki against hCRH-R1 is 14.2 nM, compared to 3.7
and 4.1 nM for SC-241 and SG-058, respectively), but was
selected because of its superior pharmacokinetic profile.
17
Michael G Zagorski
Address
Department of Chemistry
Case Western Reserve University
Millis 414SA
Cleveland
OH 44106-7078
USA
IDrugs 1998 1(1):17-18
Amongst the 4,700 presentations at the 215th National Meeting of
the American Chemical Society (ACS), were fourteen research
papers on Alzheimer’s disease (AD) and related issues. The
dementia associated with AD is a progressive and common neurodegenerative disorder producing widespread brain destruction,
with no curative therapies. The brains of AD patients have an
abundance of amyloid plaques and neurofibrillary tangles. The
major protein component of the amyloid plaques is the β-peptide
that exists in two predominant forms: the shorter, 40-residue β1-40,
and the longer, 42-residue β1-42. Recent genetic studies have
established that amyloid deposition, particularly by the longer β1-42,
is directly linked to early onset cases of AD. As a result, major
research efforts are focused on uncovering effective therapeutic
strategies to prevent or slow down the aggregation and the
associated precipitation of the β-peptide into amyloid. In the
amyloid deposits, the β-peptide adopts a β-sheet structure which is
proposed to be neurotoxic.
Introduction
Of the fourteen related papers, seven dealt with β-amyloid
and these are summarized in this report. In addition, a press
briefing was held at the Adolphus Hotel with four invited
speakers: Mony J DeLeon, EdD (Professor of Psychiatry, NYU
Medical Center, New York, USA); Regina M Murphy, PhD
(Associate Professor, Department of Chemical Engineering,
University of Wisconsin, USA); Donald F Weaver, PhD, MD
(Professor, Departments of Chemistry and Medicine, Queen’s
University, Kingston, Canada); and Michael G Zagorski, PhD
(Associate Professor, Department of Chemistry, Case Western
Reserve University, OH, USA).
Fibrillogenesis of the β -peptide
On Wednesday, Girija Krishnamurthy (Wyeth Ayerst, NJ,
USA), presented a poster summarizing measurements of
fibrillogenesis of the β-peptide. This study is the first to
correlate increased β-sheet production (as detected by
circular dichroism), with both thioflavin-T fluorescence and
neurotoxicity in rat hippocampal E18 cells. These results are
in addition to earlier findings with neuroblastoma and PC12
cells, in which the aggregated β-sheet structure is indeed
toxic, and support the idea that β-amyloid deposition is a
primary pathological event in AD.
Biophysical studies of β -amyloid
The symposium entitled ’Amyloid aggregation inhibitors’,
chaired by Corinne Augelli-Szafran (Parke-Davis, MI, USA),
began with a presentation by Jeffrey Kelly (Scripps Institute,
CA, USA). His seminar focused on the protein transthyretin
(TTR), which is a constitutively-secreted protein that transports vitamin A and thyroxin retinol binding protein in both
plasma and CSF. The three-dimensional structure of soluble
TTR was resolved several years ago by X-ray crystallography,
revealing it to be a highly β-sheet-rich homotetramer. This is
presumably the species of TTR that is involved in transport of
vitamin A and thyroxin retinol binding protein. However,
under certain diseased states, including familial amyloid
polyneuropathy (FAP), the TTR changes structure to an
insoluble amyloid fibril, similar to the amyloid deposits
produced by the β-peptide of AD. The proteins in the amyloid
deposits have antiparallel cross-β-pleated sheet structures. Dr
Kelly presented a mechanism of amyloidosis for TTR involving several alternative, misfolded intermediate structures.
TTR has a greater propensity to produce amyloid at acidic pH;
therefore, the notion that the low pH environment of the
lysosome may be a site for the early formation of amyloid was
proposed. The idea all amyloid-associated diseases may
involve a common mechanism of misfolded intermediates
was also suggested.
Dr Kelly also described several small organic compounds
that prevent amyloid formation of TTR. These compounds
were non-steroidal anti-inflammatory agents and mostly
derivatives of anthranilic acid. The X-ray crystal structure of
TTR complexed with one such inhibitor was presented; this
established that the inhibitors bind specifically to the soluble
tetrameric structure. Problems associated with inhibitor
binding to other proteins, such as albumin, and approaches
to the solution of these problems, were discussed.
Analysis of amyloid structure
The second speaker, Paul E Fraser (University of Toronto,
Canada), described improvements of fibril diffraction
methods for analyzing the structure of amyloid deposits. In
general, fibril diffraction studies have established that all
amyloid-forming proteins produce anti-parallel cross-βpleated sheet structures in the plaques. The topology of
these β-sheets appears as super-helical twists of protofilaments. To improve the resolution, the diffraction of the βpeptide was carried out in a magnetic field, since the βpeptide has a high magnetic anisotropy and therefore will
orient in a discrete fashion with an external magnetic field.
Additional high resolution pictures using atomic force
microscopy (AFM) were presented. Analysis of the fibril
diffraction and AFM images of the β-peptide complexed
with inositol derivatives (putative amyloid inhibitors)
revealed that some specific inhibition may result from
interference with the characteristic super-helical twisting
patterns. It is hoped that future studies will uncover more
site-specific interactions that will facilitate the design of
more therapeutically-useful β-amyloid inhibitors.
NMR studies
Michael G Zagorski spoke primarily about high-resolution,
solution NMR studies of the β1-40 and β1-42 peptides. In many
instances, the NMR approach can provide three-dimensional
18
structures that are comparable to those obtained by X-ray
crystallographic techniques. The three-dimensional, micellebound structure for both β-peptides was described. The
conditions under which these were obtained adequately
mimic the environment found at charged lipid surfaces such
as possibly with lipoproteins (Apo-E and Apo-J). Both
peptides have nearly identical and largely β-helical tertiary
structures when bound to micelles, indicating that the
greater propensity of the β1-42 to produce amyloid is not
related to lipid-bound state. More recent NMR studies of the
β1-40 and β1-42 peptides in aqueous solution at pH 7.4
demonstrated mostly extended chain structures. The
peptides are relatively soluble at concentrations up to 0.8
mM, and aging over several days showed that the extendedchain structures can fold back on themselves to produce an
intramolecular β-sheet structure. This is the first real
evidence for an early folded intermediate that forms during
the β-aggregation. The potential for studying the binding
interactions with β-amyloid inhibitors was illustrated with
melatonin (J Biol Chem (1998) 273:7185-7188). The NMR data
showed that melatonin binds to the β1-40 His and Asp sidechains and this interferes with the salt bridge formation that is
critical to β-aggregation.
Azo-dye analogs
A thorough examination of the inhibition of β-amyloidosis
with a series of azo-dye analogs was presented by Warren J
Porter (Eli Lilly, IN, USA). Both chrysamine-G and Congo
Red show complete neuroprotection in primal hippocampal
E18 cells, as measured by a lactase dehydrogenase assay. A
critical structural requirement for neuroprotection is the
biphenyl ring structure. Problems related to the azo-dyes
quenching thioflavin-T fluorescence were briefly described,
as this prevented measurements of amyloid formation using
the standard thioflavin-T assay. Electron microscopy was
used to establish the inhibitory effects of the different
compounds.
Peptide inhibitors
Regina M Murphy described work to establish the specific
recognition sequence of the β-peptide that is important to
aggregation and toxicity. Both static and dynamic light
scattering, together with analytical ultra-centrifugation were
employed to measure β-amyloidosis, whilst toxicity studies
used PC12 cells. Many synthetic recognition peptide
segments were prepared and analyzed, and the greatest
inhibition was observed with a Lys16-Leu17-Val18-Phe19Phe20 peptide with a Lys hexamer unit attached to the Cterminal end. The Lys segment functioned as a disrupting
element for β-aggregation, which affected the fibril
morphology as shown by light scattering. Without the Lys
hexameric unit, the Lys16-Leu17-Val18-Phe19-Phe20 peptide
inhibited toxicity but did not significantly slow down βaggregation.
Design of anti-aggregants
About 100 small organic molecules that bind to the putative
β-peptide glycosaminoglycan binding motif (His13-His14Gln15-Lys16) were described by Donald F Weaver. The idea
for such inhibitors was to mimic, and therefore prevent, the
proteoglycan and/or glycosaminoglycan induced amyloid
formation. The proteoglycans are normal cellular
components of the basement membranes of blood vessels
and other biological tissues. The majority of the compounds
described by Dr Weaver were sulfonates which inhibit
amyloid formation in mice with amyloid-infested spleens.
Following molecular modeling simulations and extensive
structure-activity relationship determinations, the Canadian
group has found a lead molecule that looks very promising.
Two pharmacophore models were ascertained, one in which
two sulfonates were positioned 5.3 Å apart, and the second
with them positioned 7 Å apart. However, Dr Weaver
cautioned that any application of this research to humans is
at least two years away, due to problems with passage of
these compounds through the blood-brain barrier.
Conclusions
Following the conclusion of the symposium, the conference
chair, Corinne Augelli-Szafran, reiterated the several new
concepts introduced at this symposium, particularly the
improvements in fibril diffraction and the use of solution
NMR. These new structural methods should provide
important new information that will facilitate the design of
more effective amyloid inhibitors.
19
Medicinal Chemistry Awards Symposium
Peter Robins
Address
Middlesex House
London
W1P 6LB
UK
drugs which are receiving considerable attention in animal
in vivo tests.
Figure 1. Irinotecan
N
CH3
IDrugs 1998 1(1):19-20
Although of perhaps limited interest to scavengers of hard
biological information, one of the undoubted highlights of the third
day of the conference was the Medicinal Chemistry Awards
Symposium. Two venerable chemists gave resumés of their life's
work, and updated the audience on their current research. This
was followed by a new award for team innovation.
N
O
N
O
OH
Dr Karle is interested in the variety of unusual
conformations that have been observed in peptides (such as
kinks, bulges and severely bent helices) and the ways in
which these may be induced in synthetic or unnatural
peptides. Several hybrid peptides containing three
adamantyl linkers were shown which exhibited 2-fold
symmetry instead of the predicted 3-fold. These insert
themselves into artificial lipid bilayers and act as nonselective cationic pores. Following her presentation, Dr
Karle was presented with a bound selection of her papers by
the Symposium Chair, AM Doherty.
H
Cl
O
CH3
Irinotecan
(Honsha)
X-rays
The first of the awardees was Isabella A Karle, currently at the
Laboratory for the structure of matter at the Naval Research
Laboratory in Washington DC. Dr Karle was introduced by Dr
Ralph H Hirschmann, who praised her "seminal contribution
to X-ray crystal structure determi-nation, and to peptide
conformational analyses in particular". In addition, he drew
attention to her "precision with great attention to detail,
combined with a wise and informed overview of the
implication of the results".
N
O
O
Team innovation
Finally, a new award for team innovation was presented to
the team at Lilly in Indianapolis responsible for the
development of the insulin prodrug Humalog. Drs JH
Andeson, RE Chance and BH Frank delivered a threehanded presentation of the development history of the
compound which will go a long way to solving problems of
patient compliance. Firstly, sc delivery of insulin is far from
satisfactory since it must be carried out 45 min before eating
to allow time for the self-associated hexameric insulin
complex to dissociate into absorbable units. This is a
problem if, for instance, a small child refuses to eat
following an insulin injection. Humalog, on the other hand,
is created by the simple expedient of reversing the
ProB28LysB29 sequence, which weakens its tendency to selfassociate into dimers. The stabilized hexameric complex in
which it exists thus dissociates immediately upon sc
injection, improving the physiologic profile tremendously.
HIV combination therapy
Anticancers
The second awardee was introduced in glowing terms by Dr
F Ivy Carroll. Dr Monroe E Wall, a natural product chemist,
made his name with the discovery of camptothecin, an
extract of the Chinese tree Camptotheca acuminata. This has
formed the basis of a whole family of anticancer
compounds, such as topotecan (SmithKline Beecham) and
irinotecan (Yakult Honsha; Figure 1), both derivatives of 10hydroxycamptothecin. However, Dr Wall is in the unusual
position of having discovered two such seminal compounds,
having added taxol to the list several years ago. In this
presentation, Dr Wall concentrated on current work with
third generation camptothecin analogs, which is being
carried out with the support of Bristol-Myers Squibb. These
new compounds, which include 7-chloromethyl-, 7-ethyland 9-amino-analogs, are water-soluble, low-toxicity pro-
Elsewhere, an interesting combination therapeutic strategy
using HIV protease inhibitors was described by HL Sham
(Abbott, Illinois, USA). Combination therapies exist that
include one of the four HIV protease inhibitors currently
approved by the US FDA and these can suppress HIV to
undetectable levels. However, the pharmacokinetics of these
compounds are not entirely satisfactory because of the low
trough concentration that is observed. If the plasma
concentration falls below the EC50 (for instance, if a dose is
missed), this creates a window of opportunity for the virus;
five to ten billion virus particles are produced/patient /day,
which, combined with the rapid rate of mutation that is
characteristic of HIV, allows the proliferation of proteaseinhibitor-resistant strains within an individual. The results
may be lethal.
20
ritonavir would provide a double protease inhibition, whilst
preventing rapid clearance, and Dr Sham showed that this
was borne out in preclinical studies. ABT-378 is also highly
active against many of the most common ritonavir-resistant
mutants, so will likely be important in dealing with new
mutants as they arise. The compound is now in early phase
II trials.
The protease inhibitor, ritonavir (Figure 2), is a potent and
reversible inhibitor of the 3A subfamily of the cytochrome
P450 enzymes, so inhibits its own metabolism. Nonetheless,
its efficacy is limited by the fact that trough plasma
concentrations are close to the EC50, requiring vigilance with
dosing. On the other hand, ABT-378 (Figure 2), is very
potent, but rapidly metabolized. Scientists at Abbot
reasoned that coadministration of this compound with
Figure 2. Ritonavir and ABT-378
S
CH3
OH
N
O
H
N
O
N
H
N
N
H
S
CH3
O
H3C
Ritonavir
(Abbott)
N
O
CH3
OH
CH3
CH3
O
H
N
O
NH
N
N
H
O
O
H3C
CH3
ABT-378
(Abbott)
CH3
21
Cytochrome P450 and drug design
Chengde Wu
Address
Texas Biotechnology Corp
Houston
TX 77630
USA
IDrugs 1998 1(1):21
This session, devoted to the consideration of drug interactions with
the cytochrome P450 family of enzymes, was presided by Professor
Patrick Woster of Wayne State University.
SAR considerations
The first speaker, Paul Ortiz de Montellano (University of
California at San Francisco, CA, USA), described work to
determine the substrate specificity of cytochrome P450
enzymes through measurement of the oxidation by P450cam of
a series of benzene substrates bearing alkyl groups of up to
four carbon atoms. By examining the three uncoupling
pathways of a complex of the aromatic substrates with ironO , the spin state shift of the complex was not found to
correlate with any other parameter, such as coupled or
uncoupled turnover.
Similar studies were carried out with the T185L and T185F
mutants, in which the active site volume was decreased.
Coupled turnover varies widely, but is maximized when the
phenyl ring bears a single large alkyl substituent. Decreasing
the size of the active site cavity increases coupled turnover
without altering the alkyl group size dependence. Finally,
substrate oxidation occurred preferentially at secondary or
tertiary C-H bonds, and aromatic oxidation was only observed
when the alternative was a primary C-H bond.
P450 versus target receptor
A mere six or so of the more than 40 known human P450
enzymes are responsible for most of the metabolic
oxidations of drugs. FP Guengerich (Vanderbilt University,
TN, USA) explained how it is advantageous to investigate
P450 interactions, in vitro, early in the drug discovery process,
since the recognition of a drug as a substrate by P450 enzymes
is a major impediment to bioavailabilty. He illustrated his
talk using the metabolism of terfenadine as an example
(Drug Metab Dispos (1993) 21:403-409), and described some of
the procedures that have been employed to investigate the
molecular determinants of P450-ligand interaction, such as
classical SAR, and amino acid substitutions.
Ronald White (Bristol-Myer Squibb, NJ, USA) gave an
industrial perspective on P450 -related considerations in drug
design. As pointed out by Dr Guengrich beforehand, the
recognition of a drug by P450 enzymes can be as important in
drug design as the structure-activity relationships for the
target receptor. After giving a short introduction to enzyme
kinetics, Cmax , t½ , and other basic pharmacological concepts,
Dr White described the various biochemical tools that may
be used to determine the affinity of a drug for P450. At
Bristol-Myers Squibb, P450 recognition data are obtained at
various stages in the drug development process, depending
on need. For instance, during the initial screening phase,
preparations of liver microsomes are sufficient to screen out
highly metabolized compounds, with no consideration of
structure, whereas more structure-related data may be
required at a later stage in order to optimize the metabolic
profile of a series of active compounds.
Substrate specificity for P450
3-D Homology models of cytochromes P4502B1 and P4503A4 have
been utilized by Professor JR Halper (University of Arizona),
together with site-directed mutagenesis, to elucidate the
molecular determinants of substrate specificity of these two
enzymes. Most of the key residues identified in 2B enzymes
fall within five substrate recognition sites (SRS) and have
counterparts in bacterial P450 residues that regulate substrate
binding or access. Analysis of the interaction of inhibitors
with models of 2B enzymes has provided a plausible
explanation for changes in susceptibility to mechanismbased inactivation that accompany particular amino acid
side chain replacements.
Based on the 3A4 model and single-site mutants, a double
mutant in SRS-2 has been constructed that exhibits normal
Michaelis-Menten kinetics. A further SRS, SRS-6, is thought
to be the activating site for P4503A4 and Professor Halper
presented the results of modeling and mutagenesis studies
which suggest that the substrate and effector binding sites in
P4503A4 are partially overlapping.
ABT-378
Finally, Dr Hing L Sham (Abbott Laboratories, IL, USA)
discussed the synergistic co-administration of Abbott’s
ritonavir and ABT-378. Ritonavir is an approved HIV
protease inhibitor but is also a 30 nM inhibitor of CYP3A4.
ABT-378, on the other hand, is a potent second generation
compound in phase II clinical trials, but with low oral
availability. Dr Sham showed that by coadministering
ritonavir and ABT-378, a dramatic, prolonged improvement
in the AUC of ABT-378 was achieved in rat, dog, monkey
and human. Abbott has recently published a patent (WO09701349) on such application of ritonavir with, for example,
cyclosporin.
22
Angiogenesis
Michael S Wolfe
Address
Department of Pharmaceutical Sciences
University of Tennessee
327 The Faculty Building
847 Monroe Street
Memphis
TN 38163
USA
IDrugs 1998 1(1):22-25
This symposium, along with one on amyloid inhibition, was
scheduled for the final day of the meeting, undoubtedly in an
attempt to keep participants in attendance. The plan worked well:
the number of symposia attendees varied from 150 to 300 on a day
typically noted for sparse attendance. The organizers guessed
correctly that the importance of this topic to drug discovery and
development would generate plenty of interest. Michael Moyer
(Pfizer) presided over the session.
Inhibition of angiogenesis as a therapeutic
target
The process of angiogenesis and possible therapeutic targets
were introduced by Jean S Beebe (Pfizer, CT, USA).
Angiogenesis, the growth or sprouting of capillaries from
existing blood vessels, is involved in a number of normal
physiological processes, including embryonic growth and
development, wound healing, ovulation and menstruation.
Therapeutic indications for inhibitors include cancer, ocular
diseases and chronic inflammation. There is a critical need
for new therapies for these conditions, and the limited role
of angiogenesis in adult physiology suggests that inhibitors
may lead to new agents with reduced toxicities.
The application of angiogenesis inhibitors to cancer
chemotherapy also offers the hope of circumventing one of
the most vexing problems in this arena: the development of
resistance. Cytotoxic therapy targets cancer cells, which are
genetically unstable, thus resistance develops frequently.
Tumor growth and metastasis are dependent on angiogenesis, and since angiogenesis inhibitors target normal cells
(eg, vascular endothelial cells), these inhibitors may be less
susceptible to the development of resistance. As an
illustration of the potential to avoid resistance, repeated
cycles of therapy with endostatin, an endogenous protein
inhibitor of angiogenesis, resulted in tumor regression in
mice. No resistance was observed and the tumors eventually
became dormant (see Boehm et al: Nature (1997) 390:404).
One strategy for inhibiting angiogenesis is blocking the
activation and initiation of endothelial cell replication.
Tumors release growth factors, such as vascular endothelial
growth factor (VEGF), that are responsible for this first step
in angiogenesis, so developing antibodies that bind these
growth factors, or small molecules that block the receptors
or downstream signaling pathways for these growth factors
are two key strategies in preventing neovascularization.
Another strategy is to target processes involved in inhibition
of angiogenesis; however, the mechanisms of action of
negative regulators, such as endostatin and angiostatin, are
ill-defined, and small molecule design will be contingent
upon determining the signaling pathways involved.
Blocking targets involved in vascular remodeling is also a key
strategy in preventing angiogenesis. The matrix metalloproteinases (MMPs) are zinc-dependent enzymes that digest
the endothelial cell matrix, thereby mediating invasion,
metastasis and angiogenesis. Considerable effort has been
directed toward inhibiting MMPs. Also, the integrin receptors
(particularly αvβ3), involved in cell adhesion, are particularly
‘hot’ targets for antagonist design.
Several angiogenesis inhibitors are in clinical trials. IFN-α,
which decreases the production of fibroblast growth factor,
has already been approved, and those in various phases of
clinical trials include thalidomide, the fumagillin analog
TNP-470 (Takeda; Figure 1), SU-5416 (Sugen; Figure 1) which
is a KDR (a distinct tyrosine kinase receptor) inhibitor, the
MMP inhibitor, Marimastat (British Biotech; Figure 1), and
αvβ3 antibody, vitaxin (Scripps Research Institute).
Dr Beebe ended her talk by raising issues that need to be
addressed in order to develop effective agents. Since it is likely
that these agents will need to be administered chronically,
they must be safe and orally active. There is also a need for
validating markers for angiogenesis in patients to determine
whether the compounds are working. Although these new
agents target normal cells, resistance may still develop if
tumors can activate alternative growth factor pathways.
Finally, combination therapies with cytotoxic agents, an idea
not yet tested clinically, may give optimal responses through
synergistic action.
Blocking the receptor tyrosine kinase Flk1/KDR
The development of novel 3-substituted indolin-2-ones as
potent and selective inhibitors of Flk-1/KDR for inhibition of
tumor angiogenesis was described by Connie Sun (Sugen, CA,
USA). Flk-1/KDR, a receptor tyrosine kinase, dimerizes upon
VEGF-binding, resulting in autophosphorylation and initializing a signal tranduction process leading to endothelial cell
replication. Sugen has targeted the tyrosine kinase domain of
this receptor.
3-Substituted indolin-2-ones (Figure 2) were initially
identified as potent inhibitors, although compound 2 was
non-selective with respect to other related receptor tyrosine
kinases. A number of structural modifications of these
1
compounds led to the conclusions that: (i) N -methylation
reduces activity; (ii) placing electron-donating groups in the
phenyl 4-position provides high potency and selectivity (in
contrast, installing electron-withdrawing substituents in this
same position results in inactive compounds); and (iii)
methylation of the vinyl group gives inactive analogs.
23
Figure 1. Angiogenesis inhibitors
CH3
H3C
O
CH3
CH3
O
H
N
O
CH3
H
N
O
H
H O
CH3
HO
O
O
N
H
N
H
OH
Cl
O
H
N
CH3
H
N
CH3
O
H3C
CH3
O
CH3
H3C
SU-5416
(Sugen)
Marimastat
(British Biotech)
TNP-470
(Takeda)
Figure 2. Flk-1/KDR inhibitors
CH3
H3C
N
CH3
H3C
N
H
O
O
N
H
1
Co-crystallization of an inhibitor with Flk-1 provided insight
into the importance of the double bond stereochemistry. In
the bound structure of SU-4984, an analog of compound 2
where the isopropyl substituent is replaced by 4methylpiperidine, the oxindole moiety occupies the ATP
binding site. Interestingly, the compound 2 analog is bound
as the Z isomer, even though it is the E isomer in solution.
Thus the E form must be converted to the Z form before
binding. Apparently, this conversion can be mediated by
acid, base or light.
The pyrrole moiety of compound 3 was replaced with other
heterocycles (thiophene, furan, pyrazole), but the pyrrole
ring was the superior compound. In this series, all the E
isomers were inactive, and the vinyl proton was again
determined to be essential for activity. In the HUVEC assay,
the 3,5-dimethyl pyrrole-analog (SU-5416) was the superior
compound, with an IC50 of 40 nM. The compound was also
highly selective for Flk-1, being essentially inactive at other
related tyrosine kinase receptors, such as PDGF (plateletderived growth factor), EGF (epidermal growth factor), and
IGF-1 (insulin-like growth factor).
N
H
2
O
N
H
3
SU-5416 was chosen for further evaluation. Cocrystallization revealed that this compound binds at the ATP
domain of Flk-1 in essentially the same manner as SU-4984.
SU-5416 displayed dose-dependent inhibition of subcutaneous growth of A375 human melanoma in mice, and in
preclinical studies, no toxic effects were noted at efficacious
doses. This compound dramatically inhibited tumor size
and tumor neovascularization, suggesting that tumor
growth is not affected directly. SU-5416 displayed a broad
antitumor spectrum: ip administration inhibited eight of ten
different cell lines grown subcutaneously in mice. Although
the plasma half-life was short (30 min), the half-life for
activity against Flk-1 was about 20 h, perhaps due to the
lipophilicity of the compound. SU-5416 is currently in phase
I clinical trials.
Orally active vitronectin receptor antagonists
The efforts to develop orally active angiogenesis inhibitors
by targeting the vitronectin receptor (αvβ3) were described
by James Samanen (SmithKline Beecham). This receptor, a
member of the integrin family, is involved in cell adhesion,
and the potential applications of antagonists include the
24
treatment of osteoporosis, restinosis and proliferative
disorders. The integrin receptors all recognize the RGD
(Arg-Gly-Asp) sequence of their respective ligands, and
selectivity of a given ligand for its integrin is thought to be
primarily due to conformational differences in the RGD
region. Thus conformationally restrained RGD-containing
peptides possess selectivity for certain integrins.
possessed no ability to inhibit migration of human dermal
micro-vascular endothelial cell (HMVEC) migration induced
by bFGF, while kringles 1-4 and 1-3 were active. Kringle 5 was
an outstanding antichemotactic agent, with an IC50 of 200 pM.
In comparison, the IC50 for kringle 1-4 (angiostatin) was > 100
nM. Kringle 5 also prevented cell adhesion onto gelatin-coated
plates, whilst kringle 4 did not.
Last year, the SmithKline Beecham groups reported an
advanced, constrained RGD mimetic, SB-223245 (Keenan et
al: J Med Chem (1997) 40:2289-92; Figure 3). Although SB223245 was αvβ3 selective and very effective in a cell
adhesion assay (IC50 = 120 nM), this compound possessed a
short plasma half-life and low oral bioavailability. One
strategy employed to improve the bioavailability was to
increase the lipophilicity: altering the benzodiazapine
moiety by replacing NH with CH2 and fusing a phenyl in
place of the amide, resulting in a carbocylic version of SB223245. Although this compound was 10-fold less active at
αvβ3, it was racemic and retained selectivity for αvβ3 over
αIIbβ3 receptors.
Kringle 5 blocked HMVEC migration stimulated by a
variety of inducers (VEGF, IL-8, HGF (hepatocyte growth
factor), PDGF (platelet derived growth factor), aFGF (acidic
fibroblast growth factor) or TGF-β1). Evidence for a receptor
for this portion of plasminogen was obtained using
125
[ I]kringle 5; the binding was saturable, with little nonspecific binding and angiostatin competed with kringle 5 for
binding.
Figure 3. SB-223245
OH
H
N
H
N
N
O
H3C
O
N
N
O
CH3
SB-223245
(SmithKline Beecham)
Further analog development resulted in the highly active 2aminopyridine-containing compounds (5 and 6). Resolution
of compound 5 revealed that the S-isomer was the more
active, and this isomer was still selective (ie, not active
against αIIbβ3). The 4’-methyl analog, compound 6, possessed
low clearance and astonishingly high oral bioavailability.
This compound was effective in a mouse air pouch model,
where angiogenesis is involved in the inflammatory
response. It also inhibited tumor growth in mice injected sc
with MOPC 315 plasmacytoma, a particularly virulent
tumor cell line. In a mouse model using the B16F10
melanoma cell line, the compound decreased tumor size, but
did not increase survival time.
Kringle 5 domain of plasminogen inhibits
endothelial cell migration
The deconstruction of human plasminogen was described by
Don Davidson (Abbott Laboratories, IL, USA). Plasminogen
possesses five triple disulfide-containing domains called
kringles, and proteolytic cleavage of plasminogen between
kringles 4 and 5 by elastase results in kringle 1-4 (angiostatin),
an endogenous angiogenesis inhibitor. Elastase was used to
isolate other plasminogen fragments in the hope of defining
the region responsible for angiogenesis inhibition. Kringle 2-3
Kringle 5 was also effective in two in vivo model systems.
Systemic (sc) administration of kringle 5 inhibited bFGFinduced angiogenesis in mouse cornea completely in five of
eight mice and partially in another two. 20 µM of protein
also completely inhibited the growth of prostate tumor cells
in rats. No blood vessels appeared to be growing in tumors
isolated from treated animals, suggesting that the inhibition
of tumor growth resulted from inhibition of angiogenesis.
The group also detected a kringle 5-like protein in human
urine: many breast cancer patients expressed this protein,
whereas control patients did not. One control did test
positive for kringle 5, but it was later discovered that she
had previously had cancer. Thus kringle 5 is a candidate
marker for disease diagnosis and progression.
Natural products as molecular probes of
angiogenesis
The ability of natural product chemistry to reveal new protein
players involved in angiogenesis was illustrated by Craig
Crews (Yale University, CT, USA). Fumagillin, isolated from
microbial sources, is an anti-angiogenic agent, and a related
analog, AGM-1470 (Takeda), is currently in clinical trials.
These compounds arrest proliferating cells in the late G1
phase with an IC50 in the pM range and display some
selectivity for endothelial cells. Although it is appreciated that
fumagillin blocks the phosphorylation of the RB protooncoprotein, its molecular mechanism of action remains
largely unresolved. The presence of the epoxides indicates
that the compounds might become covalently attached to the
target.
The Yale group synthesized an analog of fumagillin with an
extra hydroxyl group (fumagillol), which could serve as a
functional group handle for conjugation to biotin. In a
HUVEC proliferation assay, this fumagillol-biotin conjugate
was still active at nM concentrations, suggesting that the
compound might serve as a molecular hook to fish out the
immediate protein target. Visualizing the Western blot using
avidin-horseradish peroxidase conjugate resulted in a single
new band at 67 kDa; competition with non-biotinylated
fumagillol showed that this binding was specific.
Isolation, microsequencing, and cloning led to the
identification of the target protein as eukaryotic methionine
aminopeptidase-2 (MetAP-2) (Sin et al: Proc Natl Acad Sci
USA (1997) 94:6099), a known enzyme responsible for
cleaving amino terminal methionine after translation. The
two forms of this peptidase, MetAP-1 and -2, are highly
conserved, and the microbe Saccharomyces cerevisiae survives
the loss of either enzyme, but not both. Fumagillin potently
inhibits the growth of yeast with deleted MetAP-1 (IC50 < 1
nM), suggesting that the mechanism involves the inhibition
of MetAP-2. Yeast cells with deleted MetAP-2 are not
sensitive to fumagillin except at much higher concentrations,
illustrating the selectivity of the compound for MetAP-2
over MetAP-1. Using numerous fumagillin analogs, Crews
and colleagues found a correlation between potency against
MetAP-2 and HUVEC proliferation. Currently, the group is
developing a strategy for synthesizing and testing new
fumagillin analogs by releasing analogs from beads using
ammonia vapor. Thus beads with active compounds prevent
the growth of yeast with deleted MetAP-1.
The Yale group is also trying to identify the targets of two
related compounds, eponemycin and epoxomicin. Synthesis
of dihydroeponemycin and its biotinylated counterpart has
led to the detection of a putative target protein which is
apparently different from MetAP-2. Attempts to sequence
this new protein are currently underway. The group has
completed the first synthesis of epoxomicin, which should
facilitate the identification of the epoxide stereocenter.
25
Conclusion
Angiogenesis is an fascinating new area of basic science, and
controlling this process holds considerable promise for drug
development, particularly in the area of cancer chemotherapy. The identity and role of some molecular players
(eg, vitronectin receptor, Flk-1) are well defined, and
considerable effort has been spent in the development of
compounds that interact with these targets. These agents are
likely be approved for clinical use in the near future.
Meanwhile, there are undoubtedly many unknown proteins
and pathways involved in angiogenesis. Determining the
mechanism of action of negative regulators of angiogenesis,
as well as natural product inhibitors, will greatly increase
the number of viable targets for drug development.
In a conversation after the session, presiding officer Michael
Moyer emphasized that the issue of resistance to antiangiogenic drugs is not yet resolved. He also believes, like
Jean Beebe, that combination therapies with the cytotoxic
agents are certainly worth testing. It is possible that such
combinations would be synergistic and that lower (and
therefore safer) doses of the cytotoxic drugs would be
needed.
26
Aromatics, heterocycles and porphyrins
Kevin G Pinney
Address
Baylor University
Waco
TX 76798
USA
IDrugs 1998 1(1):26-27
This session covered a variety of interesting synthetic methodologies and target directed syntheses with far-reaching applications
towards the preparation of both natural and unnatural products,
as well as analogs with potentially improved biological activity.
Two presentations focused on molecular recognition of the
colchicine binding site on tubulin, and included the presentation of
molecular probes as well as lead candidates for new drug
development, whilst five sessions centered on interesting new
developments in porphyrin chemistry. The overall attendance at
the session was very good and at some points reached
approximately 250 attendees.
Directed Remote Metalation
Stephen Houldsworth from the laboratories of Professor
Victor Snieckus at the University of Waterloo, Canada
presented an interesting discussion on the further
development of their popular methodology for the Directed
Remote Metalation (DreM) of aromatic systems. This
methodology has a wide range of applications including the
preparation of dibenzo[b,d]pyranones, fluorenones, and
other classes of aromatic systems. An eloquent application of
this DreM methodology was presented which described the
synthesis of several aromatic kinamycin antibiotics such as
prekinamycin and kinobscurinone. In addition, a remote
anionic Fries rearrangement was described with application
towards the synthesis of the defucogilvocarcin family of
natural products which have antitumor activity. The
synthesis of piperolactam C, a representative benzylisoquinoline alkaloid, also with antitumor activity, was
described.
New classes of tubulin polymerization
inhibitors
Vani P Mocharla working at Baylor University in Dr Kevin
G Pinney’s research group presented the develop-ment of
several new types of inhibitors of tubulin polymerization
which were designed to interact at the colchicine binding
site on b-tubulin. Several interesting design features were
discussed, as well as details of the chemical synthesis of each
of the new inhibitors. The most prominent compounds
presented included benzo[b]thiophenes, benzofurans,
diarylethers, and a dihydronaphthalene derivative. The
dihydronaphthalene ligand is especially promising since it
demonstrates excellent inhibition of tubulin polymerization
(IC50 = 1.7 µM) as well as significant activity against human
cancer cell lines (for example, GI50 = 0.0038 µg/ml against a
prostate cancer cell line (DU-145)). Additional design and
synthetic work towards the development of ligands with
improved bioavailability was also presented. A patent
application was initially filed in March 1997 (patent
pending), and a PCT application was filed on March 6, 1998.
Molecular probes and chemotherapeutic
agents based on combretastatin A-4
A second presentation from Dr Pinney’s group at Baylor
University was given by Ma del Pilar Mejia. This talk
focused on the development of new molecular probes for
tubulin based on combretastatin A-4, which are designed to
interact with the colchicine binding site on b-tubulin.
Amongst the new probes, the most promising appears to be
a 3-azido combretastatin analog which is being developed as
a photoaffinity labeling agent for the colchicine site. This
new compound demonstrates excellent inhibition of tubulin
polymerization (IC50 = 1.3 µM) and displays significant
activity against human cancer cell lines (for example, GI 50 =
0.0033 µg/ml against a neuroblast cancer cell line (SK-NSH)). This superb biological activity suggests a strong
interaction at the colchicine binding site which is, of course,
imperative if these ligands are to be employed as molecular
probes for binding site elucidation. A new compound which
is intriguing for potential development as an anticancer
drug was also described. This 3-amino combretastatin
analog is a potent inhibitor of tubulin polymerization (IC 50 =
1.7 µM) and is extremely active against human cancer cell
lines (GI50 ≤ 0.001 µg/ml for numerous human cancer cell
lines). Synthetic details of these ligands were presented, as
well as efforts which are underway to develop prodrugs
based on the 3-amino combretastatin derivative. A patent
application has been filed on these combretastatin
derivatives.
Heterocyclic enediynes
An interesting presentation by Dr KC Russell from the
University of Miami highlighted an ongoing project aimed
at exploring the synthetic utility of heterocyclic enediynes
specifically as substrates in the Bergman cyclization. This
work is especially relevant, since a variety of antitumor
antibiotics (such as dynemicin) are known to effect their
potent biological activity through an induced Bergman
cyclization. Several new heterocyclic enediynes (diethynylpyridine and diethynylpyrimidine, for example) were
prepared and their kinetics studied in this reaction. The
heteroarenediynes react faster than the analogous arenediynes. For example, the diethynylpyrimidine derivative
shows t1/2 = 7 min at 154 °C, whereas the figure for
diethynylbenzene was 52 min at 152 °C). These results are
encouraging for new drug development utilizing heterocyclic enediynes.
Novel porphyrin ligands
Three very informative presentations were given in
sequence by members of Dr Kevin M Smith’s research group
at the University of California, Davis, USA. The first talk, by
Cinzia M Muzzi, centered on efforts to prepare a chiral
dodecasubstituted porphyrin via a Suzuki coupling reaction.
Since nonplanar distortions are thought to play a role in the
biological activity of certain tetrapyrroles, it is of interest to
prepare these sterically-congested porphyrins to investigate
their physical and electronic properties. An ultimate goal
might be to learn more about why cytochrome P450, for
example, is able to carry out complex regiospecific and
stereospecific oxidations. The second presentation was given
by Kalyn M Shea and focused on dodecasubstituted chlorins
(dihydroporphyrins). Numerous synthetic details were
presented, as well as a discussion of the non-planarity
induced by the dodecasubstitution pattern. The unique
nature of these porphyrins may have far-reaching
applications in material science and also in the development
of interesting models of biological systems. The third and
final presentation in this series by Richard G Khoury
centered on calixarenes and specifically addressed the
preparation of calix[4]arenoporphyrin. The X-ray structure
of this compound has been determined, and it is noteworthy
that the four porphyrins are all located on the same face of
the calix system. Potential applications for these new
compounds include the development of new molecular
sensors and unique molecular receptors, to name just a few.
Cobalt-precorrin-4
Patricio J Santander, working in Professor A Ian Scott’s
laboratory (Texas A&M University, College Station, TX,
USA), presented work to discover the intermediates and
enzymes beyond precorrin-3 (an intermediate on the
27
Vitamin B12 anaerobic path). He makes use of the known
13
early enzymes to prepare [ C]isotopomers of precorrin-3.
Details of the enzymes involved, as well as structural
features of a cobalt-precorrin-4 (a tetramethylated intermediate) were presented. These results have far-reaching
implications, not only because of their significance in
advancing our understanding of the biosynthetic pathway
of Vitamin B12, but also because they herald significant
advances in the use of modern NMR techniques to elucidate
complex solution structures of biomolecules.
Porphyrins and phosphorescence quenching
Two back-to-back presentations concerning porphyrin
chemistry and its applications to biological systems were
then presented by SA Vinogradov, working in conjunction
with DF Wilson (University of Pennsylvania, PA, USA). In
the first lecture, tetrabenzoporphyrins of palladium were
described as near infrared phosphors, useful for oxygen
determination by phosphorescence quenching. New
synthetic approaches resulting in the preparation of
bromine-substituted tetrabenzoporphyrins which are poised
for further functionalization were described. In the second
presentation, dendrimer-porphyrins were described, which
have strong oxygen-dependent phosphorescence. Studies
were described which address the accessibility of the core of
the dendrimer-porphyrin, especially in terms of the size of
the dendrimer constituents. Oxygen-dependent phosphorescence quenching was presented as a means of studying
and analyzing structural features of the core of these
porphyrins in terms of dendrimer branching.
28
Total syntheses of alkaloids, antibiotics, anticancers and antivirals
David Pleynet
Address
Amylin Pharmaceuticals Inc
9373 Towne Centre Drive
San Diego
CA 92121
USA
IDrugs 1998 1(1):28-29
This session, attended by approximately 120 delegates was part of
the Organic Chemistry Division. It offered relevant extensive and
interesting approaches towards the synthesis of new anticancers,
antivirals and antibiotics. The four-hour session was presided by
Patrick Harran and comprised a succession of 12 speakers, each
giving a twenty-minute delivery.
which is used in the production of a variety of blue cheeses.
The unique conformation of Roquefortine C, was
investigated by computer modeling. Its rigid and stable
structure, resulting from hydrogen bonding interactions,
was highlighted. The key steps in the synthesis of
Roquefortine C were the preparation of the prenyl pyrrolo
[2,3b] indole from tryptophan and the subsequent synthesis
of the diketopiperazine moiety by cyclodipeptide coupling.
The final step consisted of the coupling of N-trityl protected
imidazole-4-carboxalde-hyde with the previously described
diketopiperazine intermediate.
Stemfoline
A synthetic approach to stemfoline, a hexacyclic insecticidal
alkaloid was described by T Smalley (University of
Rochester, NY, USA). A key step in the ongoing synthesis is
the formation of three rings by means of a cascade reaction.
Altohyrtin
Wesley Trotter (Harvard University, USA), opened the
meeting by describing the asymmetric total synthesis of
Altohyrtin C (Spongistatin 2) in 31 steps. He described the
coupling of the various fragments of the molecules. The
most notable of which were the A-B and C-D rings coupling,
which is effected by a double stereo-differentiating aldol
reaction and, the subsequent coupling of the A-B-C-D
fragment with the E-F portion, which is achieved by means
of a Wittig reaction. This late introduction of the side chain
was achieved by diastereoselective β-C-glycosidation.
Gallotannin
The first total synthesis of a dimeric gallotannin, which is an
analog of the naturally-occurring ellagitannin Coriariin A, was
reported by Kiran Sahasrabudhe (Pennsylvania State University, PA, USA). She highlighted that a possible biomimetic
dimerization of an orthoquinone advanced intermediate by a
Diels-Alder reaction led to the formation of a key diaryl ether
linkage. It was also shown that the immunostimulatory
activity of the dimer was comparable with that of Coriariin A
and had a maximal activity after 24 h.
Saponin OSW-1
The first synthesis of the aglycone of the potent antitumor
steroid, OSW-1, was presented by Chuangxing Guo (Purdue
University, IN, USA). This occurs in nine steps, from 5androsten-3b-ol-17-one, and in an overall yield of 15%. The
key reactions involved the ene installation of the side chain,
a regio- and stereoselective dihydroxylation and the
diastereoselective reduction of the ketone function at C-16.
Roquefortine C
The conformational analysis and studies towards the total
synthesis of Roquefortine C and related compounds, were
discussed by Wei-Chuan Chen (University of Pennsylvania,
PA, USA). Roquefortine C is an alkaloid isolated from
culture filtrates of the common fungus, Penicillium roqueforti,
Fluorine-containing camptothecin analogs
The synthesis of fluorine-containing camptothecin analogs
using N-fluoro bis(trifluoromethylsulphonyl) imide, prepared
from bis(trifluoromethylsulfonyl) amine and F2 was discussed
by Weiwen Ying (Clemson University, SC, USA). The major
product, 14-fluoro-(20S)-camptothecin, was synthesized
when the reaction was carried out in 2,2,2-trifluoroethanol at
o
23 C. However two diastereomers 5-(2’-2’-2’-trifluoroethoxy)14-fluoro-(20S)-camptothecin were obtained by raising the
o
temperature to 80 C and employing 4 equivalents of N-fluoro
bis(trifluoromethylsulfonyl) imide. The fluorinated camptothecin analogs showed enhanced biological activity.
Sibirine
Masato Koreeda (University of Michigan, USA), explained
the radical cyclization of (alkoxycarbonyl-amino)methyl
radicals and its application to the total synthesis of sibirine,
which is effected in 11 steps and 13% overall yield from 3thiophenyl-cyclohexenone. He highlighted the key step,
which involved the highly stereo- and regiocontrolled 6-exotrig cyclization of the radical which was generated from an
advanced phenyl selenide intermediate and provided the
spiro-bicyclic sibirine in a yield of 60%.
A novel efficient method for opening spiroketals
The first total syntheses of cephalostatin 1, North G and the
hybrid analog, ritterostatin GN1N, were reported by Tom
LaCour (Purdue University, IN, USA). Cephalostatin 1 is an
extremely potent antineoplastic and ritterostatin GN1N is
highly active in the NC160 panel. North ritterazine G was
efficiently constructed from hecogenin acetate (30% yield, 10
steps) via a novel method for opening spiroketals and
extension of a key photolysis/Prins sequence. Installation of
the D14 moiety in the North G and South 1 units and
experimental evidence for the biosynthesis of North A were
presented. Unsymmetrical coupling provided the natural
and analog pyrazines from cephalostatin and ritterazine
components.
29
Epothilone analogs
QSAR
The design, synthesis and biological evaluation of epothilone analogs were described by Joanne Holland (University
of Pennsylvania, PA, USA). The synthesis of constrained
analogs, focusing on the eastern and western hemispheres,
were presented. Molecular modeling established that a 2carbon tether between C-2 and C-10 of epothilone, renders
the structure more rigid without changing the conformation.
The key step for the generation of the cyclic structure of
these analogs was an olefin metathesis reaction in the
presence of a Lewis acid. These analogs were tested by
Susan Horwitz and were found inactive despite a good
overlay with the X-ray structure of epothilone. This
reinforces the importance of the entire structure of
epothilone for biological activity.
QSAR and distribution studies of CADA analogs having
activity against the human cytomegalovirus (HCMV) were
presented by Meinrado Samala (University of Nevada,
USA). He performed a comparative molecular field analysis
(CoMFA) on a set of 23 compounds with EC50 values ranging
from 2 to 5 mM and is currently trying to use the predictive
abilities of the program to identify potential lead compounds.
Finally, the remaining presentations illustrated the
increasing importance of computer modeling and QSAR
programs in the generation and development of new drugs.
30
Glucosidase and fucosidase inhibitors & Advances in nucleosides and
nucleotides
Zbigniew J Witczak
Address
Department of Pharmaceutical Science
School of Pharmacy
University of Connecticut
Storrs
CT 06269
USA
evaluation of the hitherto accepted mechanism of retaining
b-glucosidases, particularly of hydrogen bonding and
electrostatic interactions. These new aspects of the inhibition
of retaining glycosidases are clearly supported by analysis
of the crystal structure of the enzyme and the synthesis of
novel, conformationally-defined neutral inhibitors from the
glyconolactone versatile synthetic intermediates.
IDrugs 1998 1(1):30-31
Professor Spencer Knapp of Rutgers University in New Jersey
also presented observations on retaining glyco-sidases, in this
case, neighboring group participation. New evidence suggests
that certain N-acetyl-β-hexa-minidases and chitinases cleave
their substrates with overall retention of configuration
through participation at C-1 by the substrate acetamido
carbonyl oxygen to form an oxazoline intermediate. For
various retaining α- and β-glucosidases, it is strongly
suggested that the enzyme active site carboxylate attack occurs
at C-1, leading to a substrate-enzyme covalent intermediate.
This leads to the question: is enzyme carboxylate attack
considered as universal or might there be retaining
glycosidases with substrate participating groups other than 2acetamido? The answer is that this is quite possible, but new,
and existing, data need to be proven and generalized.
The American Chemical Society Symposium "Glucosidase and
fucosidase inhibitors" took place on 1 April 1998 and was
organized by Professors Zbigniew J Witczak (UConn, School of
Pharmacy, CT, USA), Kuniaki Tatsuta (Waseda University,
Tokyo, Japan) and Waldemar Priebe, MD (Anderson Cancer
Center, University of Texas, USA). Professor Witczak provided
introductory remarks including the status of existing glucosidase
inhibitors, and chaired the morning session, which consisted of six
lectures. The symposium was well received, and was particularly
attractive for those interested in networking, as attendance was
about sixty. In addition, some participants and attendees presented
posters on the subject during the regular poster session organized
by the Division of Carbohydrate Chemistry.
Glucosidase and fucosidase inhibitors
Professor Steve Withers of the of University of British
Columbia, set the proceedings in motion with an excellent
overview of glucosidase inhibitors; this included new
developments in the synthesis and use of 5-fluoro-5-deoxyand 2-fluoro-2-deoxy- derivatives of glucose to obtain new
experimental evidence of transition state inhibitors via
increased interaction at the anomeric center. Kuniaki Tatsuta
gave a wonderful overview of the modern synthetic
approaches to the various classes of existing natural
glucosidase inhibitors including cyclophellitol, nagastatin
and KD16-U1. Professor Tatsuta communicated that the
analogs have different configurations and functionalities,
confirming that they serve as antagonists of the corresponding α- or β-glycosidases.
Linkage and configuration-specific inhibitors of glycosidase
may be useful in treating diabetes and metastatic cancer, as
well as lysozomal storage diseases. Professor Bruce Ganem
(Cornell University, NY, USA) discussed enantioselective
synthetic studies on novel glycosidase inhibitors, including
trehazolin, and new functionalized analogs of isofagomine.
Another extremely important aspect is their involvement in
regulating the biosynthesis of N-linked glycoproteins and
glycolipids, which play prominent roles in immune
recognition phenomena such as lymphocyte recirculation
and trafficking, and cell-cell or cell-matrix adhesion.
Retaining glycosidases
Professor Andrea Vasella, from the Swiss Federal Institute of
Technology in Zurich, discussed retaining glycosidase
inhibitors. Comparative inhibition tests have led to the re-
The morning session ended with a lecture on new
developments in the synthesis of C-glycosides as potential
inhibitors of carbohydrate processing enzymes, by Professor
Francesco Nicotra (Milano University, Italy). Two derivatives
of glycosyl phosphates as spacer-connected disaccharides
were selected as potential inhibitors of glycosyltransferases
and glycosidases. The glucosidase inhibition data, however,
suggest only weak activity.
Fucosidase inhibitors
Kuniaki Tatsuta chaired the afternoon session, which focused
on fucosidase inhibitors and new synthetic approaches to
azasugars as potential fucosidase inhibitors.
The first lecture of the afternoon was presented by Yoshiyuki
Kobayashi from the Medicinal Chemistry Research Laboratories
of Sankyo in Tokyo, Japan. He reviewed the current status of the
chemistry and biology of trehazolins, including comparative
data on both stereoisomers of trehazolin and other analogs. The
company is working on new approaches to analogs with the
same or similar type of biological activity.
Professor Olivier R Martin of SUNY, Binghampton, NY and
University of Orléans, France, then summarized novel series
of azasugars, ie, four- to seven-membered iminoheptitols
and their derivatives. These included: azetidine derivatives;
C-vinyl pyrrolidines; C-glycosyl compounds related to
nojirimycin; aza-C-glycosyl amino acids; and, substituted
azepanes derived from 7-amino-2-heptulose.
Iminosugars
The use of OH substituents to direct a chair conformation of
1-N-iminosugars and the relationship of this conformation
to inhibition of β-glycosidases was discussed by Professor
Yoshitaka Ichikawa (John Hopkins University School of
Medicine, Baltimore, MD, USA). New families of 1-Niminosugars (nitrogen at anomeric position) are extremely
potent inhibitors of the corresponding β-glycosidases,
including glucosidase, galactosidase and glucuronidase.
Inge Lundt (Technical University of Denmark, Lyngby,
Denmark) described the regioselective functionalization of
aldonolactones as a general strategy for the synthesis of
iminosugars without the use of protecting groups. 6Deoxyisofagomin, and its L-ribo analog, were prepared and
tested for enzyme inhibition. Both proved to be strong
inhibitors of almond β-D-glucosidase, with Ki = 3 µM and Ki
= 2 µM, respectively. The L-ribo analog also inhibits α-Lfucosidase, with Ki = 80 µM.
Professor George Fleet, from Dyson Perrins Laboratory at
Oxford University, began with an initial overview of natural
and unnatural fucose and rhamnose mimics. General
synthetic strategies were presented for the synthesis of bicyclic
oxa- and aza-L-rhamnose mimics. These new analogs are
commonly found in the cell walls of bacteria, such as
Mycobacterium tuberculosis, and may function as effective
inhibitors of α-L-rhamnosidases. Such new analogs have
potential as selective therapeutics for the treatment of
mycobacterial infections, such as tuberculosis.
α-L-fucosidase inhibitors
Zbigniew J Witczak presented developments from his
laboratory in the synthesis of novel (1-4)-S-thiodisaccharides
as α-L-fucosidase inhibitors. New stereoselective approaches
utilize the Michael addition of protected 1-thiosugars to a
bicyclic enone (levoglucosenone), producing target compounds in high yield. (1-4)-S-Thiodisaccharides containing
the L-fucose moiety are, however, weak α-L-fucosidase
inhibitors (Ki = 2.4 µM) with significant differences of
inhibitory activity on α-L-fucosidase from a bovine kidney
and a bovine epididymis. They are about 10-fold more
potent on α-L-fucosidase from human placenta (Ki = 0.2 µM)
than from bovine kidneys.
Finally, Khushi L Matta (Roswell Park Cancer Institute,
Buffalo, NY, USA) presented some aspects of the chemistry
and biochemistry of α-fucosylated saccharides, and the
substrate specificity of α-L-fucosidases and α-L-fucosyltransferases.
Kuniaki Tatsuta and Zbigniew J Witczak then concluded the
symposium with a summary of all lectures and highlighted
some of the new developments in this field (eg, new
iminosugars, trehazolins and azasugars) that have produced
potential leads for the development of new pharmaceuticals.
Advances in nucleosides and nucleotides
The only morning session of the last day of the meeting was
chaired by Professor Waldemar Priebe, MD, and consisted
of five lectures. First, L Ma, an associate from Professor
31
Raymond F Schinazi’s laboratory at Emory University
School of Medicine in Athens, GA, presented data from a
collaborative project with Professor CK Chu of the
University of Georgia in Athens on the separation and
biological activity of enantiomers of 2’,3’-dideoxy-5-fluoro3’-thiacytidine (FTC) and its selenium analog (Se-FDDC). In
primary human lymphocytes infected with HIV-1, (-)-β-SeFddC (mean EC50 = 0.21 µM) was 200-fold more potent than
the (+)-β-counterpart and, interestingly, demonstrated no
cytotoxicity in various cells.
Fluorinated nucleosides and oligonucleotides
Two presentations followed from the CRC Centre for Cancer
Therapeutics, Sutton, UK, on the preparation of fluorinated
analogs of nucleosides and oligonucleotides. Catherine A
Brown presented a new method for the preparation of 2’and 3’-trifluoromethyl nucleosides via SN2’ substitution of
the corresponding 2’- and 3’-difluoromethylene derivatives.
’
Interestingly, removal of the 2 - and 3’-O-trimethyl-silylethoxymethyl groups with tetrabutylammonium fluoride
resulted in unexpected SN2’ substitution at the unsaturated
difluoromethylene carbon with the formation of new
analogs, mainly 2’,3-didehydro-2’,3’-dideoxy-5’-O-dimethoxytrityl-2’-trifluoromethyl uridine. Dr Pawel Serafinowski
then discussed new studies on the synthesis of (2’-5’)-linked
oligonucleotides modified with 3’-difluoromethylene
groups. The protected dimers were synthesized by condensation of 3’-deoxy-3’-difluoromethylene-5’-O-dimethoxytrityluridine-2’-O-phosphor-amidite with 3’-O-acetyl or 3’-Olevulinylthy-midine. The synthesis of the target 2’-5’-linked
oligonucleo-tides was performed by solid phase synthetic
cycle using an oxalyl linker and t-butylhydroxyperoxide in
the oxidation step.
The final two presentations in this symposium related to
work carried out at the Howard Hughes Medical Institute at
the University of Chicago. Xiangmin Liao reported Tetrahymena ribozyme cleavage of 5’-methylene phos-phonate
and the effect of replacing the 5’-bridging oxygen of the
phosphate at the cleavage site with a methylene group. The
ribozyme unexpectedly catalyzes the cleavage of this
methylene phosphonate linkage 1000-fold faster than the
unmodified linkage in the substrates containing all
deoxynucleotides. This particular effect is attributed to a 10fold effect on docking of the substrate into the catalytic core
and a 100-fold effect on the chemical step.
Xiao-Quing Tang then discussed the synthesis and
incorporation into oligonucleotides of phosphoroamidate
derivatives of 2’-C-β-methylcytidine, which are potentially
useful for antisense oligonucleotides and for probing the
structure and function of RNA. The strategy developed for
the synthesis of the phosphoroamidate derivatives of 2’-C-βmethylcytidine, ie, protection of the 2’-hydroxy group with
either t-butyldimethyl silyl or tetrahydropyranyl groups,
permitted their incorporation into oligonucleotides by solid
phase synthesis.
32
Ernest Guenther award in the chemistry of natural products
Kevin G Pinney
Address
Baylor University
Waco
TX 76798
USA
IDrugs 1998 1(1):32-34
A marvelous symposium was held to honor Professor GR Pettit
(Arizona State University, Tempe, AZ, USA) as the 1998
recipient of the Ernest Guenther Award in the chemistry of
natural products. Attendance at the symposium was fantastic,
numbering well into the hundreds, and at various times there was
standing-room only. Three outstanding lectures given by MR
Boyd (National Cancer Institute, Frederick, MD, USA), David GI
Kingston (Virginia Polytechnic Institute and State University,
Blacksburg, VA, USA), and JP Kutney (University of British
Columbia, Vancouver, USA) preceding the superb award address
by GR Pettit. Professor Pettit was warmly acknowledged by each
lecturer who preceded him. MR Boyd gave an especially touching
tribute to Bob Pettit commenting on his professional excellence,
scientific and personal integrity, dedication to public service, and
declaring that he truly was a "National Treasure". On a personal
note, I find Bob Pettit to be a brilliant scientist and tenacious
researcher (in a sense, a true pioneer and statesman for science)
who is driven by the humanitarian purpose of discovering and
developing the most effective antitumor drugs possible and getting
them to the people who need them the most, the cancer patients
themselves. It is a further delight that Bob Pettit is a most humble
and gracious man, as well as a true gentleman, who is a world
leader in the battle against cancer.
Discovery of naturally-occurring inhibitors of
HIV
Dr MR Boyd presented a fascinating lecture describing the
current status of his laboratory’s HIV research as it pertains
to new drug development from natural sources. With 40
million people affected worldwide, there is truly an
escalating pandemic of HIV infection. This fact, coupled
with the frequent and inevitable emergence of resistance to
available drugs, mandates the need for the development of
new therapeutic agents with non-overlapping resistance
profiles and diverse mechanisms of biological activity. It
was also mentioned that an effective vaccine or vaccines has
thus far remained elusive.
New natural product drug development leads have been
discovered from both terrestrial and aquatic sources. The
discovery process employs bioassay-guided chemistry. A
cell-based bioassay using infectious HIV is utilized and the
principal source of extracts for study come from the NCI
Natural Products Repository, which is the world’s largest
collection of samples with biodiversity for drug discovery.
The lead compounds discovered from terrestrial plants have
been mostly non-peptide in nature. For example, (+)-
calanolide A, derived from the Malaysian tree, Calophyllum
lanigerumunder, is a non-nucleoside HIV-1-specific reverse
transcriptase inhibitor (NNRTI) which is active against diverse
drug-resistant strains. This drug is being developed through a
joint venture between MediChem Research (licensee to WO09320082, owned by the US government) and the government
of Sarawak in Malaysia. Calanolide seems to have favorable
bioavailability in initial clinical trials.
In addition, prostratin, a non tumor-promoting phorbol
ester, was described as a novel subclass of PKC agonist. A
variety of new drugs have also been discovered from
aquatic sources. These compounds seem especially useful
for microbicide (anti-HIV) development, and as templates
for new small-molecule inhibitors of HIV. Cyanovirin-N
(CV-N), a protein initially isolated from a blue-green alga,
Nostoc ellipsosporum, is a protective agent which targets viral
gp120. Recombinant cyanovirin is now also available, and it
has the same activity as the natural material. Low
nanomolar concentrations of CV-N irreversibly inactivate
diverse M-tropic, T-tropic, and dual-tropic primary isolates
of HIV. Possible applications of cyanovirin include virucidal
treatment of blood and topical use to avoid sexual
transmission of HIV.
Natural products from the tropical rainforest
of Suriname
Professor David GI Kingston gave an inspiring presentation
of his group’s ongoing collection, isolation, structure
determination, and synthetic analog preparation efforts in
the tropical rainforest of Suriname. Prefacing his
presentation with a factual reminder that a large number of
clinically-significant drugs have resulted directly from
discoveries in natural products chemistry, and that one-half
of all prescriptions dispensed in the United States contain
substances of natural origin, Professor Kingston challenged
the audience with the escalating problem of biodiversity loss
due, in part, to the harvesting of tropical rainforests for
timber and other industrial concerns. These natural
repositories of terrestrial life are disappearing rapidly and
currently occupy only 7 to 8% of the earth’s surface, which is
down from approximately 16% of the earth’s land surface in
the 1950s.
One effort to slow down this trend centers around the
concept of biodiversity conservation in which a host country
is rewarded financially for preserving its biodiversity.
Newly-discovered drugs would be licensed by major
pharmaceutical companies and certain royalty payments
would be returned to the host country in compensation of
their productive use of their biodiversity resources. One
mechanism for such compensation is through the "Forest
Peoples Fund" for distribution of upfront payments from
Bristol-Myers Squibb in anticipation of future discoveries.
Professor Kingston and his group, along with Bristol-Myers
Squibb, their industrial collaborator, Conservation International, Missouri Botanical Garden, and their in-country
partner BGVS, working closely with the shamans (tribal
healers) have already made significant discoveries in
Suriname. To date, approximately 1000 plant samples have
been collected, 3000 extracts have been screened for
biological activity, and 15 to 20 active compounds have been
isolated from the 80 positive hits. The remainder of
Professor Kingston’s interesting lecture focused on the
structure and activity of these new compounds, including a
new ellagic acid derivative, as well as other diterpenoids,
quinones, alkaloids, and polyketides.
The emerging role of biotechnology in natural
product drug discovery
The application of biotechnology to the field of natural
product synthesis was eloquently presented by Professor JP
Kutney, rightfully regarded as a true pioneer in the field. In
the first part of his presentation, Professor Kutney illustrated
the use of biotechnology in a synthetic application toward
vincristine and vinblastine. These two potent anticancer
drugs have been known for some time, but their cost is
prohibitive, since they are isolated from plant sources. An
intriguing biotechnological approach to this problem
involves using waste by-products from the biosynthetic
pathway as new starting materials in enzymatically-driven
reactions. Using this method, vinblastine can now be
produced in a one-pot process from catharanthine and
vindoline with an overall yield of 42%. This translates into
the ability to produce gram quantities of vinblastine in one
day. In a move that may someday replace the current
isolation of vinblastine from plants as the source of the drug,
IGT (a Vancouver company) has licensed this biotechnological process for the production of vinblastine. A new
drug, anhydrovinblastine (AVLB), has resulted from this
work and is currently in preclinical development.
In the second part of the presentation, Professor Kutney
described the useful combination of synthetic organic
chemistry with biotechnology. Synthesizing various precursors from biosynthetic pathways paves the way for the use
of these compounds in combination with enzymes for the
economical production of drugs. Various applications were
discussed including the antimitotic agent podophyllotoxin
and chiral analogs of podophyllotoxin, as well as etoposide.
These processes can be carried out in "biological factories"
which are large reactors operating in a semicontinuous
fashion. In one particular example, the same cell line
remained active for four months, allowing 22 batches of the
product to be produced. Relatively inexpensive reagents can
be employed to generate kilogram amounts of material.
Aspects of this biotechnology have also been transferred to
IGT for further development and commercialization.
In the final portion of the lecture, an informative discussion
of the use of plant tissue cultures for the production of high
levels of products (when compared to living plants) was
presented. A Chinese herbal plant, Tripterygium wilfordii, has
been known to have medicinal value for over 2000 years and
is used to treat various skin disorders, arthritis, and chronic
hepatitis. Over the years, a variety of compounds, especially
diterpenes and diterpene epoxides, have been isolated from
the plant. Since some of these compounds have pronounced
antitumor, immunosuppressive, and/or antispermatogenic
activity, the development of high-yield production processes
33
is warranted. The remainder of the lecture centered on the
application of plant cell cultures as a means not only of
addressing this availability problem but also to discover
new and novel analogs.
Adventures in the discovery of naturally
occurring anticancer drugs
The afternoon concluded with a brilliant award presentation
by Professor GR Pettit. Professor Pettit began his address by
stating that he was deeply honored by the award and that he
wished to dedicate his lecture to the memory of Dr Kenneth
Paull (National Cancer Institute) and Sir Derek Barton
(Texas A&M University) who both passed away recently
and were especially good friends of his. Continuing with a
comment that summarizes his tenacity as a researcher,
Professor Pettit stated, "when I get up each morning, the
chemistry of natural products is so exciting that I hardly
know which way to run first!"
With the cancer problem continuing to escalate (nearly
600,000 people will lose their lives to cancer in the United
States this year), it is paramount that new second, third, and
fourth generation drugs be developed to treat the over 200
types of human cancer. Beginning with a discussion of
natural products isolated from terrestrial sources and
amphibians in the early days of his academic career,
Professor Pettit discussed work which has so far spanned
more than four decades and has involved terrestrial and
marine sources throughout the entire world.
Pettit and coworkers collected the spongistatins from a red
sponge off east coast of South Africa in the early 1970s.
Extracts of these sponges more than doubled the lifetime of
certain laboratory animals afflicted with the leukemia P388
cancer cell line. Often forced to collect several tons of source
material to yield very small amounts of purified drugs,
Professor Pettit and his group have remained steadfast in
their dedication to the cancer problem. In 1982, for example,
the first of the spongistatins had been purified resulting in
approximately 800 mg! This corresponds to isolation yields
-6
-8
in the range of 10 to 10 % for certain natural products from
these sponges. The spongistatins have been shown to have
spectacular cytotoxicity and antitumor activity, and the
recent total synthesis of spongistatin 1 (Professor Kishi) and
spongistatin 2 (Professor Evans) herald the future development of new and biologically significant analogs of these
spongistatins.
Professor Pettit continued his world tour with a description
of the isolation and characterization of many natural
products including halichondrin B, cribrostatin 1, and
cephalostatin 1. The lecture shifted to a fascinating account
of the isolation, characterization, and synthesis of dolastatin
10, and related compounds, isolated from Dolabella
auricularia, a sea hare. These natural products are superb
antineoplastic substances. When in vivo studies at the NCI in
the 1980s confirmed that extremely low doses of dolastatin
10 could essentially cure certain cancer cell lines, it became
essential to be able to quickly provide enough of the
compound for clinical trials. This was not feasible due to
low isolation yields of the material from the sea hare, as well
as the conservation questions associated with removing
34
many tons of this particular sea hare from the ocean.
Professor Pettit and coworkers therefore set out to prepare
the compound by total synthesis. Being unsure of the
absolute configuration of the natural product, they were
prepared to carry out the synthesis of all 512 possible
stereoisomers; fortunately, they arrived at the correct analog
on the fifteenth total synthesis (still an impressive feat,
considering that their synthetic route encompassed approximately 28 steps).
The lecture continued with the discussion of myriad other
interesting and biologically valuable natural products,
including bryostatin 1, a fascinating compound with superb
antitumor activity. Professor Pettit concluded his lecture
with an intriguing account of combretastatin A-4 which he
originally isolated from the South African tree, Combretum
caffrum, in 1972. This compound (as a prodrug) will enter
phase I clinical trials this summer. In addition to being a
potent inhibitor of tubulin polymerization, it has the
remarkable ability to selectively inhibit the blood supply to a
tumor; in an in vivo blood flow study, the blood supply to
the tumor in a mouse was essentially completely stopped,
approximately 4 h after administration of a combretastatin
A-4 prodrug. Professor Pettit has now established the
Cancer Research Institute at Arizona State University, which
promises many more similar, exciting discoveries.
35
Combinatorial carbohydrate chemistry - where are we now?
Mike Panigot
Address
P O Box 419
State University
AR 72467-0419
USA
IDrugs 1998 1(1):35-37
This symposium to highlighted the uses of combinatorial chemistry
in the synthesis of complex polysaccharides possessing biological
activity. Approximately 50 people attended, and most were involved
in carbohydrate chemistry using either classical synthetic approaches
or combinatorial methods. The invited speakers were from both
academia and industry and the session was aimed both at academic
researchers and those who employ combinatorial syntheses in the
industrial production of biologically relevant polysaccharides.
Progress in solid phase synthesis of
oligosaccharides: impact in combinatorial
synthesis
The first address on the use of solid phase synthesis in the
preparation of complex carbohydrates was presented by
Professor Richard R Schmidt (Universitat Konstanz,
Germany). The advantages of solid phase synthesis include
the ability to modify the functionality of the carbon
scaffolding of the sugar bonded to the resin, the highly
functionalized carbohydrate structure (which may be viewed
as a polyhydroxylated aldehyde or ketone and selectively
protected or functionalized), and the defined chirality at each
of the carbon atoms of these molecules. In the examples that
Professor Schmidt discussed, D-glucosamine was bonded to
the resin as the carbon scaffold. As a preliminary example, a
polysaccharide found in human milk containing several
galactose, fucose, and N-acetylglucosamine residues was
prepared. This molecule was found to be an antigen
determinant of the lacto- and lactono-series. This glycoform is
linked to asparagine derivatives as part of a glycoprotein.
Professor Schmidt then discussed the various types of
polysaccharides and carbohydrate derivatives that exist, as
well as the advantages and difficulties found in the chemical
synthesis of each. Naturally-occurring compounds consist of
homoglycans, heteroglycans, and glycoconjugates, such as
glycolipids, glycosphingolipids, and glycoproteins. Efficient
preparation of these molecules by combinatorial methods on
a solid support requires that versatile chemical building
blocks be available in the form of selectively-blocked or
selectively-reactive carbohydrates, and that the desired
reaction takes place in high yield for a resin-bound
carbohydrate using chemistry developed for reactions in
solution.
Trichloroacetimidates were used as glycosyl donors, which
Professor Schmidt has also used in the classical solution phase
synthesis of glycosides. This compounds are easily prepared,
near neutral in their pH and in the pH of the by products
formed, exhibit a great deal of stereoselectivity in the
glycosidic bond formed, and form no reactive intermediates
or water during the glycosidation.
The oligosaccharides which were synthesized were
analyzed by MALDI-TOF MS, TLC and HPLC. As the length
of the polysaccharide chain increased, the yield of each step
decreased. Therefore, this method may be best suited for the
synthesis of small oligosaccharides. As a demonstration of
the feasibility of these methods, a β-1,3-linked homoglucan
was prepared in an overall yield of 28% over 12 steps or
90%/step in the reaction sequence. Similarly, more complex
N-glycan structures could be prepared in 20% overall yield
or 82%/step.
Strategy for the convergent assembly of
oligosaccharide libraries
Matthew Johnston (University of Birmingham, UK) discussed
chemistry which was performed in the solution phase on
protected carbohydrates that could act either as glycosyl
acceptors or as glycosyl donors. These sugars were derived
from a 3-buten-2-yl glycoside bearing benzyl protecting
groups at the 2, 3 and 4 positions, and an acetate-protecting
group at the 6 position. This, in turn, could be prepared from
acetobromoglucose by Koenigs-Knorr glycosylation with 3buten-2-ol. It could also be prepared in a more environmentally friendly manner by reaction of D-glucose and 3buten-2-ol with almond β-glucosidase immobilized on
Amberlite XAD-4 resin. The enzymatic process also provides
some stereoselectivity in the side chain, leading to a 3:2 ratio
with the R isomer predominating. Four glycosyl acceptors
were reacted with four glycosyl donors to form 16
disaccharides, which were reacted with four glycosyl donors
to form 64 trisaccharides, and so on.
Initial compounds were partially protected glucose and
galactose derivatives that could be reacted at the 4-position
or at the 6-position. These were reacted with each other to
form 16 possible disaccharides. Once the 16 disaccharides
were prepared, they were mixed, deacetylated, split, and
reacted with a fucosyl donor. After hydrolysis with
trifluoroacetic acid and analysis by HPLC, the percentages
of the different trisaccharides formed were in agreement
with what was theoretically anticipated, indicating that this
combinatorial method was successful. Emphasis then shifted
towards the synthesis of sialyl Lewis X analogs utilizing
acetate and 4-methoxybenzyl as orthogonal protecting
groups. A series of trivalent carbohydrate ligands were
prepared and screened. This process is currently being
expanded to build libraries of oligosaccharides.
Solid phase combinatorial synthesis of
"carbohybrids"
The biological importance of carbohydrates in molecular
recognition and cell signaling events and the interactions of
cell surfaces and carbohydrates in recognizing viruses,
36
bacteria, enzymes, antibodies, and toxins, was discussed by
Gerd Hummel (University of Alberta, Canada). The concept
of what they have termed a "carbohybrid" is based on the
observation that oligosaccharides are very complex
structures - too complex to be adapted by the pharmaceutical industry on a large scale. The "carbohybrid" would
contain a part of the oligosaccharide that is recognized and
necessary for efficient binding and biological activity. The
remainder of the oligosaccharide would be replaced by a
small organic molecule, simpler in structure, which would
provide some binding as well.
A number of different leaving groups in glycosyl donors
have been investigated including thioglycosides, sulfoxides,
glycosyl halides, phosphates, and trichloro-acetimidates.
From determination of the rates of glycosidation of a
number of glycosyl donors and the chemical shifts of the
1
anomeric proton in the [ H]NMR spectra of these donors, it
was found that the smaller the chemical shift, the more
reactive the glycosyl donor. This strategy was used to make
a tetrasaccharide by combining the glycosyl donors and
acceptors initially and allowing them to react based on their
rate of reactivity. The desired product was obtained in 35%
yield.
GM1 ganglioside
As an example of this combinatorial approach, the GM1
ganglioside was investigated. This molecule binds cholera
toxin and heat labile toxin found in Escherichia coli. The key
structural feature required for the binding of heat labile
toxin is a galactose residue. An anomeric thioacetate was
prepared, converted to the thiogalactose, and reacted at the
sulfur atom with a series of Michael acceptors, particularly
cyclic unsaturated ketones. Once addition had occurred, the
ketone could be reduced to the alcohol or converted to an
amine by reductive amination. Conversion to the amine
permitted the introduction of groups that could bind by
interaction of opposite charges. An extensive series of
Michael acceptors were reacted and processed, and their
activity against heat labile protein was measured.
In the combinatorial approach, the thiosugar was reacted
with ten different Michael acceptor aldehydes and ketones.
Each of these was processed to give 10 compounds for a
total of 100 compounds. Screening of these compounds was
carried out with ELISA and chemical analysis was
performed by MS. The compounds were tested as a mixture
of four diastereomers.
Similar chemistry could be performed in the solid phase on
a polymer-bound trityl chloride. Initially, the tetraacetate of
thiogalactose was converted to the ethyl disulfide, deacetylated, bonded to the trityl chloride resin, and treated with
dithiothreitol to liberate the thioglycoside. The chemistry
described above worked equally well in the solid phase and
the final products could be removed from the resin using
TFA/TIS in dichloromethane.
Programmable one pot synthesis of
oligosaccharides
Dr Chi-Huey Wong (Scripps Research Institute, CA, USA)
began his presentation with a brief description of the
classical glycosidation reaction in which a glycosyl acceptor
is reacted with a glycosyl donor in the presence of an
activating agent to form a disaccharide. The limiting step in
this sequence is activation of the glycosyl donor to form a
glycosidic bond with the glycosyl acceptor. Structural
features that affect the rate of glycosidation were discussed
for both the donor and acceptor carbohydrates. For the
glycosyl donor, these features are stereochemistry of the
carbohydrate, the structure(s) of protecting groups present,
the leaving group to be displaced by the glycosyl acceptor,
and the nature of the promoter being used. For the glycosyl
acceptor, the structural features that affect glycosidation are
the stereochemistry of the acceptor and the protecting
groups present.
Next, the use of orthogonal protection strategy in the
synthesis of complex polysaccharides was discussed. By
using four different protecting groups (4-methoxybenzyl,
TBDMS, chloroacetyl and levulinate) that could be removed
individually, a carbohydrate library was prepared containing
a number of different carbohydrate branches attached to a
common carbohydrate core. This was performed on a solid
support and glycoside incorporation was determined by gated
13
decoupled [ C]NMR using Cr(acac)2 as relaxation agent.
Generation of carbohydrate libraries for drug
discovery
The synthesis and uses of carbohydrate libraries in drug
screening were discussed by Dr Michael J Sofia (Transcell
Technologies, NJ, USA). It has been known for some time
that combinatorial chemistry to form oligopeptides and
small molecules greatly increases the number of biologicallyactive compounds that may be screened in a given time, but
the use of such techniques in carbohydrate research is much
less common.
Moenomycin A is an inhibitor of cell wall biosynthesis with
a pharmacophore that requires three structural units: a
carbamate portion; an amide; and a phospho-glycerate. A
lipid moiety and an N-acetylglucosamine group may also be
necessary for biological activity. In the chemistry discussed
by Dr Sofia, the carbamate was replaced by a dialkylurea
and an orthogonally protected carbohydrate was prepared
and bonded to a solid support as an amide through a uronic
acid group. The required carbohydrate with the necessary
functionality was prepared on a solid support, then
glycosidated using the sulfoxide donor approach developed
by Kahne. Quality control and analysis was through
1
[ H]NMR spectroscopy after cleavage of the oligosaccharide
from the resin. Additional analysis of these compounds was
obtained by electrospray MS.
Next, primary screening libraries were investigated. These
were broken down into cases in which there is an interaction
of a small molecule with a protein and the molecular
recognition sites are close and instances in which proteinprotein interactions predominate and the recognition sites
are farther apart. The rationale for the use of carbohydrates
in this particular area arises from their rigid structure,
enantiomeric purity, defined stereochemistry, and high
combinatorial density. The molecules also have the drug
characteristics of small molecules.
As a result of the three-point pharmacophore, the
carbohydrate molecule was trifunctionalized as a uronic acid
containing a free hydroxyl at C-4 and an Fmoc-NH group at
C-3 in one series and at C-2 in another. The uronic acid was
bound to a solid support as an amide and the nitrogen
functionalized by reacting it with amino acids, isocyanates,
acids and aldehydes. An IRORI Accutag 100 system was
employed with a microcan system which allowed the library
to be built by a mix and split technique. This permitted the
incorporation of unnatural sugars to investigate nonstandard binding sites. The overall purity of the compounds
being tested was greater than 80%.
Lastly, a comparison was made in diversity space of several
trisaccharides that had been prepared with several
trifunctional small molecules and several tripeptides. These
particular carbohydrates were found in a different diversity
space from the small molecules and tripeptides. It is hoped
37
that this will lead to different biological activity for this
particular class of molecules
Conclusion
The use of combinatorial chemistry in academic and
industrial research laboratories has been widespread in the
syntheses of small molecules and oligopeptides for quite
some time. Similar methodology is now encompassing the
field of carbohydrate chemistry. This should permit the
rapid discovery and testing of many new carbohydratebased materials. Given the importance of cell signaling and
molecular recognition by carbohydrates, this should allow
more carbohydrate-containing pharmaceuticals to enter the
marketplace more rapidly and at lower cost than by classical
solution phase synthesis of individual compounds.
38
Selected state of the art topics - organic chemistry poster session
Eusebio Juaristi
Address
Cinvestav
PO Box 14-1470
07360 Cd
Mexico
IDrugs 1998 1(1):38-39
Nearly 400 papers were accepted for presentation in the Division
of Organic Chemistry during the 1998 Spring ACS National
Meeting. From these, 38 poster papers were selected by Program
Chairperson, SS Hall, as most representative of the state of the art,
and ten of these selected papers related to drugs or drugs discovery.
A brief report on these posters, which attracted a large crowd of
interested attendees, is presented here.
Design and synthesis of enediyne-based
ADEPT prodrugs
Samuel A Brown and Carmelo J Rizzo (Vanderbilt
University, TN, USA) showed that the biological toxicity of
enediynes is generally dependent upon conformational
changes. As an example, the bioreduction of the trisulfide
unit of calicheamicin, followed by addition of the resulting
free thiol to the enone, afforded a cytotoxic agent presenting
1000-fold greater activity than adriamycin in murine tumor
models. Synthesis of enediyne-cephalosporin hybrids using
ADEPT (antibody-directed enzyme prodrug therapy)
technology was also presented [ORGN 053].
Solid phase syntheses of turn structures
The finding that the synthesis of cyclic peptides and
peptidomimetics by means of disulfide, amide, or ester bond
formation is usually complicated by racemization and other
side reactions was discussed by Yangbo Feng, Zigi Wang
and Kevin Burgess (Texas A&M University, USA) [ORGN
054]. These difficulties are a serious drawback for combinatorial chemistry applications.
The goal of the presented work was the development of new
methodologies for the synthesis of cyclic peptido-mimetic
libraries on solid supports. In particular, SN2 and SNAr
reactions were successful in the cyclization step leading to
C-O, C-S or C-N single bonds. These reactions were
sufficiently clean for library syntheses on solid supports,
affording macrocyclic structures (20-membered rings or
smaller), with purities above 90%.
Conjugate addition of nitrogen nucleophiles
to an unsaturated pyrrolidinone
Functionalized pyrrolidines have a wide range of biological
activities. Philip Chan, Ian Cottrell and Mark Moloney
(University of Oxford, UK) [ORGN 058] revealed how a
bicyclic lactam derived from pyroglutamic acid was a useful
template for conjugate additions of nitrogen nucleophiles, in
good yield and with high diastereoselectivity [for a
preliminary communication, see Chan P et al: Tetrahedron
Lett (1997) 38:5891]. The above protocol provides a route to
β-amino- and α,β-diamino-acids, compounds which are
recognized to be of substantial biological importance, and
whose synthesis has recently attracted considerable attention [see Juaristi E: Enantioselective Synthesis of β-Amino Acids,
Wiley-VCH, New York, 1997].
A novel approach to the synthesis of taxolchain analogs
The continuing need for the development of efficient largescale syntheses of taxol was discussed by Marko Mihovilovic, Margaret Kayser, Sonia Rodríguez and Jon Stewart
(University of New Brunswick, NJ, USA and University of
Florida, USA) [ORGN 064].
The group has employed enantioselective reduction of αketo-β-lactams, which was accomplished with engineered
strains of the yeast Saccharomyces cerevisiae. The main
advantages are their low cost and easy access, and the fact
that yeast are environmentally compatible and pathogenically benign. However, the authors report solubility
problems and possible toxicity of the substrate for yeast. The
applicability of the method was demonstrated with the
preparation of a precursor to the side chain of taxol.
Total synthesis of a potent proinflammatory
5-oxo-ETE
Subhash Khanapure, Xiao-Xin Shi, William Powell and
Joshua Rokach (Florida Institute of Technology, USA and
McGill University, Montreal, Canada) showed that 5-oxoETE, a novel arachidonate metabolite, is a potent chemotactic agent for human neutrophils, being about 100-fold
more potent than its precursor, 5(S)-HETE [ORGN 065].
In order to study the biochemical pathway for the oxidation
of 5-oxo-ETE, the authors undertook the total synthesis of a
potent proinflammatory 5-oxo-ETE and its radiolabeled
derivatives [Rokach J et al: J Org Chem (1998) 63:337]. A
convergent synthesis was accomplished via a bisdienyl
phosphorium bromide and a dithiolane aldehyde. The
acetylenic precursor made the incorporation of four tritium
atoms possible.
Synthesis of constrained cyclic peptide
helices by ring-closing metathesis
Helen Blackwell and Robert Grubbs (California Institute of
Technology, USA) reported that ring-closing metathesis
(RCM) [Grubbs R et al: J Am Chem Soc (1996) 118:9606] has
recently been applied to the synthesis of cyclic peptides
exhibiting a β-turn secondary structure. Indeed, ruthenium
complexes catalyzed ring-closing metathesis reactions to
form five-, six-, seven-, and eight-membered carbocycles and
heterocycles.
The aim of this research is to establish whether the acyclic
precursors access a helical conformation in solution.
Heptapeptide analogs of a known helical peptide were
synthesized and found to adopt helical conformations in
chloroform. The ruthenium olefin metathesis catalyst was
highly active in these media, affording the macrocyclic
peptides of interest [ORGN 072].
Biomimetic mineralization
The widespread interest in the preparation of organized
nanoscale, microscopic and bulk inorganic materials under
mild conditions was discussed by Haimanot Bekele and
Jeffrey Kelly (Scripps Research Institute, CA, USA).
Inorganic composites of uniform size, shape and morphology may have applications as catalysts, as well as magnetic,
optical and electrical materials.
In order to achieve controlled crystallization of inorganic
minerals, the authors employed an amphiphilic selfassembling peptidomimetic that adopts a two-dimensional
β-sheet structure at an air-water interface to nucleate calcite
into crystals of uniform size that are distinct from nonnucleated crystals [ORGN 075].
Synthesis of γ-lactams
Lactams are known to be essential constituents in a diverse
range of natural and synthetic products of therapeutic
interest. For example, the β-lactam family has acquired a
status of unparalleled importance in the treatment of
infectious diseases caused by bacteria. Although γ- and δlactams have been investigated less extensively, they are of
considerable interest as antitumor agents. Diana Danca and
James Rigby (Wayne State University, MI, USA) reported a
39
general method for the synthesis of γ-lactams, which is
based on the reaction of selenium-phenylselenocarbamates
with [(CH3)3Si]3SiH and subsequent addition of the incipient
free radical onto an appropriate double bond. This radical
cyclization reaction displays high regioselectivity, with a
preference for exo addition [ORGN 079].
Synthesis and application of γ,δ-unsaturated
amino acids
Unsaturated amino acids are an important class of natural
products which display an array of interesting biological
properties. Mark Burk, John Allen and William Kiesman
(Duke University, NC, USA) reported a procedure for the
highly regio- and enantio-selective catalytic hydro-genation
of enamides in conjugated diene systems (α,γ-dienamide
esters) to afford γ,δ-unsaturated amino acids [ORGN 085].
This new methodology was applied to the synthesis of the
natural product, bulgecinine, a component of β-lactam
synergists bulgecin A-C [see also Burk M et al: J Am Chem
Soc (1998) 120:657].
Progress toward total syntheses of
epothilones A and B
Epothilones are 16-membered ring macrolides which have
exhibited excellent antimitotic activity. Indeed, epothilones
operate by the same mechanism as paclitaxel. Emily Reiff,
Sajiv Nair, Ashok Tunoori, Sam Victory and Gunda Georg
(University of Kansas, USA) presented pathways for the
synthesis of the C1-C6 segment of the epothilones [ORGN
086]. These synthetic approaches are amenable to the
synthesis of attractive analogs [for related work, see
Tetrahedron Lett (1997) 38:1703].
40
An interview with the Chair of the ACS MEDI division
Peter Robins
Address
Middlesex House
London
W1P 6LB
UK
followed by three talks that summarized inhibitors of this
enzyme, which are potential anticancer agents. There have
been significant advances in this field in the last two or three
years, now that pharmacokinetic and potency problems are
beginning to be solved. In the next few years we should
obtain information on the potential of the newer compounds
in human cancer.
IDrugs 1998 1(1):40-41
The schedule for Tuesday morning was very full,
with the Awards Symposium and further general
oral sessions.
Following the conclusion of the 215th National Meeting of the
American Chemical Society Meeting in Dallas, Current Drugs
Editor, Peter Robins, spoke to the Chair of the ACS Division of
Medicinal Chemistry, Annette Doherty, of Parke-Davis/ Jouveinal
in Fresnes, France.
The awards session took place on Tuesday morning and due
to a large number of oral submissions we scheduled
additional presentations for the Tuesday morning sessions.
How did you come to be the Organizer of the
Medicinal Chemistry Section?
Medical chemists who are members of the division vote for
Vice-Chair and Councillor positions each year. The members
of the division voted for my appointment in 1996. This is a
four-year commitment to the medicinal chemistry division.
During the middle two years as Secretary of the Division,
the responsibility involves organization of the medicinal
chemistry schedule for ACS National Meetings.
The conference began for the Medicinal Chemistry
section with the two General oral sessions on
Sunday. Could you tell me a little about those, and
the poster session that followed.
On the first day of the conference, we always have general
oral papers. We usually receive between 30 and 40 of these
submissions on a wide range of subjects. These particular
sessions have become increasingly popular in the last two or
three years, so we schedule two on Sunday in general. This
year, both general sessions were very well attended. A
poster session is always held on Sunday evening, again
covering a wide range of medicinal chemistry topics.
The adenosine symposium was remarkably
popular as well.
We decided to focus on this area because in recent years,
with advances in the molecular biology of the adenosine
receptors, a number of subtypes have emerged and the
clinical indications for selective antagonists and agonists of
the various adenosine receptors are becoming clearer. A
number of different indications were discussed for the A1,
A2A, A2B and A3 receptors and there were many important
compounds disclosed. Selectivity has been a major issue in
this area and significant advances have occurred in recent
years.
The session on Monday afternoon with Dr Gibbs presiding,
was focused on protein prenylation and in particular, on Ras
farnesyltransferase inhibitors. The first two talks were
biochemical in nature, focusing on yeast, as well as the
human Ras farnesyltransferase enzymes. These were
The first presentation was for the Ralph Hirschmann
Award, presented to Dr Isabella Karle. Her fascinating talk
focused on her contributions to the field of peptide/ protein
crystallography. Next was Monroe Wall, who spoke on the
subject of camptothecins. He discussed the mechanism of
action of these compounds as topoiso-merase inhibitors, and
highlighted a number of other, newer camptothecin
compounds with potential for anticancer therapy.
What about the final award, the ACS Award for
Team Innovation?
This is a new award presented to teams of people who have
developed a product in the laboratory and taken it to
market, intended to highlight the value of multi-disciplinary
teamwork in the development of a product in commercial
use. The recipients this year were the group from Eli Lilly
responsible for the development of the rapid-acting insulin
analog, Humalog. Three members of the team gave a very
well integrated presentation, illustrating how, in industry,
the various departments work together to advance a
successful new product. Humalog has the ability to
normalize levels over a prolonged period of time and will
likely reduce the long-term complications of diabetes.
Overall, there seemed to be a large number of
presentations that discussed angiogenesis, cancer
or oncology in general.
Well certainly, protein prenylation, ATP-directed kinase
inhibitors, angiogenesis are all proliferative-type strategies,
but these were not the sole focus of the program. For
example, the anticoagulation strategies symposium was
interesting, because the Factor Xa inhibitor area is one which
has advanced significantly in recent years and there are
many new, non-peptide inhibitors being reported. This
strategy may have advantages over the thrombin inhibitors
in terms of bleeding tendency. Also, we had a session
relating to the application of amyloid aggregation inhibition
to cognition.
Thrombosis was covered quite recently at the
ACS, I understand.
The ACS planning committee for the Medicinal Chemistry
division was unsure whether to have a thrombin/ Factor Xa
inhibition symposium, because of the antithrombotic
symposium in Orlando in 1996, but there have been many
recent advances in this particular field. We decided,
therefore, to schedule a late-breaking session to cover new
developments in anticoagulation. In particular, the field of
factor Xa inhibitors is attracting interest for either deep vein
thrombosis or acute thrombotic events. As a result, there
were many new compounds revealed at this symposium,
particularly in the factor Xa area.
What was the rationale behind the Cytochrome
P450 symposium?
This is a really interesting area because we don’t fully
understand how to design drugs that do not inhibit P450. This
is an important consideration because drug interactions are
likely, if P450 inhibition occurs in vivo at plasma levels close to
efficacious doses. In this session, there were several talks
showing recent advances in our understanding of design
features associated with avoidance of P450 inhibition.
41
What about ’Amyloid aggregation inhibitors’?
Amyloid inhibition is an important area of research
receiving increased attention and we felt that this would be
a topic of interest to the medicinal chemistry community.
Given my earlier point about antiproliferative
strategies, would you say that there is a common
theme to these symposia?
Not really, because topics are voted for national meetings
based on suggestions from division members. We try to
provide a wide range of topics including a balance of new
technologies and therapeutic areas of broad interest. Clearly,
there are sessions devoted to cardiovascular medicine
(Thrombosis), CNS diseases (Amyloid aggregation), design
issues (P450 inhibition) and proliferative strategies. There
were a number of sessions focusing on growth factormediated strategies at the meeting, including ATP-site
directed kinase inhibitors, protein prenylation and
angiogenesis, but we did not select this as a specific theme.
42
American Association For Cancer Research 89th Annual Meeting
Cancer gene therapy
28 March - 1 April 1998, New Orleans, LA, USA
Debabrata Banerjee
Address
Molecular Pharmacology and Therapeutics Program
Sloan-Kettering Institute For Cancer Research
1275 York Avenue
New York
NY 10021
USA
IDrugs 1998 1(1):42-44
The annual meeting of the AACR, the largest meeting devoted to
cancer research, covered all aspects of cancer research currently
being conducted all over the world. The present report attempts to
highlight some of the more interesting presentations dealing with
cancer gene therapy. The field of cancer gene therapy has made
tremendous progress in the last few years, as witnessed by the
burgeoning numbers of proffered papers on the subject. The variety
of presentations, from novel forms of gene therapy in vitro to
clinical trials, attest to the maturity of the field from concept to
practice. This is the first of three AACR reports.
Gene transfer to hematopoietic progenitors
The ’meet the expert at sunrise’ sessions provided an
opportunity to learn about recent progress in the particular
fields thought to be important by the scientific program
committee.
An authoritative lecture on the state of gene transfer to
hematopoietic progenitors was delivered by Dr Mavilio (San
Raffaele Institute, Milan, Italy). Gene transfer to
hematopoietic stem cells (HSCs) will be useful for gene
therapy of immune deficiencies, thalassemias, leukemias,
lymphomas and AIDS, and for protecting the bone marrow
from chemotherapy-induced toxicity during high dose
treatment of solid tumors. Although many viral and nonviral vectors have been developed for successful gene
transfer, HSCs remain particularly difficult cells to transduce. HSCs have been transduced with modest success
using retroviral vectors but not with other vectors such as
adenoviral and adeno-associated viral vectors. Improvements in retroviral vectors are constantly being made in
order to increase the transduction efficiency of HSCs. These
improvements include better pseudotyping of retrovirus
particles with receptors that will possibly result in better
transduction, such as the gibbon ape leukemia virus (GALV)
receptor. Most of the retroviral vectors being used presently
are derived from the Moloney murine leukemia virus whose
genome has been modified to serve as a replication
incompetent, but infective, particle carrying the gene of
interest into the target cell genome.
Dr Mavilio explained that retrovirus infection and
integration requires that the HSCs be in cycle and many
improvements have been made in culture conditions,
including addition of growth factors to stimulate division of
the HSCs ex vivo. On the other hand, it is becoming evident
that certain types of growth factors such as interleukin 3 (IL3), while being able stimulate cell division, somehow
adversely affect the ’stemness’ of the HSCs, ie, the IL-3stimulated cells are compromised in their ability to
repopulate the hematopoietic compart-ments when reintroduced into suitable recipients. Research has indicated that
stimulation with certain growth factors, such as
thrombopoietin and the Flk3/Flt2 ligand, in place of IL-3,
results in equal or better transduction efficiencies with no
loss of ’stemness’ of the HSCs. Improvements in infection
conditions such as the use of fibronectin-coated plates
during the infection process improve the transduction
efficiency. Evaluation of successful gene transfer has
traditionally relied on the use of the neomycin
phosphotransferase gene (neo) to confer resistance to the
antibiotic, G-418, in culture. More recently, a cell surface
marker, the mutated nerve growth factor receptor (mNGFR),
is being increasingly used as it permits enrichment of
successfully transduced cells by fluorescence activated cell
sorter (FACS) technique. One can thus overcome the low
transduction efficiency by sorting successfully transduced
cells.
Since the purpose of gene transfer is to introduce a
therapeutically useful gene, the retroviral vector has to
deliver a second gene besides the selectable marker (neo or
mNGFR), explained Dr Mavilio. The second gene can be
placed in the vector under the control of either a promoter
separate from the one governing transcription of the marker
gene, or the internal ribosomal entry site (IRES) element, to
generate a bicistronic message, using the same promoter as
the marker gene. Success has been reported using both
systems although no systematic comparison has been made.
Several clinical trials involving retrovirus-mediated gene
transfer into HSCs have been conducted. Marking studies
with the neo, as well as the mNGFR gene, have reported
long-term expression in marked cells although the transduction efficiencies have been low.
Two groups have reported on transduction and long-term
expression of the adenosine deaminase (ADA) gene in
severe combined immunodeficiency (SCID) children transplanted with bone marrow stem cells transduced with the
ADA cDNA. Although these children received the ADA
gene, and it was proven to function in the transduced cells
these children have not been taken off the polyethylene
glycol-ADA enzyme therapy which is the standard of care
for SCID children.
Transfer of the multidrug resistance gene, mdr-1, into HSCs
has also been carried out. Although expression of the mdr-1
gene in retrovirally transduced cells has been documented
in vivo, protection against myelotoxicity of chemotherapeutic
drugs, such as taxol, is yet to be demonstrated. There appear
Meeting Report 89th AACR
to be several major hurdles that need to be overcome before
HSC transduction for the purposes of clinical gene therapy
can become a routine treatment. These hurdles include:
efficient transduction; maintenance of self-renewal and longterm stem cell potential, when reintroduced into the
recipient; and regulated and sustained expression of the
introduced gene.
Dr Mavilio also summarized the ongoing efforts of many
laboratories to develop superior retroviral vectors based on
lentiviruses, such as the HIV virus. These viruses are being
re-engineered so that they are not pathogenic and yet retain
their ability to infect hematopoietic cells easily. This theme
was further developed by Dr Luigi Naldini (Cell Genesys
Inc, Foster City, CA, USA) who presented two lectures on
development of lentiviral vectors for in vivo gene delivery.
Third generation lentiviral vectors, derived from the HIV
virus, have recently been developed that are entirely safe
and are efficient for in vivo gene delivery. The current
generation of HIV-based vectors are self-inactivating, lack
almost all viral sequences and yet retain good viral titers.
These vectors have proven to be useful for in vivo gene
delivery, especially by the intraportal vein and have shown
long-term integration and expression of the transferred gene
with no abnormal pathology. These viruses have greatly
reduced chances of generating replication competent
recombinants, as the number of events required to generate
such a recombinant have been increased to an unlikely
number. A major advantage of these vectors over the
conventional Moloney-based vectors is that these HIV-based
vectors can infect non-dividing cells thus eliminating the
need to bring the target HSCs into cycle.
Non-viral gene delivery into hematopoietic
cells
Retroviral vectors have been used for hematopoietic
progenitor cell gene transfer as other methods, including
non-viral methods, have not yet proven useful for such gene
transfer work. This has not prevented laboratories from
exploring non-viral means for transducing HSCs, both
murine and human, for gene therapy purposes.
Two posters described the use of liposome-based delivery
systems for transduction of murine (Dr S-C Zhao, Memorial
Sloan-Kettering Cancer Center, New York, USA) and human
(Dr KA Winters, City of Hope National Medical Center,
Duarte, CA, USA) HSCs. Interestingly both groups used
liposome-based delivery systems to transfer drug resistance
genes into HSCs. Dr Zhao et al compared the efficiencies of
retroviral vectors and liposome-based systems to transfer a
mutated human dihydrofolate reductase gene into murine
bone marrow progenitor cells. They further compared the
myeloprotection, imparted by bone marrow cells transduced
retrovirally, with the myeloprotection imparted by the
liposomally transduced cells in vivo. Their results suggest
that liposome-based (DOTAP formulation; BoehringerMannheim) delivery systems are capable of transducing the
murine HSCs as efficiently as the retroviral vector system.
The second paper presented evidence for successful gene
transfer of a bicistronic cDNA encoding the green fluorescence protein (GFP) and the human Mdr-1 P-glycoprotein
43
into murine bone marrow cells, human KB31 cells and
+
human CD34 cells. In this case the liposome used was the
polycationic liposome DOSPER (Boehringer-Mannheim). Dr
Winters showed that PCR analyses revealed presence of the
GFP and the mdr-1 genes in the transduced cells.
Approximately 50% of the transduced colonies were found
to contain the exogenously delivered genes. More-over,
these colonies were resistant to docetaxel, providing further
evidence of Mdr-1 expression. Immunohistochemical staining
of the colonies growing in presence of drug also indicated that
the transduced cells expressed the P-glycoprotein.
Both these papers suggested that liposome-based delivery
systems need to be re-evaluated for their ability to transduce
bone marrow progenitor cells.
Cancer gene therapy
The papers in this category could be subdivided into the
following: direct antitumor effects as well use in bone
marrow purging to eradicate contaminating tumor cells;
gene therapy by restoring wtp53 activity, direct effect and
chemosensitization; gene therapy by targeting angiogenesis.
These papers were presented in two minisymposia entitled
’Human cancer gene therapy’ and ’Cytoreductive gene
therapy of cancer’.
Gene transfer of the suicide gene cytosine
deaminase
Papers given by Dr SQ Fu (Yale University, New Haven, CT,
USA), Dr M Hirai (University of Nebraska Medical Center,
Omaha, NE, USA) and Dr T Nanakorn (Yale University
School of Medicine, New Haven , CT, USA) all dealt with
purging of tumor cells from bone marrow by using
adenoviral vectors (carrying either cytosine deaminase (CD)
or wt p53 genes) which theoretically should only infect the
tumor cells and not the bone marrow cells. Tumor cells
transduced with, and expressing, the CD gene are then
eliminated by treatment with the prodrug 5-fluorocytosine
(5-FC) which is converted to the cytotoxic drug, 5fluorouracil (5-FU), by enzymatic action of CD. All groups
reported success using either wt p53 or the CD gene for
purging studies. Interestingly, Dr Nanakorn also
demonstrated that bone marrow that has been purged of
tumor cells by the CD-5-FC treatment maintain their ability
to repopulate recipient mice for periods ranging from 4 days
in vitro to 14 days after in vivo purging. This is important
information for investigators wishing to use these systems
for eradicating contaminating tumor cells from bone
marrow samples prior to transplantation.
Gene therapy with wt p53
Two interesting papers presented by Dr J Horwitz and Dr J
Dell (Schering-Plough Research Institute, Kenilworth, NJ,
USA) dealt with preliminary results of a phase I clinical trial
where 64 patients with various cancers including hepatic
metastases of colon cancer, head and neck cancer, ovarian
cancer and melanoma were evaluated for transgene
expression after administration of Ad-p53 (Sch-58500) by
either intrahepatic, intraperitoneal or intratumoral routes.
Transgene expression was documented by reverse
transcriptase-PCR analyses and found to be dose-
44
dependent. With increasing doses of Ad-p53, increasing
numbers of patients were found to express the transgene
and pre-existent antibodies to adenovirus did not affect such
transgene expression.
Dr Dell reported on a different aspect of the same work. It
was observed that introduction of Ad-p53 into tumor cells
increased the chemosensitivity of the tumors to standard
chemotherapy agents including, 5-FU, cisplatin, etoposide,
methotrexate, doxorubicin and paclitaxel. Tumor-bearing
animal models showed enhanced tumor cell kill when the
combination of Ad-p53 and paclitaxel was used, rather than
either agent alone in equivalent doses. True synergism was
demonstrated by means of statistical analyses using
isobolograms for two drug combinations. An explanation
for the observed synergism was the demonstration that
paclitaxel enhanced the uptake of Ad-p53 into tumor cells,
thereby increasing the effective p53 concentration in tumor
cells.
Gene therapy targeting angiogenesis
A rather novel means of using angiogenesis inhibitors for
cancer gene therapy was reported by Dr R Pawliuk
(Harvard Medical School, MA, USA). Murine bone marrow
cells were transduced with retroviral vectors containing a
bicistronic construct encoding either endostatin-IRES-GFP or
angiostatin-IRES-GFP and were transplanted into recipients.
Donor and recipient cells could be distinguished by the
CD45.1 marker on the recipient cells and CD45.2 on the
donor cells. Transduced cells were analyzed by FACS
sorting for CD45.2 and GFP expression. Engraftment of the
recipient cells was demonstrated by detection of dual
positive cells for CD45.2 and GFP for up to 12 weeks post
transplant, with the earliest time of angiostatin/endostatin
production being 5 weeks post transplant. These mice were
then challenged with B16 melanoma cells. No untoward
toxicities were observed and wound healing in the
recipients was not compromised. Mice that received bone
marrow cells transduced with the construct bearing the
endostatin cDNA did not show tumor growth as compared
to control animals or animals receiving cells transduced
with the angiostatin gene. This raised the possibility that
these angiogenesis inhibitors may have different tumor
specificities. The authors went on to discuss future plans
regarding transduction of bone marrow cells with a
construct bearing a fusion protein that produced both
angiostatin and endostatin in the same recipient. In the
discussion following the paper it was pointed out that
systemic bone marrow transduction for the in vivo
production of angiogenesis inhibitors may not be entirely
safe in the long run and a better alternative would be to
implant depot cells producing these inhibitors within
hollow fibers or capsules, so that they can be removed once
the need for production of these angiogenesis inhibitors in
vivo is over.
Targeting tumor angiogenesis directly by introducing the
suicide gene, HSV-TK, into the tumor-associated endothelial
cells and killing them by ganciclovir (GCV) treatment, was
discussed by Dr T Tanaka (Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical
School, Boston, MA, USA). Tumor-associated endothelial
cell kill was found to be dose- and MOI- (multiplicity of
infection) dependent. The HSV-TK transduced cells
continued to grow in culture, as indicated by positive PCNA
(proliferating cell nuclear antigen) staining, thus ensuring
sensitivity to GCV which can kill only dividing cells.
HUVEC (human vascular endothelial cells), a popular cell
system with investigators in the angiogenesis field, were
shown to be particularly sensitive to the HSV-TK/GCV
combination therapy.
Cancer gene therapy has come of age.
45
Cell-based gene therapy and late-breaking news
Ezzie Hutchinson
Address
Middlesex House
London
W1P 6LB
UK
IDrugs 1998 1(1):45-46
New concepts in cell-based gene therapy
Dendritic cells pulsed with prostate tumor
antigens
A phase II trial with prostate cancer patients, using
dendritic cells pulsed with prostate tumor antigens, was
described by Dr Michael Salgaller (Northwest Therapeutics,
WA, USA). Firstly, dendritic cells, a type of antigenpresenting cell, were isolated from blood samples taken
from patients with very advanced prostate cancer. These
cells were then exposed to prostate tumor antigens
(prostate-specific mem-brane antigens-P1 and P2) in cell
culture, and injected back into the patients in six infusions
given every six weeks. Half of the patients also received
granulocyte-macrophage colony stimulating factor (75
µg/m2/day), to help boost the immune response. So far,
104 patients have been enrolled and 33 have completed
therapy. Significant acute or chronic toxicity was not
observed in any of the patients. Of the evaluable patients
27% achieved partial response, while 33% had stable
disease. Some of these responders have lived to two years,
which is impressive considering that the average survival
time for these patients is only 6 to 12 months.
SC-1 antibody
Preliminary results of a phase I/II trial using a human
monoclonal IgM antibody, SC-1, as an adjuvant therapy to
treat gastric cancer, was reported by Professor H Peter
Vollmers (Institute for Pathology, University of Wuerzburg,
Germany). The SC-1 antibody was originally isolated by
extracting B-cells from the spleen of a patient with a diffuse
type adenocarcinoma of the stomach and testing the
antibodies produced by these cells for antitumor activity.
SC-1 was demonstrated to induce apoptosis of tumor cells.
In the phase I/II clinical trial, 40 patients with poorly
differentiated adenocarcinoma have been injected iv with
20 - 30 mg purified SC-1 two days prior to gastrectomy. Of
these patients, 90% showed apoptosis in their tumor, 60%
showed tumor regression and 80% had tumor-free lymph
nodes, as opposed to only 10% in the control group.
Adenovirus wt p53
A phase I study of replication-deficient, E1 deleted
adenovirus re-engineered to deliver normal p53 (Sch-58500)
into patients with various types of cancer, was reported by
Dr M Rybak (Schering-Plough, NJ, USA). The rationale is
that injection of functional p53 into cancer cells expressing
mutant p53 will induce the cells to undergo apoptosis. Sch58500 was administered intratumorally in brain cancer and
head and neck cancer, intraperitoneally in ovarian cancer,
and via the hepatic artery in colon cancer. 74 Patients have
had a single administration of Sch-58500, ranging from 7.5
x 109 to 7.5 x 1012 particles/dose. Transgene expression was
evaluated in 69 tumors using a reverse transcriptase-PCRbased assay. Results showed that 33 tumors expressed the
transgene and expression increased with increasing doses
received.
Her2/neu inhibition by E1A
Dr Naoto Ueno (MD Anderson Cancer Center, TX, USA)
described exciting work carried out with colleagues from
Targeted Genetics and the University of Pittsburgh. Her2/neu is an oncogene overexpressed in a number of
cancers, in particular, 20 to 30% of breast and ovarian
cancers overexpress this oncogene. Expression of Her-2/neu
is correlated with poor prognosis, increased tumor
formation, increased metastasis and resistance to chemotherapeutic agents. The adenoviral tumor suppressor gene,
E1A, is able to inhibit the promoter activity of Her-2/neu,
thus downregulating expression. Delivery and expression
of an E1A gene in human cancer and normal cells using a
propri-etary liposomal delivery mechanism has been
demonstrated. Dr Ueno outlined the phase I dose-escalation
study which is underway with 12 patients with metastatic
or recurrent breast and ovarian cancers. Patients are given
weekly intra-abdominal injections of the E1A gene cationic
liposome complex over 4 to 6 weeks. The first three patients
have been evaluated and show E1A gene expression in both
cancer and normal cells. These patients also exhibited
decreased levels of Her2/neu and surrogate tumor markers.
The maximum tolerated dose has been identified and phase
II trials are planned which are hoped to confirm the
antitumor activity of the E1A gene.
Late-breaking news
This session consisted of papers submitted after the closing
date of October 1997, but were included at the conference
due to their significant interest. The abstracts for these
papers, therefore, are not to be found in the AACR abstract
book (AACR Proceedings (1998) 39).
Arsenic trioxide
A presentation on the use of arsenic trioxide (As2O3) for
treating cancer patients was given by Dr S Soignet
(Memorial Sloan-Kettering Cancer Center, NY, USA).
Arsenic was originally discovered to be of use as a drug in
the 1800s, but the promise of efficacy in treatment of
leukemias is a very recent finding. Arsenic trioxide homes
in on sulfahydrols present in cancer, and induces apoptosis
in these cells. In preclinical studies, Dr Soignet explained
that As2O3 exhibited unexpectedly broad anticancer activity
against a variety of acute and chronic myeloid and
lymphoid cell lines. This drug was able to induce apoptosis,
without the presence of the promyelocytic leukemia (PML)
46
gene, in a variety of myeloid cell lines. In a phase I study of
patients with relapsed and/or refractory acute promyelocytic leukemia (APL), all of whom had received extensive
prior treatment with all-trans retinoic acid or cytotoxic
chemotherapy, were treated with daily infusions of 0.07 to
0.21 mg/kg As2O3 until a bone marrow complete remission
was achieved, up to a maximum of 60 days. Responding
patients then received an additional 25 days of therapy after
a rest of 4 to 6 weeks. So far, of seven patients evaluable for
response, all have achieved complete remissions ranging
from 14 to 39 days. Four of these patients no longer showed
the characteristic markers for APL as demonstrated by
reverse transcriptase-PCR. The drug was also very well
tolerated. Following these striking results, an international
multicenter study will be initiated using a daily dose of 0.15
mg/kg.
CGP-57148B
CGP-57148B (Novartis), a competitive inhibitor of a protein
kinase, was the subject of a paper by Dr P le Coutre
(Instituto Nazionale Tumori, Milan, Italy). This compound
specifically inhibits the kinase which blocks proliferation of
human bcr/abl+ leukemic cells and induces apoptosis in
vitro. Nude mice received CGP-57148B, 50 mg/kg ip or 160
mg/kg po, every 8 h for 11 days. 87.5% control animals
developed leukemia within 10 days, whereas 87.5% ip
treated animals and 100% po treated animals remained free
of cancer for more than 90 days. The toxicity of this
treatment was low.
Designer T-cells
A new therapy to target tumor cells expressing carcinoembryonic antigen (CEA), a tumor-associated antigen, was
discussed by Dr RP Junghans (Harvard Medical School,
MA, USA). Preclinical evaluation has produced convincing
data which has led to an IND approval by the FDA. This is
the first anticancer gene therapy to be supported by the
National Gene Vector Laboratory at NIH. In the phase I
trial, which has recently begun, patients' T-cells are
collected
and
retrovirally
transduced
with
an
immunoglobulin T-cell receptor construct which has
fragments of a humanized anti-CEA antibody attached to it.
The T-cells are then expanded in culture and re-infused into
the patients. The hypothesis is that on contact with CEA+
tumor cells, the anti-CEA designer T-cells will be activated,
leading to IL-2 production and lysing of tumor cells by
cytotoxic T-lymphocytes.
Recombinant Listeria monocytogenes expressing
E6 and E7 proteins
Cervical cancer is often associated with human papilloma
virus (HPV) infection. More than 90% of HPV-16 transformed cervical cells express the viral proteins, E6 and E7,
constitutively, so these proteins make ideal targets for a
cervical cancer therapeutic. Dr A Zubair (University of
Pennsylvania, PA, USA) presented in vitro data to show
that a recombinant Listeria monocytogenes, which expresses
and secretes E6 and E7 proteins, can elicit a potent E7
restricted cytotoxic T-cell response against murine tumor
cells. Study of the ability of the vaccines to protect mice
from transplanted E6 and E7 expressing tumors is ongoing.
Telomerase inhibitors
This year at the AACR, as last year, there was much
excitement about telomerase as a target for anticancer drug
development. A recent paper in Science (Bodnar AG:
Science (1998) 279(5349):349-52) showed that elongated
telomeres cause a huge increase in the life-span of cells in
culture. In cancer, activation of telomerase causes
elongation of telomeres which enables a cell to by-pass
cellular
senescence. Telomere shortening is thus a powerful tumor
suppressor mechanism. Now that the controversy over the
role of telomerase has been largely solved, researchers
should be able to move forward in the quest to find
telomerase inhibitors. A paper presented by Dr D Corey
(University of Texas Southwestern Medical Center, TX,
USA) in the late-breaking news session, discussed the
development of a new class of agents to inhibit telomerase.
The 2'-O-methyl RNA oligonucleotides have IC50 values as
low as 1 nM, explained by the fact that these inhibitors have
very slow rates of dissociation from the telomerase
molecules. These potent sequence-specific inhibitors are
promising lead compounds for drug development.
Final news briefing
Professor Donald Coffey (President AACR 1998, Johns
Hopkins University School of Medicine, MD, USA)
concluded the meeting by selecting the papers from the
conference which he thought were the most exciting. The
first paper he discussed involved research on dendritic cells
pulsed with prostate tumor antigens by Dr M Salgaller,
mentioned above under 'New concepts in cell-based
therapy'. He also found the paper on arsenic trioxide by Dr
S Soignet, outlined in the late-breaking news section, of
particular merit.
One of the major goals of current cancer research is to
develop targeted chemotherapy strategies. A method for
targeting endothelial cells in prostate cancer was described
by Dr E Ruoslahti (The Burnham Institute, CA, USA) in this
final news briefing. Endothelial cells in blood vessels within
tumors are a promising target as they do not mutate like
the cancer cells themselves. In vivo selection of phage
display libraries was used to isolate peptides which bound
specifically to endothelial cells. The anticancer drug,
adriamycin, when coupled to these peptides, demonstrated
increased efficacy against human prostate cancer xenografts
in nude mice, as well as reduced toxicity.
Results of a study of the role of insulin-like growth factor-I
(IGF-I) in prostate cancer were presented by Dr JM Chan
(Harvard School of Public Health, MA, USA) and
published earlier this year (Chan JM: Science (1998)
279(5350):563-6). Dr Coffey summarized this interesting
paper. A nested case-control study was conducted on
prospectively collected plasma from cancer patients and
healthy volunteers. It was found that the risk of developing
prostate cancer increases 4-fold with high IGF-1 levels. This
early marker is possibly as good as prostate-specific antigen
(PSA) as a detector of prostate cancer cells. Antibodies to
IGF-1 are already available, so possible use of this factor as
therapy could be studied.
Conclusion
Donald Coffey ended the news briefing by noting that this
AACR, organized by Frank Rauscher III, had been a
particularly exciting one. The number of new drugs which
are reaching the clinic for cancer treatment is expanding
rapidly: a few years ago 30 to 50 new drugs would enter
the clinic per year, whilst this year the number will be 320.
47
47
Highlights of selected symposia
Ann F Chambers
Address
Department of Oncology
University of Western Ontario
790 Commissioners Road East
London
Ontario N6A 4L6
Canada
IDrugs 1998 1(1):47-48
trials in establishing optimal treatments in breast cancer. Ernst
Wynder (American Health Foundation, New York, NY, USA)
was the recipient of the American Cancer Society Award for
Cancer Epidemiology and Prevention. Dr Wynder presented
a summary of his research, beginning in the 1950s,
identifying smoking as a significant risk factor in lung
cancer, and his systematic and persistent efforts in
establishing smoking as a primary cause of lung cancer.
Tumor angiogenesis and microcirculation
The AACR annual meeting was attended by approximately 8000
international scientists, and consisted of more than 200 invited
presentations, a plenary session, 28 symposia, two controversy
sessions, multiple "meet-the-expert" sessions, two methods
workshops and several educational sessions. In addition, more than
4500 submitted abstracts were accepted for presentation in 34
minisymposia, 30 poster discussion sessions and 109 poster
sessions. The submitted abstracts are published in the 1998 AACR
Proceedings (1998) 39, and are also available online at the AACR
website (http://www.aacr.org). More than 30 organizations and
foundations provided support for a variety of the sessions. In
addition, over 250 commercial firms and other organizations
exhibited this year. This report is thus, by necessity, only a small
snapshot of selected portions of this large meeting.
Major lectures and awards
The meeting was opened with the presidential address, given
by the outgoing AACR President, Donald Coffey (Johns
Hopkins University School of Medicine, Baltimore, MD, USA)
and was entitled "Cancer cell structure: aberrations in the
decoder of DNA". Dr Coffey reviewed his and others’ work on
the role of nuclear matrix structure in regulating gene
expression in a tissue-specific fashion, with examples drawn
from changes that occur during progression from benign
hyperplasia to cancer of the prostate, and described the
importance of nuclear morphology functionally, in regulating
gene expression, as well as a prognostic indicator. Speakers in
the plenary session (sponsored by Novartis) included Erkki
Ruoslahti (The Burnham Institute, La Jolla, CA, USA), who
described a strategy of using in vivo phage display to identify
av integrin-binding peptides, for use in specific targeting of
cytotoxic drugs, such as doxorubicin, to endothelial cells in
newly formed vessels within tumors. Also in the Plenary
Session, Waun Ki Hong (MD Anderson Cancer Center,
Houston, TX, USA) summarized studies on strategies for
chemoprevention using retinoids in patients at high risk of
developing initial or second tumors of the aerodigestive
system, including studies showing phenotypic reversion
accompanied by persistence of "genetic scars" (eg, chromosomal changes such as loss of heterozygosity) in the tissue.
Bernard Fisher (Allegheny University of the Health Sciences,
Pittsburgh, PA, USA) was the recipient of the Joseph
Burchenal Clinical Research Award (sponsored by a grant
from Bristol-Myers Squibb Oncology). Dr Fisher summarized
the history of the development of current concepts on breast
cancer as a systemic disease, and the crucial role of clinical
This symposium, chaired by Rakesh Jain (Massachusetts
General Hospital, Boston, MA, USA) and sponsored by
Janssen Pharmaceutica and Janssen Research Foundation,
presented new information on the role of angiogenesis in
cancer and described possible therapeutic approaches. George
Yancopoulos (Regeneron, Tarrytown, NY, USA) summarized
the cloning of angiopoietin genes, Ang-1 and Ang-2, which can
function as activators and antagonists, respectively, of the Tie2 receptor gene in regulating vessel development, as well as
Ang-3 (in mouse) and Ang-4 (human). Dr Jain described
results from transgenic mice which overexpress Ang-1 in skin
(giving rise to hyper-vascularity), Ang-2 knockout mice
(which have fragile and leaky vessels), and Ang-3 knockout
mice (currently being characterized). The relationship between
angio-genesis and oncogenes, such as ras, was summarized by
Robert Kerbel (Sunnybrook Health Science Centre, Toronto,
Canada). He stressed the importance of cell growth in threedimensional versus monolayer culture. Studies on the
microvasculature within tumors, and the roles of both the
target organ and the tumor type in determining vessel
properties, such as permeability to macromolecules, were
presented by Dr Jain. The role of interferons in regulation of
angiogenesis and tumor metastasis was discussed by Isaiah J
Fidler (MD Anderson Cancer Center, Houston, TX, USA). He
described how angiogenesis can be affected by a balance
between factors that promote angiogenesis and those that
inhibit it, and stressed that while acute diseases may be
appropriately treated with acute therapies, metastasis, as a
chronic disease, may be more appropriately treated with
chronic therapies, such as those limiting angiogenesis. Finally,
James Pluda (National Cancer Institute, Rockville, MD, USA)
summarized the status of a series of anti-angiogenic
compounds and strategies that are in, or will soon enter,
clinical trials. He also described an evolving concept of how
anti-angiogenic strategies may be used, from the initial hope
that they would be useful as adjuncts to stabilize tumor
growth after chemotherapy, to their potential use as inducers
of tumor dormancy, to the current belief that anti-angiogenic
strategies may have a possible role as front-line therapies with
the potential for complete tumor eradication.
Extracellular matrix proteases and invasion
This minisymposium was chaired by Lynn Matrisian
(Vanderbilt University School of Medicine, Nashville, TN,
USA). Ruth Muschel (University of Pennsylvania School of
Medicine, Philadelphia, PA, USA) summarized much of the
current research on the role of proteases in cancer growth,
48
invasion and metastasis. She presented an overview which
focused particularly on the matrix metalloproteinases
(MMPs), their association with malignancy, and data
suggesting that they may play functional roles in tumor
invasion and metastasis.
Results indicating that neutrophil-derived serine prote-inases
can activate proMMP-2 produced by endothelial cells, and
that this process requires the membrane-type MT-MMP, were
described by J Schwartz (New York University Medical
Center, NY, USA). A Strongin (La Jolla Institute for Experimental Medicine, La Jolla, CA, USA) described how MT1MMP and the αvβ3 integrin cooperate in the activation of
MMP-2. Studies suggesting that the propeptide region of
MT1-MMP is important in the activation of proMMP-2, and
that this region also is involved in tissue inhibitor of
metalloproteinase-2 (TIMP-2) binding were presented by J
Cao (SUNY Stony Brook, NY, USA). Using stromelysin-1 (Str1) null mice, Howard Crawford (Vanderbilt University School
of Medicine, Nashville, TN, USA) assessed the role of Str-1 in
tumorigenesis, and reported an increase in tumor formation
and metastasis following chemical induction of tumors,
supporting the idea that host stromal cell expression of Str-1
can restrict tumor growth. Results from in vivo videomicroscopy and histology, showing that the MMP inhibitor,
batimastat (BB-94; British Biotech), inhibited growth of liver
metastases of murine melanoma cells by inhibiting
angiogenesis and not by inhibition of extravasation, were
presented by Ann Chambers (London Regional Cancer
Centre, Ontario, Canada). Finally, JM Müller (Novartis, Basel,
Switzerland) presented results using the MMP inhibitor, CGS27023A, which showed that this compound had antitumor
and anti-angiogenic activity in vivo and anti-angiogenic
activity in vitro; however, development of this compound has
been discontinued.
Stromal-epithelial, cell-cell, and cellextracellular matrix interactions in normal
development and cancer
This symposium, chaired by Mina Bissell (Lawrence
Berkeley National Laboratory, Berkeley, CA, USA),
examined the interactions between different cell types in the
context of three-dimensional tissues. Dr Bissell summarized
her studies on the importance of the microenvironment in
gene expression and behavior of mammary epithelial cells,
and proposed the idea that 'form is the ultimate regulator of
gene expression'. Keld Dano (Finsen Laboratory, Copenhagen, Denmark) reviewed his research on components of
the plasminogen activator (PA) cascade (urokinase-type PA,
urokinase-type PA receptor and PA inhibitor-1), including a
description of clinical studies, where patterns of expression
in tumor versus stromal tissue of various components of the
cascade are characteristic of specific tumor types. Lynn
Matrisian (Vanderbilt University School of Medicine, Nashville, TN, USA) summarized research from her laboratory on
the role of various MMPs in cancer, and also described
studies with the multiple intestinal neoplasia (min)
adenomatous polyposis coli (APC)-mutated mouse, in which
colocalization of matrilysin and increased β-catenin was
found in adenomas, raising the possibility that matrilysin is
a downstream gene in the APC signal pathway.
Models and methods for invasion and
metastasis
This poster discussion session was chaired by Meenhard
Herlyn (The Wistar Institute, Philadelphia, PA, USA) and
Mary Hendrix (University of Iowa College of Medicine, IO,
USA). A quantitative study on metastatic inefficiency of
melanoma cells in mouse liver, using in vivo videomicroscopy, showed that the rate-limiting steps in this model
were not cell survival in the circulation or failure to
extravasate, but failure of most extravasated cells to begin
and continue growth, was presented by Vincent Morris and
Ann Chambers (University of Western Ontario, London,
Canada). The generation and characterization of a series of
cancer cell lines transfected to stably express green
fluorescent protein, for use in studying tumor metastasis
was described by Robert Hoffman (AntiCancer, San Diego,
CA, USA). In two presentations, T Tachikawa, Y Tamura
and colleagues (Fuji, Tokyo, Japan) described procedures
they are developing to assess MMP activity using film-based
in situ zymography. Finally, Claus Kristensen/Rakesh Jain
(Massachusetts General Hospital, Boston, MA, USA)
described an in vivo study in mice, in which cells shed from
a primary tumor were collected and assessed for in vitro
clonogenic ability; the shed cells were found to be less
clonogenic in soft agar than the parental tumor cells.
Alternative research models for cancer: case
studies for consumer involvement
This unique session explored various aspects of the changing
role of cancer patients, advocates and survivors in research
and public policy. The session was introduced by Donald
Coffey, outgoing AACR President, who stressed the crucial
and growing role played by such advocates in many aspects
of academic and corporate cancer research, and cancer
research funding. Frances Visco (National Breast Cancer
Coalition, Washington DC, USA) chaired the session. The role
played by Frances Visco and the coalition in lobbying for
significant funding for breast cancer research was described
by Anna Barker (Oxis, OR, USA). The positive role played by
patients and advocates in Genentech's development and
clinical trials of the company's anti-Her2 antibody for breast
cancer treatment was described by Susan Hellman
(Genentech, CA, USA). Finally, Carol Hochberg (SHARE/SelfHelp for Women with Breast or Ovarian Cancer, NY, USA), a
breast cancer survivor, shared her own personal experiences
in dealing with cancer.
Conclusion
This report can give only a small suggestion of the
presentations available to the participants. The AACR website
can be consulted for details on any of the submitted abstracts.
The presentations spanned basic and clinical aspects of
oncology, and provided information for researchers interested
in nearly any aspect of current oncology research.
Meeting Report 9th Med Chem East England
49
Medicinal Chemistry in Eastern England - Ninth Symposium
20 April 1998, University of Hertfordshire, Hatfield, UK
Peter Norman
Address
Norman Consulting
18 Pink Lane
Burnham
Bucks
SL1 8JW
UK
IDrugs 1998 1(1):49-54
This meeting is sponsored by the Royal Society of Chemistry’s
(RSC) Biological and Medicinal chemistry sector and is now an
annual event. Around 100 delegates representing a broad
spectrum of the British pharmaceutical industry attended to hear
lectures from seven invited speakers. The meeting included the
presentation of the RSC’s Biological and Medicinal Chemistry
Sector award to a team from SmithKline Beecham, UK, for their
work on 5-HT4 antagonists, especially SB-207266. This was
presented by the group’s chairman, Dr Derek Buckle, to Dr Paul
Wyman.
Transition state analogs as cytosolic
phospholipase A2 inhibitors
Cytosolic PLA2 is believed to the primary form of
phospholipase (PLA2) involved in the pathogenesis of
inflammatory disease. It selectively liberates arachidonic acid
from phospholipids, and recent studies in knockout mice have
implicated a role in the induction of airway hyperreactivity.
Dr Tony Meete (Astra Charnwood, UK) described progress
towards the identification of transition state analogs of PLA2
as inhibitors. High-throughput screening (HTS) has so far
failed to identify any potential lead structures, but a
designed inhibitor - AR-C69611 (1-(4-decyloxyphenoxy)-3methoxypropan-2-one; Figure 1) - was described (IC50 = 5
µM).
Replacement of the methoxy group with substituted phenyl
rings, especially p-benzoic acid (Figure 1), led to compounds
with IC50 values in the 100 nM range, and activity in HL60
cells at 2 µM, but very poor aqueous solubility. Replacement
of the decyloxy group by phenylalkoxy or phenylalkylthio
groups yielded comp-ounds with better solubility and
comparable potency. Enzyme activity was found to be a
function of drug lipophilicity. AR-C73346 (4-(2-oxo-3-[4-(5phenylpentyl-thio)phenoxy]-propoxy)benzoic acid; Figure
1) was the most potent compound described (IC50 = 77 nM).
The presence of both the proximal phenyl ring and the
ketone is critical to activity, and replacement of either of the
ether linkages reduced, or abolished, activity. Enhanced
potency was achieved by oxidation of the thia linkage to a
sulfone, to give a compound, AR-C71089 (Figure 1), with an
13
IC50 of 38 nM. Studies with [ C]-labeled inhibitor confirmed
that the bound form is the hemiketal and that the binding is
reversible. It was shown that inhibition is not timedependent, and that both on and off rates are rapid.
This class has no effect against 5-lipoxygenase or cyclooxygenase, but the compounds inhibit the release of both
prostaglandins and leukotrienes in whole cell assays, with
potencies slightly lower than against isolated enzyme. Data
were presented on one compound demonstrating inhibition
of leukotriene B4 production in rat whole blood ex vivo at 2
µM, and inhibition of TPA-induced inflammation in the
mouse ear. More potent compounds than those described
are apparently under development for rheumatoid arthritis
and asthma, and are active in rat paw edema models of
inflammation.
Orally active carbapenems
Carbapenems offer a potential means of addressing the
problems of increasing antibiotic resistance in communityacquired infections. The currently available carbapenems
(iminipenem and meropenem) are unsuitable for oral
administration, partly because of their susceptibility to
cleavage by renal dihydropeptidase-1 (DHP-1). Dr Steve
Coulton (SmithKline Beecham, UK) described the outcome of
a program to identify orally active carbapenem derivatives.
SB’s approach was first to identify a potent carbapenem with
good stability against DHP-1 and second to prepare suitable
crystalline prodrugs which were well absorbed. 2-Phenylcarbapenems are known to be stable to DHP-1, so a range of
heterocycles as the 2-substituents was investigated. The
synthesis of such penems has recently been published in the
Journal of Antibiotics (1998) 51:210.
From a range of 5-membered rings, only 2,3-disubstituted
pyrazole-5-yl derivatives were found to combine the desired
potency with sufficient stability against DHP-1 cleavage.
Five compounds, substituted with methyl, ethyl or
hydroxyethyl groups, were evaluated in vivo. SB-221691 (6(1-hydroxyethyl)3-([2-(2-methyl-3-ethylpyrazol-5-yl)-7-oxo1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid; Figure 2)
showed the best duration of action and superior in vivo
activity to tribactam in a rat model of respiratory tract
infection.
The compound had good potency against resistant strains of
Streptococcus pneumoniae and Haemophilus influenzae (MIC90
values of 1 and 0.25 µg/ml) and good potency against
Staphylococcus aureus (S aureus), Streptococcus pyogenes and
Escherichia coli; oral absorption was, however, poor (8%).
Two crystalline ester derivatives of the hydroxymethoxy
ester of SB-221691 were described. The bioavailability of SB223328 (the cyclohexyloxycarbonyl ester; Figure 2) and SB224617 (the isopropoxycarbonyl ester; Figure 2) in the rat
was 45 and 41%, respectively. Both compounds were as
potent as augmentin in a rat model of respiratory tract
infection, at 5 and 50 mg/kg, respectively. Like meropenem
50
Figure 1. Astra PLA2 inhibitors
O
O
O
R1
R2
X
AR-C70484 =
R1
CH3(I)
R2
CH3(CH2)9CH3(CH2)9-
AR-C73346 =
(I)
AR-C71089 =
(I)
Ph(CH2)5CH3(CH2)9-
AR-C69611 =
X
O
O
OH
S
SO2
(I)
O
Figure 2. Carbapenems
OH
OH
H
H
H
CH3
H3C
N
N
O
HO
H
CH3
H3C
N
N
CH3
N
O
O
O
O
SB-221691
O
N
CH3
O
O
SB-223328
OH
H
H
CH3
H3C
N
N
O
CH3
H3C
O
O
O
N
CH3
O
O
SB-224617
they are inactive against methicillin-resistant S aureus. Both
compounds are currently under clinical evaluation.
ZD-1839 - an EGF receptor-tyrosine kinase
inhibitor
The EGF receptor-tyrosine kinase is heavily expressed in a
variety of cancer cells. Dr Keith Gibson (Zeneca, UK)
described the identification of a novel, reversible, inhibitor
of this kinase which is now in phase I trials. 4-Arylamino-
quinazolines were initially identified as lead structures by
3D structural searching based on a transition state model,
which was later found to be erroneous. Simple derivatives
such as 4-(3-methyl-phenyl)aminoquinazoline (Figure 3) had
reasonable potency in both enzyme and cellular assays (IC50
= 0.18 and 2.4 µM, respectively). Such compounds were
readily prepared in four steps, and robotic synthesis was
used to generate a compound library to delineate the SAR of
the series. The presence of the secondary amine was critical,
and 6- and/or 7- substitution with electron-donating groups
51
Figure 3. Zeneca tyrosine kinase inhibitors
F
HN
CH3
O
HN
N
O
N
N
screening lead from HTS
(Zeneca)
Cl
N
H3C
O
N
ZD-1839
(Zeneca)
enhanced the activity. The 6,7-dimethoxy derivative was the
most potent (IC50 = 5 nM), but was rapidly metabolized in
vivo. Replacement of the toluyl group by a 3-chloro-4fluorophenyl ring improved the pharmacokinetic properties
but compounds were only moderately effective in a tumor
xenograft model.
action. Re-introduction of the carboxyl group gave UK142773 ([3-(3-aminoiminoph-enyl)-2-[2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)sulf-onyl]amino-1-oxopropyl]-4-methyl-2-pipe-ridinecarboxylic acid; Figure 4). This compound was
well-tolerated, orally active and 18-fold selective for
thrombin (Ki = 3.1 nM) compared to trypsin.
Introduction of an amino functionality in the 6-substituent
dramatically improved absorption. Data were presented on
a variety of substituents, and whilst potency was greatest
with methylamino or dimethylamino substituents, drug
absorption was better with 6-aminopropoxy substituents.
ZD-1839 (4-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)-quinazoline; Figure 3) has IC50 values of
23 and 80 nM in enzymatic and cellular assays, respectively,
and has little activity against a range of other kinases (eg,
IC50 = 1.2 µM against erbB2). It displays 30-fold selectivity
for EGF relative to other growth factors in human umbilical
vein endothelial cell assays.
UK-156406 (Figure 4) employs a flattened piperidine ring - 4methyl-1,2,5,6-tetrahydropyridine-6-carboxylic acid - and is a
more potent compound (Ki values for thrombin and trypsin =
0.26 and 26 nM, respectively). The compound had an
improved duration of action, due to reduced hepatic clearance, by both iv and id routes. It has a bioavailability of 45% in
the dog and 15% in the rat; plasma half life = 0.8 h in the dog.
In man there was a dose-dependent inhibition of ex vivo
thrombin time at doses of 25 to 200 mg. Doses of 10 mg were
ineffective, but higher doses had a duration of action of 4 to 5
h, and were well-tolerated. The compound is now in clinical
trials.
ZD-1839 produces sustained plasma levels (5.7 µM 24 h after
a dose of 200 mg/kg). In mice it is orally effective at doses as
low as 12.5 mg/kg and will inhibit tumor growth or cause
regression of existing tumors (eg, AS49) over a sustained
period. On cessation of drug treatment, tumor regrowth
occurs.
A back-up compound was prepared by designing compounds
to interact with the lipophilic cleft between Ala-190 and Val213. Replacement of the benzamidine group by a 1-aminoimino-1,2,5,6-tetrahydropyridin-3-yl group gave a compound
(UK-239326; Figure 4) with dramatically improved potency
and selectivity and a similar profile of action in vivo.
UK-156406 - an orally active thrombin
inhibitor
Large scale synthesis of chiral reagents
Dr Alan Brown (Pfizer, UK) described the development of
an orally active thrombin inhibitor which is now in clinical
evaluation. Compounds were derived from the structures of
the thrombin inhibitors argatroban and MD-205, and X-ray
data of the argatroban-thrombin complex. The X-ray data
pointed to an interaction with the Trp-60 portion of the
enzyme and to a hydrophobic collapse of the quinoline ring
onto the piperidine ring.
These initial approaches led to the introduction of
benzamidine rings, and to the selection of a 2-methyl-1,2,3,4tetrahydroisoquinolin-7-yl ring in place of the tetrahydroquinoline ring of argatroban. Early compounds were poorly
selective and short-acting. Introduction of a 4-methyl group
in the piperidine ring gave a compound (UK-141364) which
was poorly tolerated but had a reasonable duration of
Dr Jon Cummins (ChiRex) addressed some of the problems
of obtaining large quantities of chiral reagents without using
long and/or expensive synthetic routes. In particular, he
illustrated how the catalysts recently described by Jacobsen
may be applied to prepare, or resolve, chiral epoxides (eg,
epichlorohydrin) and diols (eg, propanediol) in multikilogram quantities in high yield (> 98%). These catalysts are
reusable and he predicted that the processes can be applied
on the tonne scale.
A novel serine protease pharmacophore [5,5]trans-fused lactones and lactams
Dr Harry Kelly (Glaxo Wellcome, UK) described studies on
[5,5]transfused lactones as inhibitors of the serine protease,
thrombin. The program has generated orally active
inhibitors of elastase and potent, orally active thrombin
inhibitors with encouraging pharmacokinetics.
52
Figure 4. Pfizer thrombin inhibitors
HN
HN
NH2
NH2
H
N
N
O
CH3
N
S
H3C
O
H
N
N
O
HO
CH3
N
S
H3C
O
O
O
HO
O
O
UK-142773
(Pfizer)
UK-156406
(Pfizer)
HN
NH2
N
H
N
N
O
CH3
N
S
H3C
O
O
HO
O
UK-239326
(Pfizer)
An HTS program identified a terpenoid (GW-133686), found
in wild sage, as a potent inhibitor of thrombin (IC50 = 4 nM)
and the ability to double activated partial thromboplastin
time at 40 µM. This compound was less potent as a trypsin
inhibitor (IC50 = 70 nM), and had a sustained duration of
action in both rat and human plasma. The compound
contains the unusual [5,5]trans lactone system substituted in
the 3-position by a 2-methyl-4-oxo-pent-2-en-5-yl substituent.
The presence of the enone is critical to activity, and ring
opening of the lactone reduces activity by 400-fold. X-ray
crystallographic studies on the interaction of GW-133686
with thrombin have shown that Ser-195 opens the lactone
ring (Biochemistry (1998), in press).
The structural complexity of this lead led to the exploration
of the activity of 3-substituted-[5,5]trans lactones. The parent
compound had little activity against thrombin but was
active against trypsin, whilst 3-m-thiophenyl or 3-enone
substituted compounds had IC 50 values of 20 µM (Figure 5).
The introduction of amidine groups into the side-chain
improved the potency, with the 5-amidinopentyl derivative
having an IC50 of 130 nM (Figure 5).
Potency was further improved by preparing [5,5]trans
lactones of indanes with 4- or 5-diethylcarbamoyl
derivatives. The resulting compounds had IC50 values of 8 to
11 nM (Figure 5), but these compounds had poor stability in
plasma. Replacement of the ring oxygen by nitrogen
provided lactams with better stability but poorer potency.
Potency was restored by introduction of N-substituents,
with the N-carbomethoxy derivative having an IC50 value of
16 nM (Figure 5) and high stability in human plasma.
Further efforts, which were not described, have
concentrated on varying the S-2 and S-3 regions of the
inhibitors. This has resulted in significant potency improvements and permitted the identification of potent inhibitors
of other serine proteases. Thus the [5,5]trans lactone/lactam
group is a novel pharmacophore for serine proteases in
general.
53
Figure 5. Glaxo Wellcome serine protease inhibitors
NH
H3C
CH3
O
NH2
O
S
O
O
IC50 = 250 µM
O
O
O
lead pharmacophore
IC50 = 21 µM
O
O
IC50 = 20µM
IC50 = 130 nM
NH
NH
NH2
N
H3C
CH3
O
O
NH2
N
H3C
CH3
O
O
O
N
O
CH3
O
IC50 = 8 nM
IC50 = 16 nM
Figure 6. Knoll antidepressants
O
O
N
N
CH3
N
N
N
N
N
H2N
N
O
H2N
CH3
O
O
O
O
BTS-78554
(Knoll)
BTS-79524 - a potential antidepressant with
5-HT1A agonist and α2A antagonist activity
Dr Frank Kerrigan (Knoll, UK) outlined the need for
novel antidepressants, and briefly described how
sibutramine, a monoamine uptake inhibitor, had been
identified and originally developed as an antidepresssant.
However, when evaluated clinically it caused significant
weight loss and has subsequently been developed, and
approved in the US, as an anti-obesity drug.
BTS-79524
(Knoll)
The company’s alternative strategy for the identification
of novel antidepressants has focused on drugs that
combine 5-HT1A agonist and α2A antagonist activity. Such
compounds synergistically inhibit synaptic neurotransmission. The original lead structures were based on the
pyrimidinyl-piperazine portion of buspirone (BristolMyers Squibb), which is a potent antidepressant.
54
This led to a series of 6-amino-5-(4-arylpiperazinyl)methyl-uracil derivatives. When both ring nitrogens were
substituted, the compounds had nanomolar affinities for
the desired receptors but were found to be mutagenic.
Increasing the size of the substituents reduced the effects,
but did not reduce mutagenicity. The use of 4-(4arylpiperazinyl)-(5-amino-3-cycloalkyl-1-phenyl-pyrazole)
derivatives provided more effective compounds
(although an injudicious choice of substituents led to
enhanced α1 activity and the class retained genotoxicity).
The use of an dihydropyrazolone ring led to BTS-79524
(5-amino-4-[4-(benzodioxan-5-yl)piperazin-1-yl]methyl-1methyl -4-oxo-3-phenyl-2,3-dihydropyrazole; Figure 6).
This comp-ound has high affinity at 5-HT 1A (Ki = 26 nM)
and α2A (Ki = 29 nM) receptors with only moderate affinity
for α1 receptors (Ki = 401 nM) and was free of genotoxic
effects. Some details of the synthetic approaches to this
compound have recently been described in Tetrahedron
Letters (1998) 39:2219.
In in vivo models of depression, BTS-79524 produced 66%
downregulation of 5-HT1 receptors (cf 32% for amitryptiline) and was more potent in the rat Porsholt test
(minimum effective dose = 3 mg/kg). This compound is
not under active development, as a consequence of a
strategic decision, but has a similar profile to the
structurally distinct CP-93393 (Pfizer), that is now in early
clinical trials.
Conclusion
The organizers provided a well chosen program at a
good, easily accessible venue. The meeting covered a
broad range of therapeutic areas. One deficiency, pointed
out in the closing remarks of Paul Leeson, was that the
conference had not encompassed aspects of combinatorial
chemistry, and he suggested that next year’s meeting
should remedy this omission.
Meeting Report 71st Japan Pharmacol Soc
55
The Japanese Pharmacological Society - 71st Annual Meeting
23 - 26 March 1998, Kyoto International Conference Center, Kyoto, Japan
Trevor Wright
Address
Middlesex House
London
W1P 6LB
UK
IDrugs 1998 1(1):55-62
Angiotensin receptor subtype functions
In a presentation concerning angiotensin receptor signaling
mechanisms, T Inagami (Vanderbilt University School of
Medicine, USA) discussed the mechanisms of function of
individual angiotensin receptor subtypes and showed that
AT2 gene deletion causes blood pressure elevation. He went
on to discuss the possible mechanism of AT2 function.
Other notable oral presentations
Y-27632
This meeting was attended by over 2,700 pharmacologists and
physiologists, and hosted nearly 350 oral presentations and close to
700 posters. The topics covered a wide range of contemporary
pharmacological and physiological themes and provided an
interesting update of the development of many novel compounds
for a wide range of therapeutic indications. Antisense oligonucleotide pharmacology made an appearance this year and many
presentations concerned the safety of medicines.
Data on the pharmacological profile of a novel pyridine
derivative, Y-27632 (Yoshitomi), were presented by T
Ishizaki (Faculty of Medicine, University of Kyoto). This
compound inhibits smooth muscle contraction induced by
various agonists of the calcium sensitization pathway. It
interacts with p160ROCK, with an affinity which is about
200-fold its affinity for PKC and it is thought to directly
inhibit this Rho-associated protein kinase.
CP-99994
Clinical pharmacy and pharmacology
The six opening symposia concentrated on various aspects
of clinical pharmacy and pharmacology. Clinical pharmacy
education was not introduced into Japanese universities
until the 1970s, despite the early establishment of this
discipline in other countries, such as the US. There are
currently only six departments of clinical pharmacology
amongst the 80 medical schools throughout the country. The
general consensus of the speakers was that the role of
clinical pharmacy and pharmacology should be strengthened and that cooperation between them extended.
The role of nitric oxide
Special lectures focused on cardio- and cerebrovascular
pharmacology. S Moncada (University of London, UK), in a
slight departure from the scheduled lecture theme,
presented an overview on the discovery of nitric oxide
together with its function in health and disease. He
highlighted the broad range of functions of this simple
molecule, including its effects on immunological host
defense and its role in the pathophysiology of septic shock,
inflammation and cerebral ischemia. Professor Moncada
discussed the potential effects that selective nitric oxide
synthase inhibitors may have on protecting neurons from
damage.
Neuronal circuits
Current understanding of how neurons within the CNS
interact through both excitatory and inhibitory synapses and
how multilayered neuronal circuits within the cortical
tissues show remarkable computational functions was
discussed by M Ito (Riken, Japan). He suggested that our
knowledge of gene regulatory mechanisms will expand in
future to shed light on mechanisms of cellular function and
development which in turn will lead to the production of
brain-like computers.
A Nagahisa (Pfizer, Japan) described findings for CP-99994
(Pfizer; Figure 1), which is a centrally acting NK1 antagonist,
showing that it is a broad spectrum anti-emetic agent. It
inhibits vomiting and retching induced by morphine,
cisplatin and ipecac in ferrets and highlights the potential
advantages of neurokinin antagonists in the treatment of
chemotherapy-induced emesis.
GR-205171
The pharmacological profile of GR-205171 (Figure 1) was
reported by S Liou (Nippon Glaxo, Japan). This too is an
NK1 antagonist with potent anti-emetic effects and at 25 or
50 µg/kg iv was shown to abolish the gradual and rhythmic
bursts of firings of the central pattern generator (CPG)
concomitantly with retching discharges of both phrenic and
abdominal muscle nerves. It is thought that the CPG
neurons within the reticular area are the site
of action of this anti-emetic.
Mitragynine
S Horie (Faculty of Pharmaceutical Sciences, University of
Chiba, Japan) presented results for mitragynine, an alkaloid
extract of a Thai medicinal plant. This monoterpenoid indole
alkaloid is known to have a potent analgesic effect and is
currently being investigated for its opioid agonist effects
despite its lack of chemical similarity to morphine. It
appears to act on µ- and δ-opioid receptors and causes
antinociception.
FR-146801
The effects of FR-146801 (Fujisawa), a spontaneous NO
donor, were reported by Y Hirasawa (Fujisawa Pharmaceutical Co Ltd, Japan). This compound is a stable analog of
FK-409 (Fujisawa), and was shown to suppress intimal
thickening after balloon injury in a rat model. When given
orally, FR-146801, showed a more selective antiproliferative
effect than hypotensive effect.
56
ATZ-1993
ATZ-1993 is an orally active non-peptide endothelin
receptor antagonist and, according to H Azuma (Tokyo
Medical and Dental Engineering, Japan), it significantly
inhibits intimal hyperplasia following endothelial denudation in the rat, after dosing at 30 mg/200 ml/kg/day for 7
weeks. This compound did not affect the mean blood
pressure and heart rate and its effects on intimal hyperplasia
were at least partly due to its antagonist effects on
endothelin receptors.
Ad.CMV-cHK
K Yayama (Kobe Gakuin University, Japan) reported on the
use of an adenovirus-mediated delivery system for a human
tissue kallikrein gene. This gene delivery system was shown
to reduce blood pressure in a two-kidney, one-clip Goldblatt
hypertensive rat model. The adenovirus (Ad.CMV-cHK)
carrying this gene was under the control of a cytomegalovirus promoter and was administered iv.
significantly decreased heart rate, rate-pressure product, left
ventricular contraction and cardiac output and was 6- to 8fold more potent than esmolol on some parameters. It is
thought to be a more potent β-blocker than esmolol.
OPC-6535
OPC-6535 (Otsuka; Figure 1) is an inhibitor of leukocyte
activation, including decreased superoxide production,
lower neutrophil adhesion and decreased chemotaxis. G
Miyakoda (Otsuka Pharmaceutical Co Ltd, Japan) reported
on findings for the effect of this compound on left
ventricular function in a pig myocardial model of 30 min
ischemia and 4 week reperfusion. It was shown to
significantly improve left ventricular function and reduce
the extent of myocardial fibrosis after long-term reperfusion
when given post-ischemically.
EMD-57033
Results of using Bay-k-8644 (University of Alberta) were
presented by T Nasu (Yamaguchi University, Japan). This
dihydropyridine calcium agonist evoked strong rhythmic
contraction in normal calcium medium in taenia coli. These
results suggest that this compound allows entry of
manganese ions into the cytoplasm via calcium channels
while voltage-dependent calcium channels are activated by
Bay-k-8644.
Calcium sensitizers, such as EMD-57033 (Merck KGaA; Figure
1), are being developed for the treatment of congenital heart
failure. K Sakurai (Yamagata University School of Medicine,
Japan) reported results for this compound in a volume-loaded
rabbit model 12 weeks or more after arteriovenous shunt
formation. The cardiotonic effects of this compound do not
appear to be affected in volume-overloaded cardiomyocytes
although the response by these cells to β-blockers was
markedly decreased. It is suggested that this compound may
be of benefit in the treatment of contractile dysfunction in
congestive heart failure where β-antagonists are ineffective.
MKC-242
FR-171113
The involvement of presynaptic 5-HT1A receptor antagonist
action in the MKC-242-induced potentiation of photic
entrainment in circadian rhythms was reported by Y
Yoshinobu (Waseda University, Japan). The results obtained
indicate that MKC-242 (Mitsubishi; Figure 1) potentiates the
photic entrainment of the circadian clock via activation of
presynaptic 5-HT1A receptors, which is followed by a
reduction in serotoninergic neuron activity.
Y Kato (Fujisawa Pharmaceutical Co Ltd, Japan) presented
findings for FR-171113 (Fujisawa), a novel non-peptide
thrombin receptor antagonist, on platelet aggre-gation in
guinea pigs. Dose-dependent inhibition was observed over a
concentration range of 1 to 32 µM when aggregation was
induced by TRP-6, but not when induced by ADP or
collagen, even at 100 mM. FR-171113 inhibits thrombinreceptor mediated platelet aggregation both in vivo and in
vitro.
Bay-k-8644
OPC-14523
Data on the pharmacological effects of OPC-14523 (Otsuka)
were presented by K Tottori (Otsuka Pharmaceutical Co Ltd,
Japan). This novel antidepressant is a potent antagonist of σ
receptors and 5-HT1A receptors as well as inhibiting 5-HT
reuptake. Single doses of 3 mg/kg po, or higher,
significantly and dose-dependently antagonized reserpineinduced ptosis in mice. These results indicate that OPC14523 may be a potent antidepressant drug.
FK-409
S-8921
The hypocholesterolemic and anti-atherogenic effects of S8921 (Shionogi) were reported by S Hara (Shionogi & Co
+
Ltd). This compound is an ileal Na /bile acid cotransporter
inhibitor and was shown to produce dose-dependent
decreases in serum cholesterol levels when administered for
6 weeks in cholesterol-fed hamsters (the final concentration
of S-8921 was 0.6 to 57 mg/kg). This compound was also
shown to be > 50-fold more effective than cholestyramine.
The protective effect of FK-409 (Fujisawa; Figure 1) on
ischemic acute renal failure in rats was discussed by M
Nishiura (Osaka University of Pharmaceutical Sciences,
Japan). This compound, a spontaneous NO donor, was
shown to attenuate a decrease in renal function due to
occlusion following contralateral nephrectomy and indicates
that it may be useful in the treatment of acute renal failure
due to formation of NO.
(+)-TAN 67 (Toray; Figure 1) has antinociceptive effects in
mice according to J Kamei (Hoshi University, Japan).
Subcutaneous pretreatment at doses from 3 to 30 µg/kg for
30 min, dose-dependently attenuated the antinociception
induced by intrathecal administration of nociceptin (1
nmol). This compound may therefore act as an antagonist
for opioid receptor-like-1 receptors.
ONO-1101
S-0509
The cardiovascular effects of ONO-1101 (Ono; Figure 1)
were presented by A Sugiyama (Yamanashi Medical
University, Japan). This ultra-short acting β-blocker
Findings for a new potent and selective gastrin/CCK-B
receptor antagonist, S-0509, were presented by K Ikeda (Kyoto
Pharmaceutical University). When 1 to 60 mg/kg of
TAN 67
57
Figure 1. Structures of drugs from oral presentations
H
N
CH3
O
H
N
O
N
H
N
H
H3C
O
F
F
O
N
H3C
Cl
N
GR-205171
(Glaxo Wellcome)
MKC-242
(Mitsubishi)
OH
HO
N
NH2
N
O
H
N
O
H3C
O
H
N
F
CP-99994
(Pfizer)
O
N
O
O
N
H
H3C
H3C
O
N
H
O
N
O
O
O
O
O
ONO-1101
(Fujisawa)
FK-409
(Fujisawa)
O
CH3
O
O
S
O
N
N
H3C
N
OH
CH3
O
S
H3C
O
N
O
N
H
OPC-6535
(Otsuka)
EMD-57033
(Merck KGaA)
H
N
N
OH
TAN-67
(Toray)
CH3
CH3
58
this compound was administered orally 1 h before pyloric
ligation, it significantly suppressed basal gastric acid
secretion in rats without ulcers. It also appeared to be as
potent as famotidine (Yamanouchi) in inhibiting gastric
secretion and may be a useful anti-ulcer drug.
Highlights of poster presentations
Cardiovascular and circulatory
NTE-122
Y Azuma et al (Nissin Food Products, Japan) presented
findings for the effects of the ACAT inhibitor, NTE-122
(Nissin; Figure 2), on the metabolism and secretion of lipids
in HepG2 and CaCo-2 cells. This is a potent and selective
inhibitor of this lipid-metabolizing enzyme. It appears to
lower cholesterol levels in these cells by the inhibition of
VLDL secretion and the stimulation of bile acid secretion in
the liver, and the inhibition of lipid incorporation via the
intestinal mucosa.
JTV-605
JTV-605 is a non-peptide vasopressin antagonist and results
of its effects on diuresis were presented by K Wakitani et al
(Japan Tobacco). It shows high selectivity for V1 receptors
from rat liver and human platelets (IC50 values of 15 and 16
nM, respectively) and for V2 receptors from rat kidney and
CHO cells stably expressing human V2 receptors (IC50 values
of 22 and 19 nM, respectively). JTV-605 may be of potential
use in the treatment of patients with edema.
NS-7
The cerebroprotective effects of NS-7 (Nippon Shinyaku;
Figure 2), a novel calcium channel blocker, on ischemia
induced by transient occlusion of the middle cerebral artery
were presented by Y Aoki et al (Nippon Shinyaku, Japan).
The protective effects of this pyrimidine deriv-ative were
obvious even when it was administered 6 to 12 h after onset
of ischemia, indicating that it may be of use as a cerebroprotectant with a wide time window after ischemic insult.
SR-33805
GR-144053 (Glaxo Wellcome; Figure 2), a fibrinogen receptor
antagonist, enhances the suppression of neoin-timal formation
by losartan (DuPont Merck) according to H Matsuno et al
(Gifu University School of Medicine, Japan). This compound
was coadministered with losartan, an angiotensin II receptor
antagonist, to hamsters with intimal injury caused by the tip
of a catheter and was found to suppress neointimal formation
2 weeks after injury.
S Ieiri et al (Kyushu University, Japan) reported their
findings for SR-33805 (Sanofi Recherche; Figure 2) on the
cytosolic calcium concentrations and contractions of porcine
muscle. This indole derivative, a novel calcium channel
blocker, differs chemically from other known calcium
antagonists such as 1,4-dihydropyridines, phenylalkylamines and benzothiazepines. The results showed that it
inhibited contraction of porcine artery to a lesser extent than
would be predicted from its inhibition of calcium influx. Its
mechanism of action has yet to be elucidated.
KB-R9032
CP-060S
In a comparative study of KB-R9032 (Kanebo), K Harada et al
+
+
(Kanebo, Japan) showed that this novel Na /H ion-exchange
inhibitor was a selective NHE1 inhibitor with weak inhibitory
effects on NHE2, which were expressed in a fibroblast cell
line. Species differences were observed for the actions of KBR9032 on human and rabbit platelets according to
investigations on the attenuation of sodium proprionateinduced swelling caused by this compound. It may be of use
in the treatment of ischemic reperfusion injury.
CP-060S (Chugai) is a novel cardioprotective agent which
prevents overloading of both sodium and calcium ions, and
its effects on Arg-vasopressin-induced myocardial ischemia
in rats were presented by K Tamura et al (Chugai
Pharmaceutical Co Ltd, Japan). Orally administered CP060S, at doses of 3 and 10 mg/kg, significantly inhibited
AVP-induced ST depression and this effect was sustained
for 12 h at the higher dose. CP-060S was shown to have a
slower onset of its vasodilatory effect due to its calcium
antagonist effect than that seen for diltiazem. It may be of
use in the treatment of angina.
GR-144053
KRG-594
Y Inada et al (Kissei Pharmaceutical, Japan) presented their
findings on KRG-594 (Figure 2), a novel AT1 receptor
antagonist. This cyclopentenecarboxylate was shown to
cause a sustained hypotensive effect in SHR and renal
hypertensive rats and dogs, and was also shown to dosedependently improve glomerulosclerosis. The results
suggest that KRG-594 ameliorates diabetic nephropathy
accompanying hypertension.
E-4010
E-4010 is a novel PDE V inhibitor according to H Ishihara
(Eisai, Japan). It competitively inhibited the activity of PDE
V purified from porcine and human platelets and was
shown to be highly selective for this form compared to other
isozymes. At 100 nM, E-4010 significantly potentiated
increases in cGMP levels induced by SNP (1 µM) and ANP
(10 nM) in isolated porcine pulmonary arteries without
affecting cAMP levels.
AT-1015
The antithrombotic effects of AT-1015 and its associated
bleeding risks were presented by H Koganei (Ajinomoto,
Japan). This is a novel 5-HT2 receptor antagonist which
inhibits platelet aggregation and arterial contraction induced
by 5-HT. It did not prolong bleeding time in rat tail at a dose
of 3 mg/kg po, and it is suggested that it could be used as
an antithrombotic agent with low bleeding tendency.
OPC-33509
OPC-33509 (Otsuka) is a novel antithrombotic derivative of
cilostazol (Otsuka) and shows more potent antithrom-botic
and antihyperplastic effects than cilostazol. Y Inoue et al
(Nagoya University School of Medicine, Japan) report that
this compound has a dual mechanism of action by inhibiting
vascular intimal thickening and inhibiting thrombogenesis,
indicating that it may be of use in the treatment of
arteriosclerosis-induced ischemic diseases.
59
Figure 2. Cardiovascular and circulatory drugs
H3 C
OH
N
N
CH3
O
O
N
N
H
N
H3 C
N
H
N
N
H2 N
O
N
NH
CH3
GR-144053
(Glaxo Wellcome)
NTE-122
(Nissin Food Products)
O
S
CH3
O
N
CH3
N
N
N
N
N
N
N
OH
HN
N
O
H
K
Cl
K
F
KRG-594
(Wakunaga)
NS-7
(Nippon Shinyaku)
O
CH3
H3C
CH3
O
CH3
S
N
O
N
O
O
CH3
CH3
SR-33805
(Sanofi Recherche)
KIN-493
KIN-493 (Novartis; Figure 3), an analog of carbama-zepine,
produces marked antinociceptive actions in STZ-induced
diabetic rats according to S Kiguchi et al (Kissei
Pharmaceutical, Japan). The antinociceptive effects of this
compound, at 100 mg/kg po, were significantly reversed
by pretreatment with theophylline, bicuculline and
yohimbine, although naloxone, naltrindole, bromocriptine
and sulpiride had no effect on this compound. These
results indicate that the antinociceptive effects of KIN-493
are not mediated by opioid or dopamine receptors.
MKC-242
Findings for the mechanism of action of MKC-242
(Mitsubishi; Figure 3) were presented by P Somboonthum
et al (Department of Pharmacology, University of Osaka,
Japan). It is a potent and selective 5-HT1A receptor agonist
which has anxiolytic- and antidepressant-like effects in
animal models. Results suggest that systemic injection of
60
Figure 3. Central and peripheral nervous system drugs
O
S
NH2
O
OH
N
OH
N
Cl
N
N
H3C
HO
O
Cl
H
R-84760
(Sankyo)
CP-101606
(Pfizer)
KIN-493
(Novartis)
O
O
O
O
H
NH2
OH
N
H3C
O
O
O
N
H
Cl
N
H
F
O
Cl
F
F
MKC-242
(Mitsubishi)
KMD-3213
(Kissei)
Figure 4. Gastrointestinal drugs
O
O
HO
N
H
OH
H3C
N
N
H
N
O
S
N
H
N
H3C
HO
O
HO
OH
S
O
O
T-593
(Toyama)
BX-661A
(Nippon Hoechst Marion Roussel)
O
N
N
NH
C
C
N
H
N
KW-5092
(Kyowa)
N
this compound facilitates the release of ACh via activation of
somadendritic 5-HT1A autoreceptors and that this mechanism differs from that of 8-OH-DPAT.
KMD-3213
The potential use of a novel α1-adrenoceptor antagonist,
KMD-3213 (Kissei; Figure 3), were outlined by S Murata et al
(Kissei Pharmaceutical, Japan). This selective α1 adrenoceptor blocker has been developed for the treatment of
urinary outlet obstruction associated with benign prostatic
hyperplasia. KMD-3213 was found to be highly selective for
α1A and α1L subtypes, rather than for α1B and α1D. This
pharmacological profile differed from that of prazocin and
tamsulosin (Yamanouchi) which show high affinity for the
latter receptors.
61
mg/kg po in dogs chronically implanted with force
transducers. Z-388 improves delayed gastric emptying to the
normal level and may be useful in the treatment of
abdominal symptoms due to hypomotility and delayed
gastric emptying in patients with functional dyspepsia.
T-593
The effects of T-593 (Toyama; Figure 4) on gastric ulcer
healing were presented by Y Doi (Toyama Chemical, Japan).
This is a potent H2 receptor antagonist with gastroprotective
effects, and was shown to facilitate mucosal regeneration
and collagen fiber proliferation when administered at 30
mg/kg po for 8 weeks to a rat model of cryoprobe-induced
gastric tissue damage. It is effective in the re-epithelialization of gastric tissue and promotes the maturation of
granulation tissue.
KW-5092
Gastrointestinal
BX-661A
S Furuta et al (Nippon Hoechst Marion Roussel, Japan)
presented findings for BX-661A (Salix; Figure 4), a
compound being investigated for the treatment of ulcerative
colitis. They observed the effects of this salicylate on PMNL
functions following treatment with FMLP. BX-661A is
suggested to inhibit reactive oxygen metabolite production
and chemotaxis by inhibiting FMLP-binding to its receptor
on PMNL, both activities possibly contributing to its effects
in ulcerative colitis.
YM-905
YM-905 (Yamaouchi) is a novel muscarinic receptor
antagonist. Its effects on gastrointestinal motility and gastrocolic reflex were reported by M Suzuki et al (Yamanouchi
Pharmaceutical, Japan). It potently and competitively
inhibited carbachol-induced contractions of guinea pig colon,
with a pA2 value of 7.5 ± 0.05. It was also shown to inhibit
restraint stress-induced defecation and diarrhea over a dose
range of 1 to 30 mg/kg. It is suggested that this compound
may be useful in the treatment of irritable bowel syndrome.
Z-338
Y Matsunaga et al (Zeria Pharmaceuticals, Japan) reported
on the gastroprokinetic activity of Z-338. This thiazole-4carboxamide derivative showed a dose-dependent increase
in fed gastric motor activity at 0.3 to 3 mg/kg iv and 3 to 30
N Kishibayashi et al (Kyowa Hakko, Japan) presented
findings for KW-5092 (Kyowa Hakko; Figure 4), a novel
gastroprokinetic agent which causes the release of ACh and
also inhibits acetylcholinesterase. The EC50 value for this
compound in the potentiation of ACh release was 570 nM
and suggests that the stimulation of gastric emptying is due
to its ACh releasing effect.
Anti-inflammatory and anti-allergy
T-164
A novel anti-inflammatory compound, T-164 (Toyama),
suppresses NFκB activation from evidence presented by Y
Konishi et al (Toyama Chemical, Japan). It is known to
suppress immunoglobulin and cytokine production and to
have anti-inflammatory actions as a consequence. It was
shown that the mechanism of action of T-164 was due to the
inhibition of NFκB production.
HSR-609
The pharmacological characterization of a novel amphoteric
anti-allergic compound, HSR-609 (Hokuriku Seiyaku; Figure
5), was presented by Y Hiraoka (Hokuriku Seiyaku, Japan).
This selective H1 receptor antagonist was shown in
saturation binding experiments to significantly reduce Bmax
values when the membrane fraction from guinea pig
cerebral cortex was pre-incubated with HSR-609, before
Figure 5. Anti-inflammatory and anti-allergy drugs
OH
N
N
O
O
H
N
N
N
F
N
F
N
O
HSR-609
(Hokuriku Seiyaku)
FR-167653
(Fujisawa)
O
62
addition of tritiated pyrilamine. HSR-609 was shown to be
an insurmountable antagonist due to its slow dissociation
from H1 receptors.
FR-167653
M Kawamura et al (Kitasato University School of Medicine,
Japan) presented their results for the suppression of COX-2
expression by FR-167653 (Fujisawa; Figure 5) in a rat model
of carrageenin-induced pleurisy. This IL-1 and TNF
synthesis inhibitor at a dose of 30 mg/kg was shown to
suppress the expression of COX-2 and the accumulation of
pleural exudate 5 h after injection of carrageenin.
KB-R7785
KB-R7785 (Kanebo) is a novel matrix metalloproteinase
inhibitor and its antidiabetic effects were presented by N
Nishimura et al (Kanebo KK, Japan). It was shown to inhibit
TNF-α release from LPS-stimulated THP-1 cells in a dosedependent manner, with an IC50 of 2 µM. Subcutaneous
administration for 5 weeks at 100 and 200 mg/kg/day,
resulted in a significant reduction in plasma levels of
glucose, insulin, triglycerides, total cholesterol and creatinine. These data suggest that inhibition of TNF-α release
caused by KB-R7785 may be useful in the therapy of noninsulin dependent diabetes mellitus.
XT-611
The effects of a novel PDE IV inhibitor, XT-611, on osteoclast
and osteoblast formation were reported by K Miyamoto et al
(Kanzawa University, Japan). This is a selective isozyme
inhibitor, which relaxed tracheal smooth muscle without
affecting the heart in guinea pigs, did not cause emesis in
Suncus murinus at doses of 10 to 100 mg/kg po, unlike
other known PDE IV inhibitors such as rolipram (Schering)
and denbufylline (SmithKline Beecham) at doses of 10 to 30
mg/kg. XT-611 may be a good therapeutic candidate for the
treatment of osteoporosis due to its side-effect profile.
Antisense therapy
PA-as and PB2-as
T Mizuta et al (Rational Drug Design Laboratories, Japan)
reported their findings for two antisense phosphorothioate
oligonucleotides, PA-as and PB2-as. These were shown to be
potent inhibitors of influenza A virus replication in vitro and
were tested in a mouse model of respiratory tract infection
with this virus. Treatment of mice with a liposomally
encapsulated PB2-as 1 day before infection and 1 and 3 days
post-infection significantly prolonged the mean survival
time and increased the mean survival rate in a dosedependent manner.
Meeting Report Prostate Cancer Conference
63
Prostate Cancer - CHI Conference
Advances in Understanding Diagnostics and Therapy
23-24 March 1998, McLean, Virginia, USA
Paul B Fisher
Address
Departments of Pathology and Urology
Columbia University
College of Physicians and Surgeons
New York
NY 10032
USA
IDrugs 1998 1(1):63-66
As the title indicates, the purpose of this meeting was to highlight
new information relative to the etiology, diagnosis and potential
therapy of prostate cancer. Prostate cancer is the most prevalent
internal cancer affecting men in the USA and the second most
frequent cause of cancer-related deaths. Ongoing studies are
identifying potentially important genes and target molecules
involved in prostate cancer. These reagents will permit improved
diagnostics and offer the promise of enhanced therapeutic targets
for this cancer. The meeting was divided into four general areas.
Approximately 70 scientists representing academic, governmental
and industrial sectors attended this meeting.
Keynote address
Mr S Leslie Misrock (Pennie & Edmonds, New York, USA)
discussed his perspective as a survivor of prostate cancer.
He also presented an overview of Prostagen, a company
designed to understand prostate cancer and to improve the
diagnosis and therapy of prostate cancer. An additional aim
of Prostagen is to define strategies to prevent the development of prostate cancer.
Genetics of prostate cancer
The chairman of this session was Dr Paul B Fisher
(Columbia University, College of Physicians and Surgeons,
New York, USA).
Studies relating to familial prostate cancer in Utah kindreds
were discussed by Dr Sean V Tavtigian (Myriad Genetics Inc,
Utah, USA) and Dr Lisa Cannon-Albright (University of Utah,
USA). Evidence was presented documenting an association
between specific regions of chromosome 1q and familial
prostate cancer susceptibility. Linkage to HPC1, a region of
chromosome 1 previously shown to contain a prostate cancer
susceptibility locus, as well as additional regions of
chromosome 1q, was found to correlate with increased
incidence of prostate cancer in specific Utah kindreds.
Evidence indicating an additional region of chromosome 1q
(42.2-43) that predisposes to early onset of prostate cancer in
specific families was presented by Dr Philippe Berthon
(UroGene, France). These studies suggest that multiple
regions in addition to HPC1 may contain potential
susceptibility loci for prostate cancer, and these regions may
differ for specific subset populations.
The overall strategy being used by Human Genome Sciences
to identify genes was described by Dr Kenneth C Carter
(Human Genome Sciences, Maryland, USA). This includes
the construction of cDNA libraries, followed by highthroughput screening to identify and clone candi-date
genes, followed by labor intensive screening for biological
activity. Sequence information has now been generated for
1.4 million human cDNAs, expressed sequence tags (ESTs),
including 40% known human genes, 20% genes similar to
previously identified genes (or gene families), 10% common
sequences and approxi-mately 30% novel ESTs. A detailed
discussion was presented on one original EST, identified by
cDNA library screening that is differentially expressed in
prostate cancer, prostatic acid phosphatase.
A modified differential RNA display approach, restriction
enzyme analysis of differentially expressed sequences
(READS), for identifying differentially expressed genes was
reviewed by Dr William Munger (Gene Logic Inc, Maryland,
USA). Although not presently documented, it was suggested that this approach could be applied to prostate cancer
permitting the quantification of active genes (known and
unknown) in a prostate tissue sample.
Functional approaches for identifying and cloning human
oncogenes and tumor-associated genes with specific
application to prostate cancer were described by Dr Fisher.
Using a rapid expression cloning strategy (RExCS) combined
with differential RNA display, a novel oncogene, prostate
tumor inducing gene-1 (PTI-1) was identified. PTI-1 is able to
detect one prostate cancer cell in 100 million normal cells and
has successfully identified patients with metastatic prostate
cancer. In addition, inhibition of PTI-1 expression induces a
reversion in cancer-related properties. A second approach,
surface-epitope masking (SEM), has resulted in the production
of monoclonal antibodies reacting with prostate cancer and
the identification of prostate carcinoma tumor antigen gene-1
(PCTA-1). PCTA-1 is expressed in prostate cancer and high
grade prostatic intraepithelial neoplasia (PIN) but not in lowgrade PIN, benign prostatic hypertrophy (BPH) or normal
prostate. PCTA-1 expression is also a sensitive indicator of
potential metastatic prostate cancer cells in the circulation of
patients. These two genes provide useful diagnostic markers
for staging prostate cancer and potential targets for prostate
cancer therapy. Moreover, the approaches of RExCS and SEM
represent valuable methodologies for identifying genes
relevant to human cancer.
Studies designed to develop suitable animal model systems
that replicate distinct steps in prostate cancer progression
were described by Dr Charles L Sawyers (UCLA School of
Medicine, USA). Xenografting approaches have been used to
develop models for prostate cancer metastasis and progression to androgen independence. Cells displaying specific
stages of diseases are being investigated in an attempt to
64
identify candidate genes involved in prostate cancer
progression. One candidate gene associated with prostate
cancer progression, ie, androgen-independent growth, was
identified using a cDNA library (retrovirus) transfer
approach. This gene encodes the cathepsin D protease and
its potential role in prostate cancer is currently under
investigation.
Diagnostic and prognostic markers of
prostate cancer
The chairman of this session was Dr Jeffrey Ross (Albany
Medical College, New York, USA).
Novel amplification methods for immuno- and gene-based
diagnostics were described by Dr David Ward (Yale
University, Connecticut, USA). By using Padlock probes of
defined sequence and the rolling circle amplification (RCA)
technique, a sensitive and selective procedure has been
developed for detecting molecules present at exceedingly
low levels in a complex mixture. These approaches can be
performed in solution and with immobilized targets and
DNA arrays. Applications of Padlock probes and the RCA
strategy include potential mutation detection, allele
discrimination analyses, single molecule detection of
antigens immobilized on a glass surface and in situ analyses
with cytological sections. This scheme offers significant
promise for the detection of specific target sequences and
proteins that may contribute to prostate and other cancers.
Evidence that human glandular kallikrein (hK2) may be a
valuable clinical diagnostic marker for prostate cancer was
provided by Dr Donald J Tindall (Mayo Clinic, Minnesota,
USA). hK2 shares important properties of organ localization
and hormonal regulation with prostate-specific antigen
(PSA), a major diagnostic protein used in staging prostate
cancer. Monoclonal antibodies have been developed that
show less than 0.5% cross-reactivity with PSA. Analysis of
257 radical prostatectomy samples indicated a higher
percentage of cells staining positive for hK2 in more
advanced stages of cancer. In this context, its staining
pattern was opposite to that of PSA and indicates that hK2
may provide useful diagnostic applications for staging
prostate cancer.
An approach to prostate cancer diagnosis and prognosis
testing, called Biostage, was described by Dr Stephen Strum
(Cancer Research Institute, Healing Touch Oncology,
California, USA). This scheme involves the use of multiple
parameters, including measurements of angiogenesis, to
predict pathological stage in prostate cancer patients.
Although further studies are required, this information
could prove useful as part of a systematic approach,
including CT scans, monitoring PSA levels and in more
accurately staging prostate cancer.
Emerging genetic markers, her-2/neu and glutathione Stransferase-pi (GST-pi), that may prove useful in prostate
cancer detection and prediction of disease outcome were
discussed by Dr Jeffrey S Ross (Albany College of Medicine,
New York, USA). A fluorescence in situ hybridi-zation (FISH)
approach was used to detect her-2/neu gene amplification
indicating an adverse relationship between amplification and
prognosis. In approximately 75% of nodal metastases gene
amplification or protein over-expression was present.
Amplification of her-2/neu by FISH independently predicts
disease recurrence after primary radiation therapy. Using a
second marker, GST-pi, hypermethylation of this gene was
apparent in 52 of 57 prostate cancers.
Immune-based approaches in prostate cancer
Dr Michael I Sherman (Prostagen Inc, New Jersey, USA)
chaired this session on immune-based approaches to the
therapy of prostate cancer.
An antibody targeting approach for solid tumors was
described by Dr Paul G Abrams (NeoRx Corporation,
Washington, USA). Avicidin (an antibody containing a
receptor) delivers radiation to primary and metastatic
disease by means of a Pretarget technology. Using this
approach, early phase I trials indicate efficacy against
metastasis in a subset of patients with prostate cancers who
have failed standard therapies. Further studies with larger
patient samplings are planned.
An overview of cancer vaccines was provided by Dr Lynn E
Spitler (Jenner Biotherapies Inc, California, USA). Dr Spitler
also presented the results of phase I/II trials using OncovaxP containing a recombinant PSA formulated in liposomes,
with lipid A as adjuvant. The results of four phase I/II trials
have demonstrated safety and the ability to induce immune
responses. On the basis of these encouraging results,
expanded phase II trials and a phase III trial are anticipated.
Recent studies using immunotherapy for prostate cancer
based upon prostate-specific membrane antigen (PSMA) was
discussed by Dr Michael L Salgaller (Northwest Biotherapeutics, Washington, USA). Dendritic cells were isolated from
patients, pulsed with PMSA and readministered to patients
(with and without granulocyte-macrophage colony
stimulating factor). Of the patients (n = 107) with hormone
refractory metastatic prostate cancer, 27% were classified as
partial responders. In contrast, 33% of patients showed no
change in disease status, 39% underwent disease progression
and 21% of the test patients died.
Dendritic cell immunotherapy for prostate cancer was the
subject of a talk by Dr Frank H Valone (Dendreon Corp,
California, USA). As outlined by Dr Salgaller, dendritic
precursor cells can be isolated from patients, induced to
mature with antigen in vivo (in this case prostatic acid
phosphatase) and then infused back into patients 48 h later
to stimulate an antigen-specific T-cell response in vivo. A
phase I/II trial of immunotherapy with APC-8015 in
patients with advanced, hormone-refractory prostate cancer
was promising. This study demonstrated that APC-8015 is
safe, immunologically active (T-cell responses in 12 of 12
patients, T-helper 1 response and evidence of a dose
response) and signs of biological efficacy on the tumors.
However, the studies are not suficiently advanced to assess
clinical benefit.
Studies using a recombinant replication competent
adenovirus containing a PSA response element for treatment of prostate cancer were described by Dr Daniel R
Henderson (Calydon Inc, California, USA). Virus replication
was demonstrated in cells expressing PSA. A single injection
Meeting Report Prostate Cancer Conference
discussed by Dr Robert G Croy (MIT, USA). Compounds have
been developed that exploit molecular features in prostate
cancers, such as the expression of the androgen receptor to
disable cellular defenses against DNA damage. By blocking
repair of genotoxic damage, lethality in tumor cells is
increased while sparing normal tissues. It is predicted that
these new compounds should produce greater therapeutic
benefit and fewer side-effects than conventional cytotoxic
chemotherapy. Although further research is clearly necessary,
including studies in animals, if successful, this strategy might
prove of value for prostate therapy.
of CN-706 (adenovirus containing the PSA response element
driving the adenovirus E1A and E1B genes resulting in viral
replication) into the human prostate cancer cell line LNCaP
(which is PSA positive) inhibited tumor growth in vivo in
nude mice. Phase I trials with patients with recurrent, locally
advanced prostate cancer will be initiated shortly.
Dr Lily Wu (UCLA School of Medicine, USA) provided
additional documentation of the potential use of adenovirus
vectors containing prostate-specific promoter sequences to
target viral replication and cell lysis. Specific viral constructs
have been engineered, using the prostate-specific PSA enhancer and promoter region, to produce specific therapeutic
gene products, including IL-2/TNF-α, p53, Rb, thymidine
kinase and antisense TGF-β, in PSA expressing prostate
cancer cells. Efficacy of engineered viruses for targeting
gene expression in cells expressing PSA was documented.
Studies of the apoptosis-inducing drug, FGN-1 (sulindac
sulfone), that is being developed as a pharmaceutical for the
primary prevention of cancers was described by Dr Gary A
Piazza (Cell Pathways Inc, Pennsylvania, USA). Studies have
been conducted to evaluate the effect of FGN-1 in a number
of in vitro and in vivo preclinical models to assess the extent
to which it induces apoptosis and prevents the development
of neoplasia, as well as to elucidate mechanism of action.
Preliminary evidence suggests potential efficacy of FGN-1 in
experimental model systems. Clinical trials are planned for
determining the effect of FGN-1 on premalignant lesions
and malignancy recurrence.
Small molecule and other therapies for
prostate cancer
The chair of this session was Dr William D Figg (National
Cancer Institute, Washington DC, USA).
A novel technology for improving the utility of chemotherapy, with potential application to prostate cancer, was
Figure 1. Angiogenesis inhibitors
O
O
O
HO
S
O
OH
S
O
O
O
O
S
S
OH
HO
H3C
O
CH3
O
O
S
HO
S
NH
N
H
N
H
O
O
H
N
H
N
HN
O
O
O
Suramin
(Warner-Lambert)
H3C
O
OH
H3C
O
CH3
H
OH
O
N
H
NH
O
O
S
O
S
O
TNP-470
(Takeda)
NH
O
O
O
H
N
H2N
CH3
Cl
H
N
H2N
H
O
O
H
N
O
O
H
N
NH2
O
NH
N
H
H
O
N
O
HO
CH3
H3C
Somatuline
(Ipsen-Beaufour)
CH3
65
NH
O
O
Thalidomide
(Entremed)
66
promoter region has been isolated, ligated to a luciferase
reporter gene and stable cell constructs constitutively
expressing luciferase have been generated. Cloned cells with
the PEG-3 promoter/luciferase reporter construct are being
used to screen several combinatorial small molecule
libraries. This assay offers significant promise as a general
screening method for the identification of compounds that
inhibit both oncogene function and the progression
phenotype.
The activity of several angiogenesis inhibitors (suramin,
Warner-Lambert Co; somatuline, Ipsen-Beaufour; pentosan,
Ivax; TNP-470, Takeda Chemical Industry Ltd; carboxyamido-triazole, Merck and thalidomide, Entremed) in patients
with androgen-independent prostate cancer, was evaluated by
Dr William D Figg (National Cancer Institute, Maryland,
USA). Activity was apparent with several of these agents;
however, the duration of response was minimal in the heavily
pretreated metastatic population. The present studies suggest
that there may be greater potential if the angiogenesis
inhibitors are evaluated in patients at earlier stages of disease
progression.
Conclusion
Approaches for screening small molecule libraries for
antagonists of prostate oncogenes and downstream pathway
events were described by Dr Neil I Goldstein (GenQuest Inc,
USA). A gene mediating cancer aggressiveness, progression
elevated gene-3 (PEG-3), has been identified by subtraction
hybridization. The biological effects of PEG-3 are
downstream of multiple oncogenes, including Ha-ras, src,
human papilloma virus type 18 and PTI-1. The PEG-3
This conference provided an updated and insightful
overview of prostate cancer. The talks, in general, were of
high quality and they were well received by the attendees.
The use of a panel discussion format involving all of the
speakers in each section permitted an active dialog between
presenters and the audience. The organization of the
meeting, including breaks that permitted interaction
between the speakers and the attendees, resulted in very
interactive and successful symposia.
Meeting Report 99th Am Soc Clin Pharmacol Ther
67
American Society for Clinical Pharmacology and Therapeutics
99th Annual Meeting
30 March - 1 April 1998, New Orleans, Louisiana, USA
Denise Morgan
Address
Middlesex House
London
W1P 6LB
UK
IDrugs 1998 1(1):67-72
This meeting was attended by over 1000 clinicians, and clinical
pharmacologists from academia and industry. The three-day
conference consisted of poster sessions, state-of-the-art lectures,
plenary sessions, oral presentations, symposia and workshops.
The Human Genome Project
Genomic research and gene therapy are becoming
increasingly popular strategies for the development of novel
therapeutics. Based on the relationship between an
individual’s genotype and phenotype, knowledge of the
pathophysiology of disease at the gene level would enable
one to ascertain differences in an individual’s symptoms, as
well as differences in the pharmacokinetic and pharmacodynamic response to a drug (eg, changes in efficacy and
increasing incidence of adverse events).
Professor Richard Myers (Stanford Human Genome Center,
CA, USA) opened the meeting by presenting a lecture on the
progress of the Human Genome Project. Established in 1990,
the Stanford Human Genome Center has built maps of
human chromosome 4, to a 500 kb resolution, using a panel
of 83 radiation hybrids and more than 10,000 sequencetagged sites. The center is actively involved in the Human
Genome Project, which will determine the complete
sequence of at least 17.5 million base pairs of human DNA
using a directed sequencing strategy, in the hope of
identifying genes and obtaining information on gene
expression and function, gene-gene interaction and genomic
regulation, in order to use this knowledge to advance and
improve the drug discovery pharmaceutical process.
As Chairman of the Human Genetics and Drug Therapy
symposium, Dr B Michael Silber (Groton, CT, USA)
suggested that mapping of the human genome will be
completed by 1999, with complete sequences available by
2000/2001. Utilization of genomic research and the
candidate gene approach to pharmaceutical development
has already begun in select pharmaceutical companies and
routine use of this approach is anticipated by 2000.
The consensus within the pharmaceutical industry is that
the use of genomics in drug development is a great step
forward, leading to an optimization of the drug discovery
process through retrospective analysis of discovery targets, a
change in clinical trial design and interpretation, an impact on
product labeling, faster regulatory approval, early diagnosis
and estimation of disease risk, and quicker decisions
regarding patient care. Dr Silber emphasized that problems do
exist with this approach to drug development and such major
concerns include: the notion that not all patients with the same
genetic indiscrepancy will have the same disease (eg,
differences may occur due to environmental factors, such as
the duration of disease); misinterpretation of such information
by the regulatory authorities and improper use by industry;
the lack of regulation over validation procedures and the
maintenance of confidentiality of DNA samples held in
databases and DNA banks, which are already being
developed by many pharmaceutical companies.
Novel approaches to the prevention and
treatment of acute renal failure
Acute renal failure (ARF) is a complication of 10% of hospital
administrations, 30% of renal allografts and 20% of abdominal
aneurysm repairs. As a result, the pharmaceu-tical industry
has begun looking for novel ways of treating ARF and
improving renal function in these patients.
Growth factors
An overview of the use of growth factors, in particular,
insulin-like growth factor-1 (IGF-1), for the treatment of ARF
was presented by Dr Steven Miller (Washington University
School of Medicine, St Louis, MO, USA). The mechanism of
action of the IGFs is yet to be fully elucidated but mitogenic,
hemodynamic, cytoprotective, apoptotic and cytokine
modulatory actions are known to play an important role.
Various companies are involved in the development of IGF-1
products for the potential treatment of ARF, plus a number of
other conditions including osteoporosis, cachexia and
metabolic disorders, neuropathies, neurodegenerative disease
and diabetes; several clinical trials have been successfully
completed with IGF-1 for the prevention of renal dysfunction.
Other growth factors being investigated for the potential
treatment of ARF include HGF (hepatocyte growth factor),
EGF (epidermal growth factor), OP-1 (osteogenic protein-1,
also termed BMP-7), HB-EGF (heparin binding epidermal
growth factor) and α-MSH (a tridecapeptide derived from the
post-tranlational processing).
Dr Miller presented data from studies conducted in a rodent
model of ARF involving 36 animals treated with IGF-1 and 7
treated with placebo. Results showed that IGF-1 reduces
serum creatine levels and causes an increase in body weight
via an anabolic mechanism. Mortality was decreased to 7.3%
in those animals treated with IGF-1 compared to 36.7% in
the placebo group.
One other potential use of IGF-1 seems to be as a reversal
agent for the ischemia caused by ARF. This was
demonstrated in a randomized, blinded, placebo-
68
Figure 1. Adenosine receptor antagonists
H
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H3C
O
H
N
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H3C
H
N
N
NH
O
N
N
O
N
N
H
H
N
N
H3C
H3C
N
N
CH3
KW-3902
(Kyowa Hakko Kogyo)
CVT-124
(University of Florida)
controlled study of ischemia in a canine model, where
ischemia resulted from removal of both kidneys, one of
which was subsequently stored in IGF-1 (n=8) or placebo
(n=8), followed by re-implantation of either an IGF-1- or
placebo-stored kidney. In this model, serum creatine levels
were reduced by approximately 50% over a 6-day period.
Adenosine receptor antagonists
The merits of adenosine A1 receptor antagonism for the
treatment of ARF were discussed by Dr George F Schreiner
(Scios, Sunnyvale, CA, USA). Selective adenosine A1 receptor
blockade reverses cAMP inhibition in the renal vasculature
resulting in an increase in renal blood flow and GFR and a
reduction in ischemia induced by secondary vasocon-striction.
The resulting vasodilation enables the kidney to remove
nephrotoxins and reduces the possibility of precipitation and
reabsorption of these toxins in the proximal tubule.
Dr Schreiner referred to four adenosine A1 receptor
antagonists currently in development; FR-166124 (Fujisawa)
and KW-3902 (Kyowa Hakko Kogyo; Figure 1), both for the
treatment of ARF, CVT-124 (University of Florida; Figure 1)
for the treatment of congestive heart failure (CHF), ARF and
edema, and N-0861 (Discovery Therapeutics; Figure 1) being
developed for the treatment of CHF and arrhythmia. All
these compounds are in phase II clinical trials.
The endothelin pathway, ARF and
cardiovascular disease
Drs David Brooks (SmithKline Beecham, PA, USA) and
Michael R Goldberg (Merck, PA, USA) discussed endothelin
(ET) receptor antagonism and ET converting enzyme (ECE)
inhibition. Both of these points in the ET pathway are
amenable to drug intervention and are under investigation
for the potential treatment of ARF, pulmonary and essential
hypertension, CHF and benign prostatic hyperplasia.
Endothelin receptor antagonists
Stimulation of ETA and ETB receptor subtypes, located in the
kidney, is responsible for the regulation of renal blood flow,
naturesis (via a reduction in GFR mediated by the ETA
receptor), renal secretion and downregulation of the renin-
N-0861
(UCB)
angiotensin-aldosterone system. Stimulation of ET receptors
is also responsible for ET-1 mediated vasodilation and nitric
oxide release and ET-3 mediated vasoconstriction (via ETB
receptors located in the blood vessels).
BQ-123 (Banyu, Figure 2), a selective peptidic ETA
antagonist, causes an increase in forearm blood flow and a
vasodilatory response in CHF when given as an iv infusion,
starting with a loading dose of 1 µg/kg/min. Chronic
dosing of BQ-123 in a rat model also blocks the dilation and
hypertrophy which results in left ventricular remodeling, a
prognostic marker of CHF. Takeda Chemical Industries Ltd
is developing TAK-044 (Figure 2), a mixed ETA and ETB
antagonist, which lowers diastolic blood pressure and
plasma ET at 0.15 to 10 µg/kg over a 15 min iv infusion.
Roche is conducting phase III CHF clinical trials with
bosentan (Figure 2) which has Ki values for ETA and ETB of
15 and 102 nm, respectively. SB-209670 (SmithKline
Beecham, Figure 2), a mixed, non-peptidic antagonist with
Ki values for ETA and ETB of 0.4 and 18 nM, respectively,
reduces both systolic and diastolic blood pressure and
causes a reflex tachycardia in patients with mild to moderate
hypertension.
Endothelin converting enzyme inhibitors
Novartis AG is developing CGS-26303 (Figure 3), the first
non-peptidic inhibitor of ECE and neutral endopeptidase
24.11, for the potential treatment of hypertension, cerebral
vasospasm and renal failure. Three analogs of CGS-26303
(CGS-24592, CGS-31447 (Figure 3) and CGS-36112) are in the
discovery phase of development. All four compounds have
differential selectivity for ECE and neutral endopeptidase.
In the spontaneous hypertensive rat model, CGS-26303
reduces blood pressure by 15 to 20 mmHg (an effect which
remains for approximately 2 weeks after the initial dose). In
a rabbit model of cerebral vasospasm, treatment with CGS26303 significantly increases vasodilation of the basilar
artery and reduces arterial hyperplasia. CGS-26303 also
causes a significant reduction (32%) in neointimal formation
following angioplasty. Furthermore, in a puromycininduced model of nephropathy, sc injection of CGS-26303, 1
Figure 2. Endothelin receptor antagonists
CH3
O
H3C
O
Na
Na
O
H3C
O
O
CH3
O
H
N
N
NH
O
N
H
N
H
N
H
HN
N
OH
O
N
H
N
H
O
O
O
O
OH
N
O
HN
H
N
N
H
HN
O
H3C
CH3
O
S
OH
TAK-044
(Takeda)
BQ-123
(Banyu)
O
CH3
HO
O
H3C
OH
O
O
H3C
O
O
O
N
OH
N
S
N
N
H
H3C
H3C
O
O
O
N
O
CH3
O
Bosentan
(Roche)
SB-209670
Figure 3. Endothelin converting enzyme inhibitors
HO
P
HO
N
O
N
H
N
N
N
H
CGS-26303
(Novartis)
HO
P
HO
N
O
N
H
N
N
N
H
CGS-31447
(Novartis)
69
70
h after an iv bolus of puromycin, resulted in a modest
reversal of puromycin aminoglycoside-induced injury.
Chemoprevention - NSAIDs and colon cancer
Dr Randall W Burt (University of Utah School of Medicine,
Salt Lake City, USA) conducted a workshop on colon cancer
and chemoprevention. The incidence of colon cancer is
rapidly increasing, with approximately 131,000 new cases
and 55,000 deaths reported in the US in 1997. Colon cancer
arises due to either a genetic predisposition or a series of
acquired mutations and, therefore, is an excellent candidate
for chemoprevention.
Dr Burt discussed the use of NSAIDs to aid in the regression
and prevention of colon cancer in those patients with a
genetic predisposition, mediated via the presence of the APC
gene on the long arm of chromosome five. This phenomenon was first discovered in 1980, when animal studies in
a rat tumor model showed that indocin inhibited carcinogenesis in a dose- and duration-dependent fashion via the
blockade of cyclooxygenase and cell proliferation, and
stimulation of apoptosis and immune surveillance. Dr Burt
further discussed the use of, and current clinical trials in,
FGN-1 (sulindac sulfone, Cell Pathways), celecoxib (Searle;
Figure 4) and acetyl salicylic acid for the prevention and
regression of familial adenomatous polyposis.
Figure 4. Celecoxib
O
S
NH2
O
H3C
N
N
F
F
F
Celecoxib
(Searle)
Public policy forum - science and policy
issues of tobacco addiction
Nicotine addiction is a widespread problem throughout the
Western world with approximately 3000 people a day
becoming regular smokers, of which, 70% will express the
desire to quit. The industry conceives cigarettes as a whole
package, with nicotine being the product, the cigarette pack
acting as the storage container for a day’s supply, the puff
being the vehicle for nicotine delivery and the cigarette as an
optimized dispenser for drug delivery. The American
tobacco industry has declared that nicotine is used in
cigarettes purely for taste, and not for pharmacological
effect, but patents are available for the use of organic acids
which have been developed to mask the taste of nicotine. In
1982, a study completed by the Federal Trade Commission
demonstrated that nicotine levels in cigarettes had increased
while tar levels had dropped, fueling suspicion that the
tobacco industry recognized that nicotine exerted an
intended pharma-cological effect. This study prompted the
FDA to consider its position concerning the industry and
initiate an investigation into the tobacco industry.
Evidence compiled during the FDA’s investigation proved
that the tobacco industry paid close attention to the quality
and quantity of nicotine in the cigarette blend at every stage
of manufacture. Adequate delivery of nicotine and the
requirement of a high nicotine content when choosing the
tobacco leaf have been, and still are, primary objectives in
the development of the final product. Chemical manipulation of tobacco delivery has been used by the industry to
liberate and increase the levels of free nicotine in the blend,
thereby increasing the tobacco content and increasing the
level of addiction. Other evidence produced during this
investigation included internal memos and studies, and in
particular, one project conducted by a company that was
designed to find a non-nicotine product that would exert the
same psychomotor effects in the advent that nicotine was
removed from the market.
In April 1997, after the investigation had produced sufficient
evidence, the Federal District Court in Greensboro, NC, ruled
that the FDA had jurisdiction under the Federal Food, Drug
and Cosmetic Act, to regulate nicotine-containing cigarettes
and smokeless tobacco products. Since gaining jurisdiction
over the tobacco industry, the FDA has begun to implement a
strategic plan, focused on the younger generation, which will
reduce access to tobacco products, restrict the availability of
vending machines selling these products, reduce the appeal of
smoking to children via the toning down of advertising and
banning of advertising around schools and by enforcing age
and photographic identification restrictions on people under
the age of 27 years.
The FDA is currently contracting with state officials in order
to police this plan. To date, approximately 12 states have
joined the campaign and by the end of 1998, the FDA hopes
to have implemented its plan in all US states. Only the age
restrictions are being enforced at this time and compliance
checks have already been initiated in participating states.
With a budget of $34 million, and a planned budget of $134
million for 1999, a total of 7000 inspections have already
taken place and this is hoped to increase to approximately
100,000 by the end of this year.
The FDA has set up an internet site at http://www.fda.gov to
increase awareness of its policies concerning this issue.
Poster highlights
Clinical studies
Eli Lilly & Co presented a parallel, randomized, doubleblind study of LY-303870 (lanepitant; Figure 5), a selective
NK-1 antagonist, conducted in 214 outpatients with
moderate to severe lower limb osteoarthritis pain. Patients
were randomized to an initial loading dose of 20, 60, 200 or
600 mg LY-303870, placebo, or 375 mg naproxen, followed
by 10 (n=37), 30 (n=35), 100 (n=34) or 300 (n=36) mg LY303870 bid or naproxen 375 mg bid. The study compared
71
Figure 5. Drugs from poster presentations
H
N
CH3
O
H3C
O
O
O
CH3
H3C
N
N
S
CH3
NH
N
O
CH3
N
H
O
S
N
HBY-097
(Hoechst Marion Roussel)
LY-303870
(Eli Lilly)
Cl
N
H3C
OH
N
O
Br
N
O
H
N
N
N
N
CI-1007
(Parke-Davis)
GR-117289
(Glaxo Wellcome)
H3C
H3C
O
N
O
O
N
NH2
LY-300164
(Eli Lilly)
pain intensity and relief, patient global impression scores,
adjunctive analgesic use and safety assessment between the
three groups. There were no statistically significant differences for the initial dose assessment, however, naproxentreated patients had significantly less pain and used less
adjunctive analgesics than the other two groups during the
multiple-dose period. Although safely administered, both
active drugs were associated with gastrointestinal sideeffects including diarrhea and gastric discomfort.
Pharmacokinetic studies
HBY-097 (talviraline, Hoechst-Roussel; Figure 5), a nonnucleoside reverse transcriptase inhibitor, and indinavir
(Merck & Co) are metabolized via the same metabolic path-
72
way (CYP3A4 metabolism) and may be used in combination
therapy with zidovudine. Hoechst AG, Bayer AG and the
UCSF presented a multiple-dose pharmacokinetic (PK)
study of HBY-097, when given in combination with
indinavir and zidovudine, in order to evaluate any potential
drug interactions. Results showed that HBY-097 induces
CYP3A4 metabolism of indinavir thus indicating a need for
dose adjustments when both these drugs are used
concomitantly.
UCSF also presented a second poster, in collaboration with
Aronex, of a PK study of AR-177 (Zintevir). AR-177, a
synthetic oligonucleotide HIV integrase inhibitor, is more
effective than reverse transcriptase inhibitors in the postinfected, acute phase of HIV, but less effective in the chronic
or latent phase (as it does not affect gene transcription,
protein synthesis, viral assembly or the HIV protease
enzyme). The PK study investigated the single-dose kinetics
of AR-177 in 16 asymptomatic HIV positive subjects, who
received one of four doses (0.75, 1.5, 3 or 6 mg/kg)
administered via a 2-h iv infusion. Results of the study
indicated that AR-177 exhibits complex pharma-cokinetics
with a trend towards decreasing plasma clearance with
increasing dose. A multiple-dose study is ongoing to clarify
the disposition of AR-177.
The Center for Human Drug Research, supported by Glaxo
Wellcome plc, reported on a completed PK study of GR117289 (Figure 5), an angiotensin II receptor antagonist
which was being developed by Glaxo Wellcome for the
treatment of hypertension (but has now been discontinued).
The placebo-controlled, rising-dose trial was conducted in
19 male patients with a diastolic blood pressure (DBP)
between 100-115 mmHg. Following an iv dose of up to 30
mg, regular measurement of BP, plasma renin and
aldosterone took place and treatments were compared using
the time-averaged values over a 12-h (for DBP) or 8-h period
(renin activity and aldosterone). The study concluded that
GR-117289 did not alter DBP in hypertensive patients at any
given dose, while dose-dependent increases in renin activity
and decreases in aldosterone were noted. The main adverse
event recorded during the study was headache, with two
patients withdrawning from the study for this reason.
Parke-Davis & Co displayed a report of three PK studies
conducted with CI-1007 (Figure 5), a dopamine D2 and D3
agonist with mild extrapyrimidal side-effects, under
development for the potential treatment of psychosis and
schizophrenia. The first study was a placebo-controlled,
cross-over trial designed to evaluate the effects of CI-1007 as
a single, oral dose of 0.5, 1, 2.5, 5, 10 or 15 mg (four
subjects/dose group) plus placebo. The second study was a
parallel group, double-blind, dose-escalation trial conducted
in six healthy volunteers and the third study evaluated CI-
1007 (5, 10 and 15 mg doses) in 16 schizophrenic patients.
The first study was terminated at the 15 mg dose due to
adverse events; an increase in growth hormone and decrease
in serum prolactin levels was observed in all three trials. A
safety and tolerability study in CYP2D6 poor metabolizers is
ongoing.
Results of two single- and multiple-dosing PK studies of LY300164 (Eli Lilly), an orally available, potent, selective
AMPA receptor antagonist for the treatment of epilepsy
were also presented at the meeting. The two studies
represent the first experience of LY-300164 in healthy
subjects. A total of 24 healthy males received a single dose of
up to 100 mg and 16 male subjects received multiple doses
of 20 mg tid, or escalating doses of 30 to 60 mg tid for ten
days. Adverse side-effects occurred at doses above 50 mg
and included mild drowsiness (subjects showed a decrease
in awareness 2.5 h after dosing, as measured by the Bond
Lader Mood Rating Scale), dizziness, ataxia and
paresthesiae. However, LY-300164 was shown to be safe
with respect to all of the biochemical indices measured.
Bristol-Myers Squibb unveiled the new potent, metabolically-stable, direct thrombin inhibitor, BMS-189664, which
is being investigated for the prevention of deep venous
thrombosis, venous thrombosis following hip and knee
surgery and prevention of stroke in patients with atrial
fibrillation. Intravenous infusion of BMS-189664 has been
shown to be effective in rat and monkey models of venous
and arterial thrombosis and platelet-dependent arterial
thrombosis, respectively. Doses sufficient to inhibit arterial
thrombosis can enable dissolution of preformed venous clots
in rats without increasing bleeding time. In the randomized,
double-blind, placebo-controlled clinical study conducted in
63 healthy subjects, clinically relevant prolongations in aPTT
were observed after iv administration and oral doses above
1000 mg. The drug was well-tolerated and adverse events
were seen in only four patients; these included headache,
diarrhea, gum bleeding, an upper respiratory tract infection
and forearm blister.
Conclusion
The diverse array of topics presented at the lectures, plenary
sessions and symposium resulted in a meeting that was both
informative and stimulating for those who attended. The
organizers successfully managed to bring together
representatives from academia and industry to present both
new and established research which encompassed subjects
from the entire discipline of clinical pharmacology. The
100th Annual meeting will be held in March 1999 in San
Antonio, Texas. Information regarding ASCPT can be found
on the world wide web at http://www.ascpt.org.
Review Neurokinin antagonists
73
Neurokinin antagonists as potential therapies for inflammation and
rheumatoid arthritis
Andreas von Sprecher1, Marc Gerspacher2 and Gary P Anderson3
Addresses
1
Novartis Pharma AG
K-136.3.93
CH-4002 Basel
Switzerland
pain, migraine, emesis, cancer and psychiatric disorders such
as anxiety and schizophrenia [8-10]. This review focuses on
the clinical potential of NK antagonists in inflammatory
diseases, especially rheumatoid arthritis.
2
Rheumatoid arthritis
Novartis Pharma AG
K-136.3.25
CH-4002 Basel
Switzerland
3
Department of Pharmacology
University of Melbourne
Parkville, 3052 VIC
Australia
IDrugs 1998 1(1):73-91
 Current Drugs Ltd ISSN 1369-7056
Introduction
Inflammatory diseases are major global causes of morbidity
and mortality [1]. The importance of inflammatory disease has
stimulated intense research into their pathogenic mechanisms
and better treatment strategies [2]. Considerable evidence
suggests that the very common inflammatory diseases,
rheumatoid arthritis and asthma, are the consequence of
inappropriate T-cell-mediated immunological responses that
trigger secondary effector mechanisms, such as induction of
growth factors or proteases that permanently alter tissue [3,4].
An emerging but controversial body of evidence suggests that
neurokinins may play an important role in disease induction
and progression.
Neurokinins (NKs or tachykinins) are the most important of
the peptides released from neuronal sensory afferents. They
form a family of peptides which share the common C-terminal
sequence Phe-X-Gly-Leu-Met-NH2 [5]. In mammals, three
different NKs, substance P (SP), neurokinin A (NKA), and
neurokinin B (NKB), act as neurotransmitters and neuromodulators [5,6]. SP and NKA are products of the same gene
(preprotachykinin I), which is expressed both in the central
(CNS) and peripheral nervous systems [5], whilst NKB is
produced by a distinct gene (preprotachykinin II), which is
selectively expressed in the CNS [5]. Based on the different
rank order of potency of natural NKs, three distinct NK
receptor types have been identified, which have been
termed NK1 (SP-preferring), NK2 (NKA-preferring), and NK3
(NKB-preferring) [7]. The existence of these three receptors
has been supported by the development of selective receptor
agonists (synthetic ligands), the development of receptorselective competitive antagonists, and, ultimately, the
isolation and cloning of the three receptor proteins [6]. With
few exceptions, the expression of the NK3 receptor is
confined to the CNS, whilst NK1 and NK2 receptors are
widely distributed throughout the CNS and peripheral
nervous systems [7]. NK antagonists have been proposed to
have potential utility in a wide range of pathological
conditions, including inflammatory processes such as asthma,
rheumatoid arthritis and inflammatory bowel disease (colitis),
The synovium is richly innervated in all species, including
humans, by sensory nerves [11-13]. A subpopulation of the
sensory nerves, the vagal C-fibers, and a minor population
of spinal Aδ fibers [14], contain a number of neuropeptides
including NKs and calcitonin gene-related peptide (CGRP).
The physiological function of neuropeptides in the sensory
nerve fiber endings is unknown, but it is established that
activation of the sensory nerves leads to both afferent signals
to the CNS and to the local release of neuropeptides within
the synovium. This local release of neuropeptides causes an
acute inflammatory response, termed neurogenic inflammation, characterized by vasodilation, microvascular
leakage, recruitment of inflammatory leukocytes, promotion of histamine release from mast cells and the release
of cytokines from the invading leukocytes [15]. Growth
factors, such as leukemia inhibitor factor (LIF) and nerve
growth factor (NGF) [16] are implicated in the proliferation
of NKs-containing nerves and in the induction of NK genes
[11]. It is possible that these growth factors may also directly
stimulate non-neuronal tissues to produce NKs [16]. Indeed,
in chronic disease, non-neuronal cells, such as tissue
macrophages and epidermal cells, may be the major sources
of NKs that drive disease [16]. These effects of neuropeptides released during neurogenic inflammation are
mediated through NK1 and NK2 receptors expressed by
target cells in the joint and mimic several of the typical
features of rheumatoid arthritis [14,17,18]. Whilst neuronal
versus non-neuronal sources of neurokinins may vary in
their contribution to disease, it is likely that neurogenic
mechanisms may be involved in the development of
symmetrical rheumatoid arthritis, where inflammation in
one joint is often followed by inflammation and hyperalgesia in the contralateral joint [19]. Contralateral joint
inflammation has been observed in a strict monoarthritis
model where latex spheres were injected intraarticularly to
induce disease: neuronal processes may be one form of
communication between the joints in this model. This
response was accompanied by a concurrent increase in SP-like
immunoreactivity (SPLIR) content of both the ipsilateral and
the contralateral dorsal root ganglia, and contralateral joint
inflammation was attenuated by inhibition of small unmyelinated nerve activity. Similarly several groups [20-24] have
demonstrated increased SP-ergic innervation of the inflamed
synovium in experimental arthritis. Ahmed et al also
demonstrated that whilst surgical denervation attenuated
ipsilateral joint inflammation, it did not prevent it [25].
Interestingly denervation reduced, but did not prevent,
increased levels of SP in the inflamed joint [25], strongly
suggesting local non-neuronal production. In experimental
arthritis, spinal SP content appears to peak early, perhaps
reflecting acute hyperalgesia: Garret and colleagues [26] found
74
È-preprotachykinin mRNA levels to be increased one day after
induction of experimental joint inflammation (but not at day
10) and Malcangio and Bowery [27] found no increase in
release of SP from spinal cord sections one week after
induction of inflammation. These data suggest that increases
in NK mRNA levels may be transient or subject to rapid
modulation during exacerbations of disease. Conversely,
Hanesch et al [28] used in situ hybridization to demonstrate
that the proportion of dorsal horn cells expressing
preprotachykinin A mRNA was increased, both acutely and
chronically, in experimental monoarthritis. Few studies have
addressed the role of neurokinins in human disease. Elevated
levels of SP have been detected in human rheumatoid
synovial fluid [29,30], at levels correlating with the proinflammatory cytokine interleukin (IL)-6 [31]. Anichini et al [32]
found significantly higher serum SP levels in rheumatoid
arthritis patients in comparison to healthy controls. Sacerdote
et al [33] found that the elevated levels of SP in their patients
decreased following therapy with non-steroidal antiinflammatory drugs (NSAIDs). Gold salt therapy has also
been shown to reduce synovial SPLIR in human rheumatoid
arthritis patients [34].
Pain
NKs may prove to be important mediators of pain in
inflamed joints. Early studies demonstrated that peptide
concentrations, particularly of SP, but also of CGRP, NKA
and NPY, increased in the regions of the dorsal horn
processing afferent projections from affected joints in
experimental models of rheumatoid- and osteoarthritis [3538]. More recent research suggests that hyperalgesia is
accompanied not only by increases in SP content, but also by
compensatory changes in NK receptor function and a
possible failure of GABA-ergic inhibitory pathways to
adequately suppress SP-ergic nociception [39]. The receptor
subtype(s) mediating nociception remain uncertain, with
evidence for the contibution of both NK1 [40] and NK2 [41] to
hyperexcitability of dorsal horn neurons and pain.
Effector mechanisms
The acute effects of NKs are consistent with direct promotion
of inflammation, causing at least short-term increases in
leukocyte adhesion and diapedesis, plasma exudation,
alterations in blood flow, and, probably, some contribution to
hyperalgesia through release of secondary mediators. Despite
the indications from animal models, it is not known if NKs
contribute to more chronic inflammation. The proliferation
of SP-ergic fibers is consistent with a role in long-term
inflammation. SP promotes tissue-destroying superoxide
and hypochlorous anions via an NK1 receptor-mediated
mechanism in synoviocytes and tissue macrophages [42].
Macrophage induction of coagulation products and protease
induction have also been demonstrated, at least in vitro. It
seems possible that autocrine/paracrine production of SP by
macrophages may mediate at least some of the disregulation
of matrix metalloproteinase/tissue inhibitor of metalloproteinase imbalance that contributes substantially to pannus
formation and joint degradation. Although SP is a weak
coangiogenic factor in vitro, there is little compelling
evidence to suggest that SP or NKA have an important role
in altered microcirculation, endothelial cell phenotype or
proliferation. SP is, however, almost certainly involved in
reciprocal induction of the cytokines IL-1 and IL-6, which
exert potent proinflammatory effects in joints [31,43]. The
role of NKs in driving the underlying T-lymphocytemediated immune responses that cause chronic joint disease
is uncertain. SP is a weak co-mitogen for human and animal
T-cells, but there are, however, no firm reasons to believe
that NKs fundamentally affect either primary T-cell
response or committed T-cell effector function to a great
extent. Furthermore, it is possible that SP may mediate at
least part of its effects on lymphocytes by non-receptormediated direct G protein activation in a manner analogous
to that occurring in human mast cells.
One possibility that has important implications for therapy
is that NKs may exert both pathogenic and protective effects.
Bileviciute et al [44] used a model of immunoprophylaxis of
adjuvant-induced arthritis to demonstrate that reduced
disease severity was actually accompanied by increased
SPLIR and NKA-like immunoreactivity at 24 h after injection
of Freund’s complete adjuvant. Similarly, Garrett and
coworkers [26] found that dorsal horn α-preprotachykinin
increased acutely in experimental arthritis (24 h) and in the
joint at 10 days following challenge. However, Carleson et al
[45] have consistently demonstrated increased SPLIR in the
perfused joint during sustained inflammation, whereas
Jeanjean et al [46] showed that dexamethasone, a highly
effective anti-inflammatory drug, decreased IL-1β-induced
long-term increases in axonal SP transport suggesting, on
balance, a proinflammatory effect.
Conclusions
Although the role of NKs in inflammatory disease conditions
is not fully elucidated, there is growing evidence that
neurokinins may play a pivotal role both in disease induction
and in the establishment of chronic inflammatory disease. The
advent of safe effective antagonists of NK receptors is likely to
contribute important new knowledge of disease pathogenesis.
Hopefully, these advances may lead to better therapies which
are more specific and with fewer side-effects than the treatment with currently prescribed anti-arthritis drugs (NSAIDs
and steroids). With the exception of Lilly’s lanepitant, which
had no effect in clinical trials for the treatment of ostheoarthritic pain, there are no reports of NKs antagonists in
clinical trials for the treatment of rheumatoid arthritis. It is too
early to draw any conclusions; however, it can be speculated
that non-selective (dual NK1/NK2) antagonists will be more
suitable to treat inflammatory diseases than selective NK1
antagonists like lanepitant. Further work is needed to clarify
whether additional blockade of the NK3 receptor and
penetration of the blood-brain barrier can improve the antiinflammatory profile of NKs antagonists, or whether
peripheral blockade of NK1 and NK2 receptors is preferable.
With the emergence of dual NK1/NK2 antagonists and
compounds able to unselectively block all three receptors
these questions can be addressed.
Neurokinin antagonists (Tables 1-8)
(Comments on the compounds in the tables)
Tables 1-8 depict the neurokinin antagonists currently under
investigation. With the exception of some important
compounds, this is restricted to non-peptide compounds, as
peptides often lack oral bioavailability and metabolic stability,
limiting their clinical usefulness. The list of compounds is by
no means complete but, every effort was made to include
representative compounds of each important structural class.
The neurokinin antagonists are sorted firstly according to
receptor selectivity and secondly according to the chemical
structure. A tabular format has been chosen in order to
provide quick access to the relevant in vitro (receptor binding)
and in vivo (emphasis on inflammation and pain) data. The
tables also contain available clinical data, state of development
and the most likely indications. The comments section below
does not repeat the references given in the tables.
Selective NK1 antagonists (Tables 1-4)
Quinuclidine- and piperidine- derivatives (Table 1)
The first non-peptide NK1 antagonist CP-96345, a (2S,3S)-cis- 3(2-methoxybenzyl)-amino-2-benzhydryl-quinuclidine, was
described by Pfizer in 1991. The first lead structure that was
eventually developed into CP-96345 was identified by a
random screen. CP-96345 is a highly active and selective,
competitive, NK1 antagonist. Further modifications of the
structure of CP-96345 led to the discovery of CP-99994,
where the quinuclidine ring system has been replaced by a
more simple piperidine ring and a phenyl substituent acts as
a replacement for the benzhydryl residue. CP-99994 is
highly active in vitro, but the compound suffers from poor
oral bioavailability, short duration of action and L-type
calcium channel interaction. Nevertheless, CP-99994 underwent clinical trials for asthma and pain. Whereas in the
asthma trial CP-99994 showed no beneficial effect, a small
effect could be observed in the pain clinical trial, but not
significant enough to justify further development. After
these disappointing experiences, Pfizer turned its interest to
another structure, CP-122721, which is also a piperidine
derivative, bearing an additional trifluoromethoxy substituent. This structural modification led to an increased binding
affinity to the NK1 receptor, in comparison with CP-99994. In a
protein plasma extravasation (PPE) model in vivo, CP-122721
exhibited high oral activities (ED50 values in the 10 to 50 µg
range). Despite its attractive pharmacological profile in an
emesis clinical trial, CP-122721 showed activity only when
applied in combination with a 5-HT3 antagonist. Like the
parent compounds, CP-122721 also displayed significant Ltype calcium channel interaction, which could potentially
limit its clinical usefulness. However, no side-effects were
reported in this clinical trial.
Merck has also synthesized piperidine NK1 antagonists
based on the structure of CP-99994. Replacement of the 3amino group by an oxygen and a modified benzyl
substituent (3,5-bis-trifluoro-methyl) led to the development
of L-733060, a highly potent NK1 antagonist with reduced L-
Selective NK1 antagonists
Table 1. Analogs of CP-96345, 2,3-substituted piperidine derivatives
Compound
O
Stage of development
Indication
Phase I (pain)
Suspended
NH
Tool: First non-peptidic
SP antagonist
Inflammation
H3C
In vitro pharmacology
and clinical trials
hNK1: IC50 = 0.77 nM (IM-9
cells) [47].
Pain
L-type calcium channel
interaction (27 nM)
N
CP-96345
(Pfizer)
O
H3C
Phase II (asthma)
Phase II (pain)
Suspended
Phase II (emesis)
NH
Poor oral bioavailability
and duration of action
N
H
interaction
(3 µM)
CP-99994
(Pfizer)
75
hNK1: Ki = 0.17 nM (IM-9
cells) [51].
Asthma: no effect on
bronchoconstriction and
cough induced by hypertonic
saline in asthmatic subjects
(250 µg/kg, iv)
Pain: peripheral neuropathy:
not better than placebo (0.2
mg/kg, iv).
Dental surgery: better than
placebo less effective than
ibuprofen. Short duration of
action (90min, 0.75 mg/kg,
iv) [10]
In vivo pharmacology: emphasis on
inflammation, pain, arthritis
Arthritis model: blockade of painsensitization response in the spinal cord
induced by injection of kaolin and
carrageenin into the rat knee joint.
Inhibition of IL-2 production by
lymphocytes and IL-3 production by
bone marrow cells. Inhibition of SP-,
capsaicin-, or antidromic nerve
stimulation-induced PPE in different
species and tissues. Inhibition of SPinduced decrease in blood pressure and
cigarette smoke-induced airway edema
in rats. Inhibition of nociception: acetic
acid writhing in the mouse, hot plate test
in the mouse or the reaction time after
pressure of the rat paw inflamed with
carrageenin, or with formalin [48-50].
Inhibition of capsaicin-induced PPE in
the guinea pig airway with an ID50 value
of 4 mg/kg, po.
Penetration of blood-brain barrier.
Anti-emetic activity [48].
76
Table 1. Analogs of CP-96345, 2,3-substituted piperidine derivatives (continued)
Compound
O
F
H3C
F
F
O
Indication
Phase II (emesis)
Inflammation, Pain
Migraine
Asthma
Good oral bioavailability
NH
interaction (390 nM)
N
H
CP-122721
(Pfizer)
Preclinical
Emesis
Migraine
Asthma
F
F
F
and clinical trials
hNK1: pIC50 = 9.8 nM (IM-9
cells) [52].
Emesis: Improvement of
acute cisplatin-induced
vomiting in tumor patients in
combination with a 5-HT3
antagonist and almost
complete suppression of
delayed nemesis. No effect
of 200 mg, po, of CP122721 alone on acute
vomiting [10].
hNK1: IC50 = 0.87 nM (CHO
cells) [53].
F
O
PPE model: ED50 = 0.01 to 0.05 mg/kg, po.
Inhibition of capsaicin-induced PPE in the
guinea pig lung with ID50 = 0.01 mg/kg, po
[52].
Potent anti-emetic activity [10].
Inhibition of dural PPE induced by
electrical stimulation of the trigeminal
ganglion in rats with an ID50 value of 212 µ
g/kg, complete inhibition with 1 mg/kg, iv
[54].
F
F
N
H
L-733060
(Merck)
Preclinical
Emesis
Migraine, pain
Inflammation, asthma
F
F
F
F
F
N
H
N
HN
cells) [56].
Inhibition of SP-induced dermal PPE in
guinea pigs: ED50 = 0.009 mg/kg, po
(CP-99994: ED50 = 1.6 mg/kg, po [56].
L-758298: hNK1: IC50 = 2.8
nM (CHO cells).
L-754030: hNK1: IC50 = 0.1
nM (CHO cells) [57].
Potent oral activity against a variety of
emetic stimuli. Rapid conversion of
L-758298 to L-754030 in vivo [57].
in rats and monkeys
Weak L-type calcium
channel interaction
N
L-741671
(Merck)
Preclinical
Emesis
Migraine, pain
Inflammation, asthma
F
F
O
Inhibition of SP-induced dermal PPE in the
guinea pig (ED50 = 0.037 mg/kg, po), PPE
produced by stimulation of the trigeminal
nerve in the dura mater of the rat (ED50 =
0.028 mg/kg, iv) and cisplatin-induced
retching and vomiting in ferrets (ED50 = 1.0
mg/kg, iv) [55].
F
O
O
cells) [55].
F
F
O
F
F
N
H
N
O
HN
N
L-742694
(Merck)
Phase I/II
Emesis
F
F
F
H3C
O
F
F
O
F
N
H
N
O
F
N
Water-soluble prodrug
(L-758298)
N
R
L-758298 (R = -PO3H2), Prodrug
L-754030 (R = H)
(Merck)
Good CNS penetration
(L-754030)
Clinical trials: Antagonism
of SP-induced forearm
vasodilatation in eight
healthy male volunteers
(0.25, 1, or 5 mg L-758298,
iv) [58]
77
Table 1. Analogs of CP-96345, 2,3-substituted piperidine derivatives (continued)
Compound
Indication
Preclinical
Emesis
Migraine
O
H3C
N
N
NH
N
N
N
H
and clinical trials
hNK1: pKi = 10.3 nM (CHO
cells)
hNK1: pKi = 10.5 nM (U373
MG astrocytoma cells) [59].
Dose-dependent inhibition (3 and 10
mg/kg, iv) of PPE in dura mater,
cojunctiva, eyelid and lip, induced by
electrical stimulation of the trigeminal
ganglion in rats.
Anti-emetic activity [60].
hNK1: pKi = 10.6 nM (CHO
cells) [61].
Inhibition of cyclophosphamide-induced
PPE in the bladder of pretreated ferrets
(0.3 mg/kg, sc). Synergistic inhibition in
combination with sc dexamethasone. The
combined treatment also inhibited X-rayinduced PPE in the small intestine [62].
Potent oral anti-emetic activity [61].
Selected for clinical
evaluation, but replaced
by GR-205171
Weak L-type calcium
channel interaction
GR-203040
(Glaxo Wellcome)
F
O
F
F
H3C
N
NH
N
Phase II
Emesis
Migraine, pain
Inflammation
N
N
Reduced L-type calcium
channel activity
(pKi = 5.6)
N
H
GR-205171
(Glaxo Wellcome)
PPE
hNK1,2,3
Protein plasma extravasation
Human neurokinin receptor binding affinity
type calcium channel activity. Further modifications of L733060, namely the introduction of substituents to the
piperidine nitrogen, led to even more potent NK1 antagonists
exemplified by L-741671, a 3-oxo-1,2,4-triazolylmethylsubstituted derivative of L-733060. With the aim of further
reducing the L-type calcium channel activity and thus
reducing the potential of side-effects, the piperidine ring
was replaced by the less basic morpholine acetal ring system
leading to the 3-oxo-1,2,3-triazol-5-yl substituted morpholine derivative L-742694. This compound is one of the most
potent NK1 antagonists known. When compared to CP99994, L-742694 was found to exhibit a 100-fold increase in
oral activity in a plasma extravasation model. Further
refinement of L-742694 led to the metabolically more stable
L-754030 and the water-soluble and clinically used prodrug
L-758298.
Another company actively pursuing the piperidine structures
originally discovered by Pfizer is Glaxo Wellcome. GR-203040,
one of Glaxo’s earlier compounds, was selected for clinical
trials but was replaced later by its close analog GR-205171.
This last compound is reported to be under clinical evaluation
for emesis and/or migraine. The structure of GR-205171 can
be regarded as CP-99994 with an additional 5-trifluoro-1,2,3,4tetrazole substituent attached to the 2-methoxybenzyl residue.
This apparently small structural modification led to a
compound with good oral bioavailability as well as low Ltype calcium channel activity.
Perhydroisoindole derivatives (Table 2)
Screening of compound libraries followed by lead
optimization led to the discovery of Rhône-Poulenc Rorer's
early NK1 antagonist, RP-67580. This diarylperhydroisoindole
derivative displayed potent affinity for the rat NK1 receptor
but, binding to the human NK1 receptor was rather moderate.
RP-67580 also suffered from poor bioavailability (accounted to
the amidine structural element), as well as considerable L-type
calcium channel interaction. On the other hand, RP-67580
showed activities in several animal models of inflammation.
Replacement of the amidine by an amide together with the
attachment of an additional phenyl substituent at position 4 of
the perhydroisoindole ring system resulted in the discovery of
RPR-100893 (dapitant). Dapitant was highly active in binding
to the human NK1 receptor with little binding affinity for the
rat receptor. Although dapitant displayed highly attractive in
vivo (iv, po) activities in several models of inflammation and
migraine, it proved to be ineffective in a migraine clinical trial
and was therefore suspended. The follow up compounds
RPR-107880 and RPR-111905 are close structural analogs of
dapitant. The 7,7-diphenyl substitution pattern is replaced by
either a 6-methyl (RPR-107880) or a 6-cyanomethyl (RPR111905) substitutent. Both compounds show improved
binding affinities for the human NK1 receptor and a more
attractive profile in in vivo inflammation models in
comparison with dapitant.
Analogs of FK-888, modified peptides (Table 3)
The pseudopeptide compound FK-888 was identified as a
potent NK1 antagonist through rational drug design starting
from a tripeptide lead structure. Molecular modeling studies
helped to define the essential structural elements (the
indole, the two aryl substituents) and connecting structural
elements necessary for good NK1 binding affinity. FK-888
was tested for anti-inflammatory activities in several in vivo
78
Table 2. Analogs of RP-67580, perhydroisoindole derivatives
Compound
H3C
O
H
Indication
Preclinical
Inflammation, pain
Discontinued
Selectivity for rat NK 1
receptor
N
and clinical trials
hNK1: IC50 = 39.8 nM
(U373MG astrocytoma
cells)
ratNK1: IC50 = 7 nM (rat
brain)
[63,5].
Antinociceptive activity in the mouse
phenylbenzoquinone writhing, ED50 = 0.7
mg/kg, sc, and formalin test, ED 50 = 3.7 mg/kg
locally.
Anti-inflammatory activity in the rat by
inhibition of PPE induced by SP in the urinary
bladder (0.04 mg/kg, iv), or by electrical
stimulation of the saphenous nerve in the
hindpaw (ID50 = 0.15 mg/kg, iv). PPE induced
by sc SP or septide in the hindpaw was also
inhibited [48].
hNK1: IC50 = 13 nM (IM-9
cells)
ratNK1: IC50 = >1000 nM
(rat brain) [64].
Analgesic in the formalin test in guinea pigs
(ED50 = 3.1 mg/kg sc and 11 mg/kg po).
Inhibition of septide-induced PPE in peripheral
tissues (guinea pig trachea): ED50 = 0.1 mg/kg,
iv and 0.33 mg/kg, po.
Inhibition of capsaicin-induced PPE, guinea
pig dura mater: ED50 = 8 µg/kg, po [65].
NH
H
Poor oral bioavailability
O
RP-67580
interaction (200nM)
Phase II (migraine)
Suspended
H3C
H
N
O
O
H
OH
CH3
Migraine: Ineffective in
clinical trials for the
treatment of migraine at
doses up to 20 mg [8].
O
CH3
RPR-100893, dapitant
H3C
H
H3C
Preclinical
Inflammation, pain
Migraine
hNK1: Ki = 4 nM (IM-9
cells) [66].
Inhibition of septide-induced PPE in guinea pig
peripheral tissues (ED50 = 0.02 mg/kg, iv) [66].
Preclinical
Inflammation, pain
Migraine
hNK1: IC50 = 0.3 nM (IM9 cells) [67].
Analgesic in the formalin test in guinea pigs,
ED50 = 17 mg, po.
Inhibition of septide-induced PPE in guinea pig
peripheral tissues (ureter), ED50 = 0.004
mg/kg, po.
Inhibition of capsaicin-induced PPE, guinea
pig dura mater: ED50 = 0.093 mg/kg, po [67].
N
O
H
OH
O
O
CH3
CH3
RPR-107880
H3C
H
C
N
N
H
OH
O
O
O
CH3
CH3
RPR-111905
models in guinea pigs, where it inhibited SP-induced PPE in
the lower trachea and main bronchi. FK-888 also inhibited
cigarette smoke- and vagus nerve stimulation-induced PPE
in the airways of guinea pigs. In an asthma clinical trial (2.5
mg of inhaled FK-888), however, no beneficial effect on
exercise-induced broncho-constriction was observed.
The Sandoz group (now Novartis), starting from a novel
proline thiourea lead, modified the structure to incorporate
aromatic amino acid amides of proline with improved NK1
affinity. Highly potent antagonists, exemplified by SDZNKT-343 contain the same N-methyl-N-benzylamide
residue found in FK-888. This compound exhibited good
oral activity in guinea pig pain models.
Replacement of the hydroxyproline part of FK-888 by 1aminocyclohexoyl led to Menarini’s MEN-10930. This
compound had a comparable affinity to the human NK1
receptor as the parent compound. Another structurally-related
derivative, MEN-11149, showed iv and po activities in anti-
asthma models in guinea pigs. Furthermore, MEN-11149 did
not interact with L-type calcium channels.
Servier also actively pursued the FK-888 lead structure. Its
efforts resulted in the discovery of S-18523, and S-19752
where the simple methyl substituent in the case of FK-888 is
replaced by an alkyl tetrazolyl residue. Additionally, in S19752, the naphthyl group of FK-888 has been replaced by an
indole residue. Both compounds were active in several
analgesia assays in mice and rats, and also inhibited SPinduced PPE in the bladder and bronchi of guinea pigs after
iv application.
79
A completely different peptide-based structure was discovered in the labs of Parke-Davis. The tryptophane derivative
PD-154075 was discovered after systematic modification of a
screening lead (Z-TrpPhe-NH2). The structure of this
dipeptide lead was simplified, leading to conformationally
flexible NK1 antagonists displaying micromolar binding
affinities. In the next step, conformational restraints, in this
case methyl-groups, were introduced into the backbone in
order to enhance receptor affinity and selectivity until a
sufficiently selective and potent compound was found.
Table 3. Analogs of FK-888, modified peptides
Compound
H3C
N
H
N
O
O
O
Indication
Phase II (asthma)
Suspended
Asthma, bronchitis,
migraine
and clinical trials
hNK1: IC50 = 0.72 nM
[68].
Phase I
Inflammatory pain
Arthritis
Migraine
hNK1: IC50 = 0.62 nM
[70].
Preclinical
Asthma
Inflammation
Neurological diseases
hNK1: Ki = 1 nM and 2.5
nM (IM-9 cells and
U373MG astrocytoma
cells) [72,73].
Preclinical
Asthma
Inflammation
Neurological diseases
hNK1: Ki = 2.8 nM (IM-9
cells)
No L-type calcium
channel interaction [74].
N
N
OH
H3C
FK-888
(Fujisawa)
H3C
N
H
N
O
O
O
N
H
Asthma: 2.5 mg inhaled
FK-888 had no effect on
the acute exerciseinduced
bronchoconstriction in
asthmatic patients, but
the recovery times were
shortened [10].
N
N
O
O
SDZ-NKT-343
(Novartis)
H
N
O
H
N
O
NH
Inhibition of SP-induced PPE in the lower
trachea and main bronchi of guinea pigs
(ID50 = 0.1 µmol/kg, iv; complete inhibition
with 2 µmol/kg, iv). Inhibition of PPE in the
airways induced by vagus nerve stimulation
or by cigarette smoke [69].
Withdrawal thresholds to mechanical and
thermal stimuli in a guinea pig hyperalgesia
model in both inflamed and non-inflamed
paws. ED50 values of 0.73 mg/kg, po for the
mechanical hyperalgesia and 1.75 mg/kg,
po for the thermal hyperalgesia [71].
Inhibition of PPE in guinea pig knee joint
induced by Freund’s adjuvant [70].
N
O
CH3
MEN-10930
(Menarini)
O
H
N
NH
O
O
N
H
MEN-11149
(Menarini)
CH3
Inhibition of [Sar9,Met(O2)11]SP-induced
bronchoconstriction in guinea pigs with an
ED50 value of 83 nmol/kg, iv (duration of
action > 3h) and PPE in guinea pig bronchi
with ED50 values of 0.2 µmol/kg, iv, and
0.97µmol/kg, po [74].
80
Table 3. Analogs of FK-888, modified peptides (continued)
Compound
O
H
N
O
N
CH3
O
Indication
Preclinical
Inflammation, pain
Asthma
and clinical trials
hNK1: Ki = 1.5 nM (IM-9
cells)
NK1: Ki = 9.6 nM (rabbit
vena cava) [75].
Potent analgesic activity (iv and po) in the
hot plate test in mice, the
phenylbenzoquinone-induced writhing
response in mice and the formalin test in
rats with ED50 values of 0.018 and 0.56
mg/kg, iv, and 48% inhibition at 2.5 mg/kg,
iv, respectively.
Inhibition of SP-induced PPE in the bladder
and bronchi of guinea pigs with ED50 values
of 0.18 and 0.14 mg/kg, iv [75].
Preclinical
Inflammation, pain
Asthma
hNK1: Ki = 2.4 nM (IM-9
cells)
NK1: Ki = 9.0 nM (rabbit
vena cava) [76].
Inhibition of SP-induced bronchoconstriction in guinea pigs with an oral ID50
value of 50 nmol/kg at 3 min and 70%
inhibition with 0.5 mM aerosol at 3 min.
Duration of action at least 40 min.
Reduction of capsaicin-induced PPE in the
bronchi of guinea pigs with an IC50 value of
26 µg/kg, iv [76].
Preclinical
Asthma, inflammation
hNK1: Ki = 0.35 nM (IM-9
cells) [77].
Inhibition of SPOMe-induced PPE in the
guinea pig bladder (ED50 = 0.02 mg/kg, iv)
[77].
N
N
OH
N
N
+
N
K
N
S-18523
(Servier)
F
O
F
H
N
O
F
O
O
NH
N
N
F
OH
F
F
N
N
N
K
+
N
S-19752
(Servier)
H
N
O HC
3
O
O
H
N
N
H
O
CH 3
PD-154075 (CAM-4261)
(Parke-Davis)
Selective NK1 antagonists, miscellaneous
structures (Table 4)
Sanofi is also involved in the search for NK antagonists.
Starting from a common lead structure that was identified by
random screen, SR-140333 (and also the NK2 antagonist SR48968) was developed. In terms of binding affinity SR-140333
is one of the most potent NK1 antagonists described in the
literature. SR-140333 showed long-lasting activity in antiinflammatory models. Thus it inhibited PPE in the rat paw (iv
and sc), but its anti-arthritic potential may be limited, due to
the fact that SR-140333 had no analgesic activity in rats. The
compound is reported to be under clinical phase I evaluation
(possible indications: migraine, pain, inflammation).
Another important NK1 antagonist, which reached clinical
trials (pain, migraine, suspended because of lack of efficacy) is
lanepitant from Lilly. From a series of tryptophane-based nonpeptide structures, this compound was found to be the most
suitable candidate for further development. Lanepitant
exhibited an attractive profile (potent, long-acting) in several
animal models of inflammation, pain and migraine. However,
in the clinic, lanepitant had no beneficial effects on pain in
acute migraine or chronic osteoarthritis. The absence of any
clinical activity might be attributed to low bioavailability.
Takeda reported the pyridopyridine structural class of
compounds exemplified by N-[3,5-(bistrifluoromethyl)benzyl]-7,8-dihydro-N,7-dimethyl-8-oxo-5-(4-fluoro-phenyl)-6-py-
rido[3,4b]-pyridinecarboxamide as potent NK1 antagonists.
Combination of structural elements from a cholecystokinin
(CCK) antagonist and from already known NK1 antagonists
led to the design of this class of highly potent NK1
antagonists. The specific compound mentioned had very
good oral activity in capsaicin-induced PPE from guinea pig
trachea, thus demonstrating its potential as anti-inflammatory
agent.
Merck also produced a series of tryptophane-based NK1
antagonists exemplified by L-737488. Lead compounds
(tryptophane esters) which were eventually developed into
L-737488 were again identified by random screening of
available compound pools. L-737488 exhibited good oral
activity in an anti-inflammatory model (inhibition of SPinduced dermal PPE) in guinea pigs, without having sideeffects on heart rate or blood pressure.
With the discovery of CGP-49823, Ciba-Geigy (now Novartis)
established its own structural class: 1-benzoyl-2-benzyl-4aminopiperidines were developed from a piperazine lead
structure, which was selected from a variety of other selected
compounds for the NK1 screen as a conformationally
constrained analog of the Phe-Phe-structural element of SP
(previously shown to be essential for reasonable affinity to the
NK1 receptor). Because of its ability to penetrate the bloodbrain barrier CGP-49823 was under development as potential
anxiolytic agent.
81
Table 4. Miscellaneous structures
Compound
Cl
Cl
O
N
+
CH3
N
Indication
Phase I
Migraine
Pain
Inflammation
and clinical trials
hNK1: IC50 = 0.01 nM
(IM-9 cells) [78].
Complete inhibition of mustard oil-induced
PPE in the dorsal skin of the hind paw of
rats at 0.1 mg/kg, iv and 1.0 mg/kg, sc
lasting for 24 h.
No analgesic activity after ocular application
of capsaicin in rats. No change of thermal
nociceptive thresholds, or thermal hyperesthesia induced by intraplantar PGE2 [79].
Phase II (Migraine, pain)
Suspended
Migraine, pain, asthma
hNK1: IC50 = 0.15 nM (IM9 cells) [80].
Inhibition of intrathecal (it) NK1 agonistdriven nociceptive behavior in mice with
ED50 of 0.21 nM, it, and 1.73 mg/kg, ip.
Inhibition of neurogenic dural inflammation
following trigeminal ganglion stimulation in
guinea pigs with ED50 = 15 ng/kg, iv, and 91
ng/kg, po [80].
Active in a model of persistent nociceptive
activation by tissue injury (formalin test) with
ED50 values of 4 mg/kg, ip, and 10 mg/kg,
po, 24 h blockade of pain-related behavior
by 10 mg/kg, po [82].
CH3
O
Cl
SR-140333
(Sanofi)
N
O
O
N
CH3
NH
N
O
CH3
N
H
LY-303870, lanepitant
(Eli Lilly)
Preclinical
Inflammation, pain
Asthma
Migraine
F
F
F
O
N
N
CH3
CH3
Migraine, arthritic pain:
The ability of lanepitant
(LY-303870
dihydrochloride
trihydrate) to attenuate
acute migraine pain and
chronic osteoarthritis pain
was assessed in double
blind, placebo-controlled
studies. The compound
was well-tolerated with
no serious adverse
effects. In both studies,
lanepitant had no effect
on pain intensity and
provided no pain relief
when compared to
placebo. In conclusion,
lanepitant was found to
be very safe but to have
no effect on pain in acute
migraine or osteoarthritis
[81].
hNK1: IC50 = 0.21 nM
(IM-9 cells) [83].
Capsaicin-induced PPE: guinea pig trachea:
ED50 = 0.068 mg/kg, po, and 0.017 mg/kg, iv
[83].
F
N
F
F
O
F
EP-00652218
EP-00585913
(Takeda)
F
F
Preclinical
Emesis
Migraine
Asthma
F
NH
O
F
N
H
N
F
O
L-737488
(Merck)
F
hNK1: IC50 = 0.9 nM
(CHO cells) [84].
Inhibition of SP-induced dermal PPE in the
guinea pig with an ID50 of 1.8 mg/kg, po [84].
82
Table 4. Miscellaneous structures (continued)
Compound
N
CH3
H
N
Indication
Phase I
Anxiety
and clinical trials
NK1: IC50 = 12 nM
(bovine retina) [85].
Anxiolytic activity in gerbils (social
interaction) [85].
N
H3C
O
CGP-49823
(Novartis)
Selective NK2 antagonists (Tables 5-6)
Analogs of SR-48968 (Table 5)
SR-48968 (saredutant) was described as the first selective
and highly potent non-peptide competitive NK2 antagonist
in 1992. SR-48968 and the selective NK1 antagonist SR-140333
were developed from a common lead structure, which was
identified by a random screen. In several in vivo models SR48968 inhibits a number of NK2-induced effects. SR-48968
inhibits NKA-induced bronchoconstriction (long-acting),
citric acid or capsaicin-induced cough and prevents citric
acid- and ovalbumin-induced airway hyperreactivity in
guinea pigs. In a recently-published clinical trial, SR-48968,
at an oral dose of 100 mg inhibited NKA-induced bronchoconstriction in asthmatic patients. SR-144190, a morpholine
analog of SR-48968 demonstrated a similar pharmacological
profile as the parent compound, with a greatly improved
ability to cross the blood-brain barrier.
Yamanouchi discovered another potent NK2 antagonist of
the SR-48968 type by modification of the piperidine residue
(introduction of a spiro-benzothiophene residue instead of
the N-acetylphenyl substitutuent at position 4 of the
piperidine ring). Besides the nanomolar affinity at the NK2
receptor YM-38336 also exhibits some affinity (~ 700nM) for
the NK1 receptor.
Zeneca obtained a series of highly potent NK2 antagonists
also by modifying the SR-48968 lead structure through the
introduction of different substituents at position 4 of the
piperidine ring. ZD-7944 incorporates the benzamide
residue of SR-48968 into a ring system.
Selective NK2 antagonists, miscellaneous
structures (Table 6)
Menarini identified MEN-10627, a cyclopeptide, as a selective
and potent NK2 antagonist. In vivo (iv and po) this compound
inhibited a variety of NKA-induced effects. Due to the rigid
structure, Menarini claims MEN-10627 to be resistant to
degradation by peptidases and thus the compound is
expected to exhibit a long duration of action.
Table 5. Analogs of SR-48968
Compound
O
HN
N
N
H3C
O
CH3
Indication
Phase II
Asthma
Incontinence
Pain
Inflammatory bowel
disorders
In vitro pharmacology and
clinical trials
Rat, Hamster, guinea pig,
human NK2 receptors Kd =
0.4 - 2.9 nM [86].
Asthma: 100 mg, po, SR48968, inhibited inhaled
NKA-induced
bronchoconstriction in
asthmatic patients [87].
Anxiolytic activity (rodents,
marmosets) Inhibition of NKA-induced
bronchoconstriction in guinea pigs, iv
and po.
Inhibition of citric acid- or capsaicininduced cough in guinea pigs.
Prevention of airway hyperreactivity
induced by citric acid and ovalbumin
challenges in guinea pigs [86].
Preclinical
Asthma
Incontinence
Pain
Inflammatory bowel
disorders
Rat, hamster, guinea pig,
human NK2 receptors pA2 =
9.08 to 10.10 [88].
Similar pharmacological profile as SR48968, with increased bioavailability in
the CNS [88].
Cl
Cl
SR-48968, saredutant
(Sanofi)
HN
O
N
H3C
N
N
O
CH3
O
F
F
SR-144190
(Sanofi)
Good CNS penetration
83
Table 5. Analogs of SR-48968 (continued)
Compound
O
S
O
N
Indication
Preclinical
Asthma
Urinary incontinence
Irritable bowel disease
and clinical trials
NK2 (hamster urinary
bladder): IC50 = 8.9 nM
NK1 (hamster urinary
bladder): IC50 = 680 nM
[89].
Inhibition of NKA agonist-induced
bronchoconstriction in guinea pigs:
IC50 = 20 µg/kg, iv; ID 50 = 405 µg/kg, id [89].
Preclinical
Asthma
Suspended
hNK2: Ki = 0.14 nM
(mouse erythroleukemia
(MEL) cells) [90].
Inhibition of NKA- or [β-ala8]-NKA(4-10)induced guinea pig trachea contractions:
pKB = 9.4 [90].
Inhibition of [β-ala8]-NKA(4-10)-induced
bronchoconstriction in guinea pigs: 100%
protection at 5 µmol/kg po, (- 2 h) (similar
compound disclosed in patent
EP-00630887 [91]).
N
CH 3
Cl
Cl
YM-38336
(Yamanouchi)
H3C
S
O
N
N
H
O
CH3
Cl
Cl
Cl
ZD-7944
(Zeneca)
Glaxo developed its selective NK2 antagonist GR-159897, a
fluoroindole linked via an ethyl spacer to a 4-(phenylsulfinylmethyl)-4-methoxypiperidine residue, starting from a
structurally-related screening lead. GR-159897 showed high
oral activity in a NK2 bronchoconstriction model in guinea
pigs, and in addition, anxiolytic activity has been
demonstrated in rodents and marmosets.
The structure of Rhône-Poulenc Rorer’s selective NK2
antagonist, RPR-106145, is closely related to the structure of
RPR-100893 (selective NK1 antagonist). In comparison with
RPR-100893, the perhydroisoindole backbone of RPR-106145
possesses the enantiomeric configuration at the chiral
centers. Besides an additional hydroxy group at C-5 and an
indole acetic acid residue instead of the 2-methoxypropionic acid substituent, RPR-106145 can be viewed as the
mirror image of RPR-100893.
The selective NK2 antagonist PD-147714 (Parke-Davis) was
obtained using the same strategy as described above for the
discovery of the company’s selective NK1 antagonist, PD154075.
Zeneca discovered a novel class of non-peptide NK2
antagonists. The pyrrolopyrimidine compound, ZM-253270,
exhibited high affinity to hamster NK2 receptors; however,
binding to human receptors was much weaker.
Dual NK1/NK2 antagonists (Table 7)
FK-224, a natural product from microbial cultures, showed
moderate activity at human NK1 and NK2 receptors. The
affinity to rat receptors was only weak. Nevertheless, FK-224
inhibited carrageenin-induced PPE into rat knee joints as
well as carrageenin- and antigen-induced cell accumulation
in the synovial cavity. Due to its activity in asthma models
in guinea pigs, FK-224 was chosen for clinical evaluation. In
clinical trials, FK-224 proved to be ineffective in mild to
moderate asthmatics (no effects on lung function or on
NKA-induced bronchoconstriction). On the other hand, FK224 inhibited bradykinin-induced bronchoconstriction and
cough in asthmatics. This beneficial effect of FK-224 can,
however, be explained by the known bradykinin antagonist
activity of the compound.
The credit for the first really potent dual NK1/NK2 antagonist
can be given to Hoechst Marion Roussel. MDL-105212A,
which is structurally related to SR-48968, exhibits nanomolar
affinities to both human receptors, and it also had good
binding affinity to the guinea pig NK3
receptor. In in vivo asthma models, the compound showed a
variety of beneficial effects when administered intravenously;
however, oral activity remained at a rather moderate level.
Introduction of a morpholine group into the unsubstituted
amide residue of MDL-105212A led to MDL-105172A. This
compound had more balanced binding affinities to all three
neurokinin receptors and improved activity in guinea pig
asthma models, when compared with MDL-105212A.
For the design of its dual NK1/NK2 antagonists, Merck also
used Sanofi’s NK1 (SR-140333) and NK2 (SR-48968) antagonists,
respectively, as lead structures. Modification of the piperidine
residue, as well as the introduction of trifluoromethyl groups
into the benzoyl substituent led to potent (at cloned human
receptors) dual NK1/NK2 antagonists (L-743986 and analogs).
Potent oral activity was demonstrated in an anti-inflammatory
model of asthma in guinea pigs.
Servier’s S-16476, a modified peptide structure, exhibited
only moderate dual NK1/NK2 binding affinities, but was
active in vivo, when administered iv.
84
Compound
Indication
Preclinical
Asthma
Urinary incontinence
Irritable bowel disease
HN
H
N
HN
O O
O
O
NH
NH H
MEN-11420: hNK2: Ki =
2.5 nM [110]
O
HN
O
O
H3C
and clinical trials
MEN-10627: Rabbit
pulmonary artery,
hamster trachea NK2
receptors: pKB = 8.110.1 [91].
MEN-10627: iv and po: inhibition of NKA
agonist-induced urinary bladder contractions
in rats.
Inhibition of NKA agonist- and antigeninduced bronchoconstriction in guinea-pigs.
Reduction of PAF-induced bronchial
hyperresponsiveness to histamine in guineapigs [91-92].
NH
S
N
H
MEN-11420: Improved in vivo potency
(about 10-fold) and duration of action
compared to MEN-10627 [110].
CH3
CH3
MEN-10627
(Menarini)
H
N
OH
O
HN
OH
OH
H3C
O
MEN-11420
(Nepadutant; Menarini)
N
F
S+
Preclinical
Anxiety
Asthma
Inhibition of [3H]GR100679 (NK2 agonist)
binding:hNK2: pKi = 9.5
(CHO cells) and rat
colon membranes: pKi =
10.0 [94].
Preclinical
Asthma
hNK2: Ki = 16 nM [66].
Preclinical
NK2: Ki = 1.4 nM
(hamster urinary
bladder) [95,96].
Preclinical
NK2: Ki = 2.0, 2.2, and
105 nM (hamster urinary
bladder, cloned hamster
and cloned human NK2
receptors) [97].
O
N
H
CH3
O-
GR-159897
(Glaxo)
H
Inhibition of NK2 agonist (GR-64349)induced bronchoconstriction in guinea pigs:
10 µmol/kg, po.
Anxiolytic activity (rodents and marmosets).
Bioavailability in dogs: 87% [94].
NH
N
HO
O
H
HO
O
CH3
RPR-106145
H3C
O
NH
O
O
H
N
O
H3C
O
N
H
O
NH2
N
H
H3C
O
PD-147714 (CAM-2291)
(Parke-Davis)
Cl
HN
H3C
N
N
H3C
N
O
N
H3C
OH
N
N
O
ZM-253270
(Zeneca)
Inhibition of NKA-induced contractions of
hamster trachea:
-logKB = 7.5. 90-fold less potent as a
competitive antagonist of NKA in human
bronchus [97].
85
Dual NK1/NK2 antagonists
Compound
CH3
HO
H3C
CH3
O
H
H
N
HN
H
N
NH2
N
H
O
O
Indication
Phase II (asthma)
Suspended
Asthma, bronchitis
Inflammation, arthritis
O
OH
H3C
CH3
O
O
N
H
H
N
O
O
CH3
NH
O
O
OH
FK-224
(Fujisawa)
O
Preclinical
Asthma
Suspended
CH3
O
H2N
CH3
N
N
O
O
CH3
O
Cl
H
Cl
Cl
MDL-105212A
O
CH3
Preclinical
Asthma
hNK1: IC50 = 4.3 nM (IM9 cells)
hNK2: IC50 = 2.1 nM
(HSKR-1 cells)
NK3: IC50 = 2.5 nM
(guinea pig cerebral
cortex) [102].
Preclinical
Asthma
hNK1: IC50 = 0.2 nM
(CHO cells)
hNK2: IC50 = 1.5 nM
(CHO cells) [103].
Preclinical
Asthma
[104]
O
O
CH3
N
N
N
O
CH3
O
O
Cl
H
Cl
and clinical trials
hNK1: IC50 = 190 nM
hNK2: IC50 = 190 nM
ratNK1: IC50 = 1.7 µM
ratNK2: IC50 = 1.9 µM [68].
Asthma: Inhibition of
bradykinin- induced
bronchoconstriction and
cough in 9 asthmatics (4
mg, aerosol).
No effect on baseline
lung function and no
protection against NKAinduced bronchoconstriction in 10 mild
asthmatic patients (4
mg, aerosol) [10].
No beneficial effects on
symptoms and lung
function in patients with
mild to moderate
asthma (4-week
treatment, 4 mg,
aerosol, qid) [98].
hNK1: IC50 = 3.1 nM (IM9 cells)
hNK2: IC50 = 8.4 nM
(HSKR-1 cells)
NK3: IC50 = 21 nM
(guinea pig cerebral
cortex) [100].
Cl
MDL-105172A
O
H3C
N
F
O
F
N
F
N
CH3
Cl
F
F
F
Cl
L-743986 analog
(Merck)
F
O
F
N
F
N
CH3
Cl
F
F
F
Cl
L-743986
(Merck)
Inhibition of PPE induced by carrageenin
and SP into the knee joint of rats with an
ED50 of 10 µg/knee, as well as carrageeninand antigen-induced cell accumulation in the
synovial cavity [99]. Inhibition of SP-induced
PPE in the lower trachea and main bronchi
of guinea pigs (ID50 = 1.1 µmol/kg, iv;
complete inhibition with 10 µmol/kg, iv).
Inhibition of [β-Ala]NKA- and [Sar]SPinduced bronchoconstriction in guinea pigs.
Inhibition of PPE in the airways induced by
vagus nerve stimulation or by cigarette
smoke [69].
Inhibition of SP-induced PPE in guinea pig
trachea and primary bronchi: ED50 = 0.20
mg/kg, iv. Inhibition of NKA (aerosol)induced respiratory collapse in guinea pigs:
ED50 = 5 mg/kg, iv and capsaicin-induced
increases in pulmonary insufflation pressure:
ED50 = 0.5 mg/kg, iv. Inhibition of capsaicin
(aerosol)-induced effects in guinea pigs :
ED50 = 5 mg/kg, iv and 50 mg/kg, po [101].
Inhibition of SP-induced PPE in guinea pig
trachea and primary bronchi: ED50 = 1
mg/kg, iv.
8
Inhibition of [β-Ala ]NKA 4-10 (aerosol)induced respiratory collapse in guinea pigs:
ED50 = 0.5 mg/kg, iv.
Inhibition of capsaicin (aerosol)-induced
respiratory effects in guinea pigs : ED 50 = 1
mg/kg, iv and 20 mg/kg, po. Lack of CNS
penetration [102].
Inhibition of resiniferatoxin-induced PPE in
guinea pigs (ED50 = 0.3 mg/kg, po) [103].
86
Table 7. Miscellaneous structures (continued)
Compound
Indication
Preclinical
Asthma
and clinical trials
hNK1: IC50 = 85 nM
hNK2: IC50 = 129 nM
Poor affinity for the rat
NK1 receptor [105].
Inhibition of SP-, NKA- and capsaicininduced bronchoconstriction in guinea pigs
with ED50 values of approx 4 µmol/kg, iv, 7µ
mol/kg, iv; and 30% inhibition at
20 µmol/kg, iv.
Abolition of PPE in guinea pig bronchi
evoked by endogenously released
neurokinins under vagus nerve stimulation
at 10 µmol/kg, iv [105].
Indication
and
clinical trials
hNK3: Ki = 0.21 nM
(CHO cells)
NK3: Ki = 0.11 nM
(Guinea pig brain cortex)
NK3: Ki = 0.42 nM
(Gerbil brain cortex)
NK3: Ki = 15 nM (Rat
brain cortex)
h NK1: Ki = 60 nM (IM-9
cells)
NK2: Ki = 73 nM (Guinea
pig ileum) [106].
cells)
NK3: IC50 = 3.7 nM
hNK1: IC50 = 3000 nM
(IM-9 cells)
NK2: IC50 = 790 nM
(hamster urinary bladder)
[107].
H
O
N
HN
O
O
H
N
CH3
N
N
H
O O
O
N
Na
OH
O
S-16474
(Servier)
Compound
H3C
Preclinical
Psychosis, anxiety
O
N
N
N
H3C
O
Cl
Cl
SR-142801, osanetant
(Sanofi)
Preclinical
Asthma
F
H3C
F
CH3
O H3C
O
H
N
N
H
O
H
N
H2N
O
PD-161182
(Parke-Davis)
Preclinical
CNS indications
CH3
O
NH
R
N
SB-223412 (R = -OH)
SB-222200 (R = -CH3)
SB-223412: hNK3: IC50 =
1.2 nM, Ki= 1.0 nM
(CHO cells)
NK3: IC50 = 1.3 nM
NK3: IC50 = 31 nM (Rat
brain cortex)
hNK1: IC50 = >100,000
nM (CHO cells)
hNK2: IC50 = 180 nM
(CHO cells) [108].
SB-222200: hNK3: Ki=
4.4 nM (CHO cells)
hNK1: Ki= >100,000 nM
(CHO cells)
hNK2: Ki= 250 nM (CHO
cells) [109].
Inhibition of intrastriatally-injected senktide
(NK3 agonist)-induced turning behavior in
gerbils at 3 mg/kg, po.
Oral activity, long duration of action (8 h).
Penetration of blood-brain barrier [106].
Inhibition of senktide-evoked increases in
intracellular calcium levels in CHO cells with
a Ke of 0.88 nM and inhibition of senktideinduced increases in spontaneous firing of
guinea pig habenula neurons with a Ke of 5.8
nM [107].
SB-223412: Activity against senktideinduced miosis in rabbits and behavioral
responses in mice with ED50 values of 0.44
mg/kg, iv, and 12.2 mg/kg, po, respectively.
Good oral bioavailability in rats and dogs
[108].
SB-222200: Good CNS penetration; brain
concentrations of twice the plasma
concentrations were obtained in rats [109].
Selective NK3 antagonists (Table 8)
Combination of structural elements from Sanofi’s NK1 and
NK2 receptor antagonists led to the discovery of SR-142801
(osanetant), an NK3 antagonist. Besides high affinity to the
human and to animal (brain) NK3 receptors, osanetant also
displayed considerable affinity to the NK1 and NK2 receptors.
In in vivo models, osanetant was shown to penetrate the
blood-brain barrier and to exhibit a long duration of action.
Osanetant is of particular interest for the treatment of central
nervous system disorders such as psychosis or anxiety.
PD-161182 (Parke-Davis), which has a peptoid structure, is a
potent and selective NK3 antagonist showing only weak
affinities for the NK1 and NK2 receptors.
SmithKline Beecham is pursuing a series of 2,3,4-trisubstituted quinolines as NK3 antagonists. SB-223412 and SB222200 are highly potent and selective NK3 antagonists with
some weak NK2 activity, but have no affinity for other
receptors and ion channels.
With potent and selective NK3 antagonists like the
compounds mentioned above, it will be possible to elucidate
the role of the NK3 receptor in physiological or pathophysiological processes in the central nervous system, and
to define their utility as potential drugs.
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92
Plant-derived anticancer agents currently in clinical use or in clinical trials
Hui-Kang Wang
Address
Natural Products Laboratory
Division of Medicinal Chemistry and Natural Products
School of Pharmacy
University of North Carolina
Chapel Hill
North Carolina 27599
USA
IDrugs 1998 1(1):92-102
Throughout history, plant products and their modified analogs have
been rich sources of clinically useful drugs, including anticancer
agents. This review covers those agents that are currently in clinical
use or in clinical trials as cancer chemotherapeutic drugs, including
vinblastine, vincristine, Navelbine, etoposide, Teniposide, Taxol and,
most recently, Taxotere, topotecan, and irinotecan.
Introduction
Current estimates indicate that there are about 250,000
species of flowering plants on earth, and of these,
approximately 155,000 are found in the tropics [1]. Using
plants as resources for drug discovery can be traced back to
prehistoric time, and has been very efficient. Medicinal
plants used in Chinese traditional medicine and other ethnic
medicine have been employed for centuries in many
countries for treating cancers, and are great treasures for
new drug discovery. However, only recently have systematic efforts been made to isolate and characterize the active
principles from antitumor-active plant extracts. In the past
three decades, numerous cytotoxic antitumor agents and
their analogs have been discovered and developed in many
laboratories, and these have been reviewed [2-7]. This
review covers only those agents that are currently in clinical
use or in clinical trials as cancer chemotherapeutic drugs.
Alkaloids
Camptothecin and its analogs
Camptothecin (CPT, 1) and 10-hydroxycamptothecin (OPT,
2; Chiba) were first isolated from the wood, bark, and fruits
of the Chinese tree Camptotheca acuminata (Nyssaceae) [8].
Sodium CPT was tested for treatment of gastric carcinoma
and other malignancies in the US in the 1970s, but its use
was halted due to the severe side-effects. However, in
China, an effort was made to decrease its toxicity and
increase its activity. The monoammonium glycyrrhizinate of
CPT was found to be effective in decreasing the toxicity of
CPT. Also, a CPT suspension with a particle size of
approximately 1 mm was made for intravenous use. Clinical
trials involving 450 patients showed that this CPT
suspension had a definite therapeutic effect on liver
carcinoma, increasing the percentage of successful surgical
operations from 18% to 49%. The one year survival rate was
raised from 39% to 54%. Clinical studies on 250 cancer
patients revealed that OPT was effective against primary
liver carcinoma, cancer of the head and neck, leukemia, and
gastric carcinoma, with less adverse effects than those
caused by sodium CPT [9].
Camptothecin has drawn great attention worldwide as a
potent inhibitor of DNA topoisomerase I. (Topoisomerases
are responsible for the winding and unwinding of the
supercoiled DNA composing the chromosomes. If the
chromosomes cannot be unwound, transcription of the DNA
message cannot occur and the protein cannot be synthesized.) Structure-activity studies have revealed a direct
relationship between the ability of CPT analogs to inhibit
topoisomerase I catalytic activity and their potency as
cytotoxic agents [10-13]. These studies support the view that
the interaction between CPT and the topoisomerase I-DNA
complex is responsible to the cytotoxicity of CPT and its
analogs. Among the numerous camptothecin derivatives,
topotecan (SKF-104864, TPT, 3; SmithKline Beecham), CPT11 (4; Yakult Honsha), and 9-aminocamptothecin (5;
Research Triangle Institute) are being tested clinically as
anticancer drugs against colon, ovarian, and other cancers in
Europe, Japan, and the US, respectively. These drugs have
shown significant activity in advanced malignancies. On
May 29, 1996, the US Food and Drug Administration (FDA)
approved topotecan for use in patients whose ovarian cancer
had progressed after the failure of first-line chemotherapy.
Topotecan is marketed by SmithKline Beecham under the
name of Hycamtin®. CPT-11 received accelerated approval
in June 1996, one day after the FDA Oncologic Drugs
Advisory Committee recommended approval. The drug is
®
marketed by Daiichi (Irinotecan ), Pharmacia & Upjohn
®
®
(Camptosar ), and Rhone-Poulenc Rorer (Campto ) [14].
Vinca alkaloids
Vinca alkaloids, including vinblastine (6; Gedeon Richter)
and vincristine (7), were first isolated from Cantharanthus
rosea in the 1960s. Since then, more than 90 unique bisindole
alkaloids have been isolated from the genus Cantharanthus
[15]. Vinblastine and vincristine are currently used in the
treatment of various cancers in the US and other countries,
while semisynthetic vindesine (8; Eli Lilly) is now in a phase
II trial for the treatment of leukemia, hepatocellular cancer
[16], and non-small cell lung cancer [17] in South Africa,
Canada, and Europe, respectively. S-12363 (Vinfosiltine, 9;
Servier), another semisynthetic vinca alkaloid derivative,
which has an a-aminophosphate moiety at the C-23 position
of O-deacetylvinblastine, has entered a phase II study for the
treatment of advanced malignant melanoma [18] and
advanced breast cancer [19].
Vinorelbine (5’-noranhydrovinblastine, 10) is a semisynthetic analog that was initially developed by Pierre Potier of
France; it gained approval in France in April 1989 and April
1991, for the treatment of non-small cell lung cancer and
advanced breast cancer, respectively [20]. In 1989, Burroughs
Wellcome acquired the license to develop and market
vinorelbine in North America. Since 1990, Burroughs
Wellcome has been conducting studies of vinorelbine to
meet US FDA requirements for approval and to investigate
its potential for treating other cancers. To date, these have
Review Plant-derived anticancer agents
93
Figure 1. Structures of camptothecin and analogs
CH3
N
CH3
HO
N
HO
O
N
N
O
N
N
H3C
O
H3C
HO
O
H3C
HO
O
O
3 Topotecan
2 10-Hydroxycamptothecin
(Chiba)
N
NH2
CH3
N
O
HO
O
1 Camptothecin
O
N
O
N
O
N
O
N
O
N
H3C
H3C
O
O
HO
HO
O
O
4 CPT-11
(Yakult)
confirmed the earier results obtained from the European
studies. More than 3,000 patients worldwide have received
vinorelbine in clinical studies. The US FDA approved a
®
semisynthetic vinorelbine (Navelbine , Pierre Fabre) for the
treatment of advanced non-small cell lung cancer in 1995 [21].
5 9-Aminocamptothecin
(Research Triangle Institute)
breast cancer [25], acute myelogenous leukemia [26], acute
myelogenous leukemia [27], and in patients with
myelodysplastic syndrome (MDS) and MDS evolving to
acute myeloid leukemia [28].
Colchicine
Homoharringtonine
Cephalotaxus alkaloids, including harringtonine, isoharringtonine, and homoharringtonine (11; Chinese Academy of
Medical Sciences) were isolated from Cephalotaxus
harringtonia [22]. Cephalotaxus alkaloids are inhibitors of
protein synthesis. The effects of harringtonine and homoharringtonine on protein synthesis are: degradation of
polyribosomes; release of completed protein chains; and,
delayed inhibition of initiation of protein synthesis (compared to cycloheximide) without affecting chain elongation
[23]. Research progress on Cephalotaxus alkaloids has been
summarized, including their discovery, nature of antitumor
activity, isolation, structural characterization, synthesis,
biogenesis, and mechanism of physiological action [24].
Homoharringtonine has been clinically tested in advanced
Colchicine (12), an antimitotic alkaloid isolated from
Colchicum autumnale and is a classical drug used in the
treatment of gout and familial Mediterranean fever (FMF)
[29]. Recent research has revealed that colchicine and its
analogs also show anti-flammatory [30], antitumor [31], and
anti-HIV activities [32]. It has been suggested that most of
the biological effects of colchicine are related to its tubulin
binding action. High toxicity has limited the clinical usage of
colchicine. However, colchicinamide (13), a semisynthetic
derivative of colchicine in which the C-14 methoxy group is
replaced by an amino group, showed a therapeutic index
which was 1.75-fold greater than that of colchicine against
various solid tumors. Colchicinamide has been used in
China for the treatment of breast cancer and other solid
tumors [8].
94
Figure 2. Structures of vinca alkaloids
OH
OH
N
HN
N
HN
CH3
CH3
O
O
H3C
H3C
N
O
O
H3C
CH3
O
H
N
H3C
N
O
O
O
H
OH
O
O
H3C
CH3
O
H
N
CH3
H
CH3
6 Vinblastine
O
H
OH
O
O
O
CH3
CH3
7 Vincristine
OH
OH
N
HN
H3C
O
H3C
N
O
O
N
CH3
H3C
CH3
H3C
H
H
N
N
H3C
H
O
CH3
O
CH3
O
O
N
HN
H3C
OH
OH
H
H
OH
O
NH2
NH
H3C
H3C
8 Vindesine
9 Vinfosiltine
N
HN
CH3
O
H3C
N
O
O
H3C
CH3
O
H
N
H3C
O
H
OH
O
O
10
CH3
CH3
O
CH3
O
CH3
P
O
Indirubin
Figure 3. Structure of homoharringtonine
Indirubin (19) is an antileukemic compound isolated from
the Chinese medicine Indigo naturalis (Qing-Dai), a blue
pigment made from the leaves of Baphicacanthus cusia,
Indigofera tinctoria, Polygonum tingctorium, or Isatis tingctoris
[40]. Qing-Dai has been used in a traditional Chinese prescription "Dang Gui Lu Hui Wan", which is used in the
treatment of chronic myelocytic leukemia. Indirubin and its
more potent water-soluble synthetic derivative, N-methylindirubin oxime (20), have been used for treatment of chronic
myelocytic leukemia in China [41]. Interestingly, indirubin
and indigo have been identified from the urine of an acute
myelomonocytic leukemia patient after admin-istration of
mitoxantrone (Immunex) and etoposide [42].
O
N
O
O
H3C
H
O
H3C
OH
H3C
OH
O
CH3
O
95
O
11 Homoharringtonine
(Chinese Academy of Medical Science)
Terpenes
Taxol
Deacetylcolchicine (14), which showed effectiveness against
Hodgkin’s lymphoma, chronic granulocytic leukemia, and
melanoma, has been in phase II clinical trials [33].
Ellipticine
Ellipticine (15) is an antitumor alkaloid isolated from
Ochrosia elliptica [34] and subsequently from several other
species of Ochrosia, including O acuminata [35], and Bleekeria
vitiensis [36]. Ellipticine is a topoisomerase II inhibitor which
has demonstrated anticancer activity in several animal and
human tumor systems. Three analogs of ellipticine have
been tested clinically. DHE (N-2-(Diethylaminoethyl)-9hydroxy-ellipticinium chloride, 16) is an intercalating agent,
which is currently in a phase II trial in Europe for the
treatment of breast cancer using a weekly regimen [37]. A
phase II trial of NMHE (2-N-methyl 9-hydroxy-ellipticine,
17; Sanofi) was conducted in 57 patients with advanced
2
metastatic breast cancer given as 100 mg/m weekly, and
two complete regressions (of 3 and 12 months) and seven
regressions of over 50% were observed. A total regression
rate of 19% regression was mainly observed in cutaneous or
subcutaneous metastases [38]. The compound has since been
launched. SR-95325-B (retelliptine dihydrochloride, NSC-D626717-W, 18; Sanofi) is another ellipticine derivative having
a very high level of antitumor activity in resistant murine
solid tumor models and was in a phase I trial [39], before
being discontinued.
Taxol (21; NIH), a highly promising chemotherapeutic diterpene which is active against ovarian and breast cancers, was
isolated from the bark of the Pacific yew tree, Taxus brevifolia
[43]. Since yields of taxol are very low and collection of the
bark destroys the tree, which is an endangered species,
worldwide attempts have been made to identify other
species as sources of taxol or related compounds for
semisynthesis. A recent review has covered all aspects of
taxane diterpenoids, including their isolation, total
synthesis, and biological and SAR studies [44]. With a new
synthetic process [101], baccatin III (22), extracted from a
renewable source, Taxus baccata, is converted into taxol, and
then formulated. The FDA approved the natural form of
taxol (paclitaxel®) in December 1992 for treatment of
metastatic ovarian cancer after failure of first-line or
subsequent chemotherapy. The FDA has further determined
that the semisynthetic paclitaxel® is bioequivalent to that
produced from Pacific yew bark and has granted marketing
approval for a semisynthetic form of paclitaxel® (BristolMyers Squibb) for treating of certain cancers of the breast
and ovary. The use of taxol for treating metastatic breast
cancer received marketing approval in April 1994. A shorter
course of infusion for metastatic ovarian cancer (3 h instead
of 24 h) was approved in June 1994 to reduce the incidence
of neutropenia.
Figure 4. Structures of colchicine and derivatives
O
O
H3C
H3C
CH3
O
O
H3C
NH
H3C
NH
CH3
O
O
O
H3C
O
H3C
12 Colchicine
H3C
NH2
O
O
O
H3C
H3C
O
H3C
NH2
O
H3C
13 Colchicinamide
14 Deacetylcolchicine
96
Figure 5. Structures of ellipticine derivatives
CH3
CH3
CH3
HO
N
N
N
H
N
H
H
CH3
CH3
N
+
Cl
CH3
15 Ellipticine
16 DHE
CH3
N
+
CH3
O
H3C
CH3
O
CH3
N
H3C
N
H
N
HN
CH3
HO
O
N
H
CH3
H
Cl
CH3
17 NMHE
18 SR-95325B
(Elf Sanofi)
Figure 6. Structures of indirubin and derivatives
O
N
H
N
OH
H
N
N
H
N
H3C
O
O
19 Indirubin
In a separate action, an FDA advisory panel recom-mended
against immediate approval for a semisynthetic compound,
docetaxel (23, Taxotere®; Rhone-Poulenc Rorer). Docetaxel
has greater cytotoxic effects than taxol on human tumor cells
[45]. Although docetaxel is effective against advanced breast
cancer and certain types of lung cancer, the FDA committee
stated that further studies of side-effects, especially effects
on the immune system, are required. Docetaxel is
apparently more toxic to patients than taxol. However,
Rhone-Poulenc Rorer officials have said that side-effects
could be controlled through additional medication.
Taxol possesses a 6-8-6 tri-ring taxane skeleton, with nine
chiral centers; it was immediately chosen as a total synthesis
target by competent organic chemists after its discovery.
After a challenge of more than 20 years, the goal was finally
20
achieved independently in 1994 by Holton et al and
Nicolaou et al [46-47].
Taxol exhibits an unique mechanism of action. It promotes
polymerization of tubulin and stabilizes the structure of
intracellular microtubules. This process effectively inhibits
the normal dynamic reorganization of the microtubules that
is necessary for interphase and mitotic functions [48].
Lignans
Podophyllotoxins
An important anticancer lignan is podophyllotoxin (24;
Hafslund Nycomed), isolated from Podophyllum peltatum or
Podophyllum emodii (Berberidaceae). The aqueous extracts of
the roots or rhizomes of Podophyllum peltatum were called
podophyllin and were included in the first US
97
Figure 7. Taxol derivatives
O
O
O
O
N
H
H3C
H3C
O
H3C
H3C
CH3
O
O
CH3 OH
CH3
HO
HO
HO
O
O
O
CH3 OH
CH3
CH3
OH
O
H
O
O
O
O
H
O
O
O
O
H3C
H3C
21 Taxol
22 Baccatin III
O
O
O
O
CH3
H3C
H3C
H3C
H3C
N
H
CH3
O
CH3 OH
O
CH3
OH
HO
O
O
H
O
O
O
H3C
23 Docetaxel
(Rhone-Poulenc Rorer)
Pharmacopoeia (USP 1820). It was used hundreds of years
ago as a cathartic and anthelmintic by the American Indians
and by natives of the Himalayan mountain region. The
American colonists subsequently used podophyllin as an
emetic. The antimitotic properties of podophyllin were first
discovered in 1946, and chemical analysis of podophyllin
revealed a number of cytotoxic lignan compounds,
including podophyllotoxin [49]. Due to the high toxicities of
these natural products, Sandoz initiated a semisynthetic
podophyllotoxin derivative program. The two most
successful anticancer analogs from this program were
etoposide (VP-16 or VP-16-213, 25) and teniposide (VM-26,
26). VM-26 was synthesized in late 1965 and VP-16 about
nine months later. VM-26 was first tested in man in 1967,
and VP-16 in 1971. Sandoz commercialized VM-26 in some
countries in 1976 under the names of Vumon® and
Vehem®. Etoposide, which was approved by the US FDA
for the treatment of testicular cancer in 1983 and was
introduced to the US market as VePesid® by Bristol-Myers
Squibb, is one of the most active anticancer agents in the
treatment of testicular teratoma, Hodgkin's and nonHodgkin's lymphomas, small-cell lung cancer, and a variety
of other malignancies. Both VP-16 and VM-26 appear to be
useful compounds in the treatment of certain tumors.
Although they differ somewhat in their pharmacokinetic
profiles, there is no evidence that one drug is superior to the
other in a specific tumor type; their clinical toxicities are also
similar [50].
A number of reviews have been published and cover all
aspects of VM-16 and VM-26, including their chemistry,
biology, pharmacokinetics, and clinical applications. Among
these papers, two excellent reviews have detailed the
discovery of etoposide and the development of podophyllotoxin derivatives [51-52]. Two recent reviews have
summarized hundreds of newly synthesized VP-16 analogs,
their biological evaluation and their bioanalysis [53-54]. Due
to problems encountered with VP-16 or VM-26, such as
98
Figure 8. Structures of lignans
O
H
H 3C
O
O
H
O
OH
H
H
OH
HO
O
O
O
O
O
O
H
O
H3C
O
O
CH3
O
H3C
O
OH
24 Podophyllotoxin
(Hafslund Nycomed)
O
H
O
O
N
H
O
O
O
CH3
26 Teniposide
H
H3C
O
OH
25 Etoposide
H
O
O
CH3
H3C
H3C
H
O
O
H
O
O
O
H
H 3C
H
O
H
O
OH
HO
O
O
O
H
H
O
H
S
H
H
OH
HO
O
O
N CH3
HO
H
NH
O
H 3C
O
H
H
O
O
O
O
O
O
H
H 3C
O
H
O
O
HO
H
CH3
H3C
O
O
O
O
CH3
H3C
O
O
CH3
OH
O
OH
P
HO
O
O
27 Etoposide phosphate
(Bristol-Myers Squibb)
28 NK-611
(Nippon Kayaku)
toxicity, poor water solubility, and the development of
resistance by tumor cells, investigators are exploring new
synthetic analogs for new lead compounds with an
enhanced therapeutic index, extensive therapeutic scope and
higher water-solubility. From these endeavors, three analogs,
etoposide phosphate (27; Bristol-Myers Squibb), NK-611 (28;
Nippon), and GL-331 (29; University of North Carolina), have
entered clinical trials.
Etoposide phosphate
Etoposide phosphate (BMY-40481, 27; Bristol-Myers Squibb)
is a new water-soluble analog of VP-16. It acts as a prodrug,
probably through activation by plasma phosphatase. In vitro,
27 was less potent than VP-16; however, in animals, 27 is
converted to VP-16 within a few minutes, regardless of
mode of administration [55]. The phase I clinical and
29 GL-331
(University of North Carolina)
pharmacokinetic study of oral etoposide phosphate has been
reported [56-59]. The recommended oral dose of 27 is 25
2
mg/m /day for 5 days, every 3 weeks in high-risk patients
[59].
NK-611
NK-611 (4’-demethylepipodophyllotoxin-9(2-deoxy-2-dimethylamino-4,6-O-ethylidene)-b-D-glucopyranoside, 28) was
developed by Nippon Kayaku (Tokyo, Japan). It is a new
water-soluble derivative of VP-16 with potent antineoplastic
activity [60]. A dimethylamino group was introduced into
the sugar moiety to make the water-soluble amine salt. The
simultaneous determination of 28 and its metabolite (DeNK611) in dog plasma, by column-switching HPLC, has been
reported [60] and the clinical pharmacokinetic studies of 28
have been performed [61,62]. The maximum tolerated dose
of NK-611 administered daily for 21 consecutive days was
12.5 mg/day. The dose-limiting toxicity is granulocytopenia.
The recommended doses for further phase II clinical trials
2
are 10 mg/day [61] or 20 mg/m for 5 consecutive days
every 4 weeks [62] for patients who had never been treated,
or have undergone previous chemotherapy only once.
GL-331
Recently, GL-331 (29), a new derivative of epipodophyllotoxin, discovered and developed in Dr Lee’s laboratory
and licensed to Genelabs, CA, USA, completed phase I
clinical trials as an anticancer drug at MD Anderson Cancer
Center, TX, USA in May 1993, and entered phase IIa trials in
Taiwan in January 1997. GL-331, a novel topoisomerase II
inhibitor of the epipodophyllotoxin family, has been
evaluated in a variety of in vitro and in vivo systems, and has
shown activity in both VP-16-sensitive and resistant cell
lines and in animal models. The selectivity and potency of
GL-331 against tumors were evaluated by the National
Cancer Institute (NCI) using the in vitro screening of drug
cytotoxicity against a human tumor panel, consisting of 60
tumor cell lines derived from nine histological types. Results
indicated that leukemia, non-small cell lung cancer, colon
cancer and renal cancer exhibited preferential susceptibility
to GL-331. GL-331 achieved optimal therapeutic effect
against L1210/0 (13.5 mg/kg) in terms of ILS, long-term
survival, and tumor burden reduction at approximately half
the dosage of VP-16. GL-331 also outperformed VP-16 in
terms of ILS, long-term survival, and tumor burden. The
final publication of data from phase I clinical trials of GL-331
is not available yet, however, some details have been
discussed in a review article [54].
Flavones
Flavone-8-acetic acid
Numerous flavonoids have been isolated as cytotoxic
antitumor agents. Flavone-8-acetic acid (FAA, NSC-347512,
30; Merck & Co) is a synthetic derivative of the basic
flavonoid skeleton with a unique form of preclinical
antitumor activity, but its mechanism of action is still not
known. The phase I clinical trials of FAA were conducted in
the US [63], France [64], UK [65], and New Zealand [66] for
the treatment of advanced malignant melanoma. One result
of these trials is evidence that treatment of patients with
FAA and rIL-2 induces the synthesis of nitric oxide, a
physiological mediator and potential cytotoxic agent [66].
Development of FAA has now been discontinued.
99
Proteins
Ricin
Ricin is a plant protein isolated from Ricinus communis [67].
This widely used toxin consists of two 30,000 to 32,000
molecular weight polypeptide chains, A and B, linked by a
disulfide bond. After binding to the cell via the B-chain, the
A-chain subunit crosses a cell membrane by an ill-defined
mechanism to reach the cytosol where it inactivates the 60S
ribosomal subunit and, thus, terminates protein synthesis. A
single A-chain molecule can inactivate 1500 ribosomes/min
and, thus, very few A-chain molecules, perhaps as few as
one, are sufficient to kill a cell. This extreme potency makes
this protein an attractive candidate for monoclonal antibody
targeting. The majority of recent research has concentrated
on the use of A-chain immunotoxins in which the
monoclonal antibody is attached to purified A-Chain via a
disulfide bond [68]. To date, phase I/II clinical trials of some
monoclonal antibody-ricin A chains have been conducted by
Xoma for the treatment of metastatic melanoma [69], colon
cancer [70], and B-cell lymphoma [71] in the US.
Conclusion
The new plant-derived anticancer agents and their analogs
that are currently in clinical use or in clinical trials as cancer
chemotherapeutic drugs have been briefly reviewed. To
develop these anticancer agents, the cooperation between
botanists, chemists, and biologists is necessary and should
be further encouraged and strengthened. However,
compared to the huge number of plants which exist on
earth, only a small number of species have been explored,
and even then incompletely. In most cases, the polar or
water-soluble constituents, or the macro molecules of the
plant extracts have not been thoroughly investigated and are
worthy of further study. Recent significant advances in
biological assay methodology, isolation technology and
spectroscopic structural determination, will be helpful in
solving these problems. Nine anticancer drugs isolated or
developed from plant sources have been approved for
clinical use in the US; these include vinblastine, vincristine,
Navelbine, etoposide, Teniposide, Taxol and, most recently,
Taxotere, topotecan, and irinotecan (Yakult). This continuing
success guarantees an optimistic future. New anticancer
agents, especially anti-solid tumor agents, will be continuously discovered. Continuing searches among medicinal
plants and the semi-synthesis of analogs of lead active
principles will undoubtedly lead to further examples of
novel plant-derived anticancer agents.
Figure 9. Structure of flavone-8-acetic acid
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•• Conversion of taxol from baccatin III.
Review Mucosal immunity 103
Mucosal immunity elicited by DNA vaccines
Taff Jones
Address
Experimental Vaccines
Centre for Applied Microbiology & Research
Porton Down
Salisbury
Wilts SP4 0JG
UK
Tel: +44 198 061 2615
Fax: +44 198 061 1096
E.mail: [email protected]
IDrugs 1998 1(1):103-108
The induction of mucosal immunity could improve vaccine
prophylaxis by preventing pathogens from colonizing their host.
As a consequence, the delivery of vaccines to mucosal surfaces has
become a crucial issue. DNA vaccines, administered by intramuscular injection or by particle bombardment of the epidermis,
have revolutionized vaccine research in the last few years, but
these routes of delivery are unlikely to elicit mucosal immunity
efficiently. This article reviews the progress being made towards
meeting the vital challenge of efficiently eliciting mucosal
immunity through the appropriate delivery of DNA vaccines.
Introduction
Over the last two decades, several factors have given fresh
impetus to the development of novel vaccines. Drugresistant strains of an increasing number of pathogens
continue to give worldwide cause for concern. In both the
developed and developing world, large populations
continue to die from vaccine-preventable diseases, new
diseases continue to emerge and ’old’ diseases are reemerging. Vaccine research has flourished as a consequence,
and in these stimulating times, there have been some
significant developments.
It is now generally accepted that systemic humoral immune
responses are both insufficient and inadequate to combat the
range of different pathogens which we are exposed to. For
example, it is thought that over 95% of human and animal
pathogens gain access to their host cells across a mucosal
surface. It has been argued that the induction of immune
responses at the appropriate mucosal surface could at least
limit, if not abolish, the transmucosal passage of pathogens,
minimizing colonization and access to host cells. Although
most vaccines are currently delivered by intramuscular (im)
injection, the most efficient way of inducing mucosal
responses is by immunizing the mucosal surfaces. Thus
there is now an emphasis on finding alternatives to im
injection as a means of administering vaccines [1].
Further, it is known that humoral (circulating antibody)
responses are ineffective against viruses and other intracellular pathogens, which require cell-mediated immunity
(CMI) to eradicate the pathogen and infected host cells [2].
Whilst live, attenuated organism vaccines are most efficient at
stimulating CMI, they carry their own risks of eliciting sideeffects in a small number of cases and occasionally, a
worrying reversion to virulence. The current subunit
vaccines, adsorbed onto aluminum salts, induce only weak
CMI, if at all [3].
Newer adjuvants have demonstrated an ability to induce
vigorous CMI and these will ultimately improve subunit
vaccines. Perhaps the most promising development in vaccine
research in recent years, has been the astonishing finding that
im injection of ’naked DNA’ induces long-lasting, potent CMI,
as well as humoral immunity. This finding has opened up a
plethora of possibilities for vaccines, as well as raising some
novel safety and regulatory issues [4]. These latter issues have
been dealt with in a previous review [5]. This review
addresses the induction of mucosal immunity by appropriate
delivery of DNA vaccines.
DNA immunization
DNA vaccines are plasmid DNAs, comprised of a gene
which encodes a defined protein, and expression vectors
which contain the necessary components to allow the gene
of interest to be expressed in eukaryotic cells. Compared
with inactivated pathogens or subunit vaccines, which do
not endogenously synthesize proteins, there are several
advantages in expressing antigens within a cell. The loss of
antigenicity through chemical inactivation or adjuvanting is
avoided; intracellular synthesis of the protein allows for
conformational and post-translational integrity; and intracellular processing of the antigen permits the presentation
+
by MHC class I molecules, which elicit CD8 cytotoxic Tlymphocytes (CTL), which are a vital component of CMI. In
these respects, DNA vaccines resemble live, attenuated virus
vaccines, but with the major safety feature that they cannot
revert to virulence. For these, and for the more practical
reasons that plasmid purification is a generic process and
plasmids are generally more stable than proteins or whole
organisms (which usually require a cold chain for storage
and transport), research into DNA vaccines has increased
rapidly since the first seminal publication in 1990 [6]. There
is now a large amount of published data reporting the
induction of immunity and protection in a variety of animal
models, all of which support the efficacy of DNA vaccines
[4]. Plasmid DNA ‘vaccines’ are also finding application in
the treatment of autoimmune disorders, allergies and
cancers [7,8].
Most of the literature has reported the administration of
DNA vaccines by im, iv or intradermal injection, or by genegun delivery of DNA-coated gold particles to the epidermis.
As demonstrated with subunit or whole-organism vaccines,
the most efficient way of inducing mucosal immune
responses is by direct immunization of a mucosal surface [1].
There are now a few studies which report the successful
induction of mucosal immunity with DNA vaccines; these
are discussed in this review.
Induction of mucosal immunity by the genegun
Herrmann and colleagues [9] compared the protection elicited
by gene-gun immunization of a non-mucosal (abdominal
epidermis) and a mucosal (perineal/anal mucosa) route in
mice, using a plasmid encoding a rotavirus antigen. The
animals were subsequently challenged with homologous
virus and protection was assessed by determining the levels of
fecally shed virus. Complete protection was achieved by
immunizing mice perineally with a fifth of the dose used to
immunize epidermally, suggesting that the mucosal route of
immunization elicited immunity against a mucosal pathogen
more efficiently than the non-mucosal route. Intestinal IgA
was not detected prior to challenge in either case.
Mucosal responses were also elicited in the female genital
tract using the gene-gun [10]. A plasmid containing a
reporter gene was used to transfect vaginal mucosa in rats.
Control animals were immunized via abdominal epidermis
and in both cases, a boost was administered by the same
route. A third group received a primary dose epidermally
followed by a vaginal boost. All immunization regimes
resulted in significant titers of specific IgG and IgA in serum
which persisted for 14 weeks post-boosting. All immunization regimes also elicited substantial titers of IgA and IgG
in vaginal secretions at six weeks post boosting, but IgG and
IgA antibodies were only detected thereafter in animals
primed and boosted vaginally.
Thus epidermal immunization with the gene-gun can elicit
mucosal immunity and protection against a mucosal
pathogen, though the mechanisms of protection remain
unresolved. Epidermal immunization may directly elicit
immune responses at mucosal surfaces distant from the site
of immunization, or transudation of serum antibodies may
occur either spontaneously or from disruption of epithelial
barriers as a result of infection. However it is clear from both
these studies that direct immunization of mucosal tissue is
the more efficient means of inducing mucosal immunity.
Direct application of DNA to mucosal tissues
A number of studies have attempted to induce mucosal
immune responses through the direct application of DNA to
mucosal surfaces. Although not as effective in inducing
protective efficacy against lethal challenge as im immunization (which resulted in 95% survival in immunized
animals), the intranasal instillation of a flu hemagglutininencoding DNA vaccine to mice resulted in 76% survival,
although the dose used was half that of the im dose [11].
Both routes of immunization elicited detectable but low
serum antibody titers. The gene-gun immunization was as
effective as im injection in protecting epidermallyimmunized mice, and elicited serum antibody titers which
were 3- to 5-fold higher.
Similar studies, though with a different outcome, have been
reported. The intranasal instillation of mice with a plasmid
encoding flu hemagglutinin, with or without coadministered cholera toxin (CT), a mucosal adjuvant, failed
to elicit detectable antibodies in serum, saliva or nasal
washes, although antibody forming cells (AFCs) were
detected in both spleen and lung cell suspensions when CT
was included in the instillate [12]. Although im injection of
the same plasmid elicited full protection against challenge
with live virus, mice immunized by intranasal instillation
were not protected.
The ability to induce mucosal immune responses by
intranasal instillation of a plasmid encoding Herpes Simplex
Virus glycoprotein B, with or without CT, was compared
with im injection of the same plasmid [13]. The former
regime induced a specific IgA response in vaginal
secretions, which was enhanced by co-administration of CT,
whereas im injection induced specific IgG in serum, and
specific IgG but not IgA in vaginal secretions.
Although the intranasal route of DNA administration
elicited CMI in vivo, as assessed by delayed-type hypersensitivity (DTH), it was less efficient than the im route in
protecting immunized mice against vaginal challenge [13].
Furthermore, and contrary to current dogma, the intranasal
administration of DNA failed to prevent the transmucosal
passage of HSV, despite successfully eliciting mucosal
immunity.
Inoculation of plasmids by the intranasal route has been
directly compared with other mucosal routes, namely po,
intrajejunally and via the buccal mucosa, [14]. Using a
measles virus hemagglutinin, CTL activity in restimulated
spleen cells was assessed after inoculating mice with a single
dose of unformulated plasmid or plasmid plus CT.
Unformulated plasmid given by the intranasal or buccal
routes elicited the most vigorous CTL responses, with the po
and intrajejunal routes eliciting CTL responses which were
approximately 50% lower. The formulation of the plasmid
with CT slightly diminished the CTL response following
administration intranasally or via the buccal mucosa, but in
contrast, more than doubled the responses to plasmid given
po and significantly enhanced the response to plasmid given
by the intrajejunal route.
Other studies have demonstrated that intravaginal
inoculation of mice with a plasmid encoding the HIV
envelope glycoprotein, gp160, yielded detectable anti-gp160
IgA and IgG antibodies in vaginal washes which were
capable of neutralizing HIV in syncytia-formation neutralization assays [15].
Thus direct application of DNA vaccines to mucosal tissues is
feasible and elicits systemic and mucosal immunity and
protection. Although DNA is taken up relatively efficiently by
myocytes following im injection, the transfection of epithelial
cells may be less efficient in the absence of a transfectant or
facilitator, and the variability of some of the data emerging
from the studies described are consistent with this.
Lipid formulated DNA
Lipids can facilitate the entry of macromolecules into cells,
presumably through perturbation of the cell membrane.
Lipids have been widely used to transfect cells with DNA,
either as classical liposomes using phospholipids or as
complexes with novel cationic lipids. Both formulations have
proved successful in the immunization of mucosal epithelia.
Most applications of lipid-complexed DNA have used the
intranasal route of immunization to elicit mucosal immune
responses. The formulation of a plasmid DNA-lipid complex
containing the reporter gene, luciferase, and the cationic
lipids, DMRIE/DOPE, when administered intranasally to
mice, showed a 30-fold higher expression of the enzyme in
nasal mucosa compared to that achieved by naked DNA,
and elicited a vigorous, broad spectrum, immune response
[16]. Anti-luciferase IgA and IgG were detected in serum, as
were specific IgA and IgG in vaginal fluids, indicating that
intranasal immunization with a lipid-complexed plasmid
induced systemic and mucosal antibody responses. An
assay with a monoclonal antibody to the secretory
component confirmed that the vaginal IgA was mucosally
derived. Lymphoproliferation of in vitro stimulated spleen
cells was observed in the immunized animals, while spleen
and iliac lymph node effector cells also showed a
substantial, specific CTL activity against MHC class I
restricted target cells expressing luciferase. Thus this study
demonstrated that the intranasal instillation of lipid-DNA
complexes was a simple and potent mechanism of
stimulating humoral and cellular, systemic and mucosal
immunity, and these studies have now been extended to
non-human primates with similar results [T Jones, personal
communication].
In addition to assessing the induction of CTL activity
following inoculation of nasal or gut mucosae with a
plasmid encoding the measles virus hemagglutinin, the
effects of adding the cationic lipid, DOTAP, to the plasmid
prior to administration were also investigated [14]. The
authors had previously observed a decrease in the CTL
activity following nasal administration, and a substantial
increase in CTL activity following po or intrajejunal
administration, of CT- adjuvanted plasmid. Using DOTAP
as the adjuvant instead of CT, these effects were enhanced.
DOTAP complexed plasmid diminished the CTL activity
even more than CT when administered intranasally, but
enhanced the CTL activity nearly 3-fold over unformulated
DNA when given po or intrajejunally.
In another study, the induction of humoral and cellular,
systemic and mucosal immune responses were compared by
im or intranasal administration of a plasmid encoding an
HIV-1 protein [17]. An im injection elicited serum IgG and
fecal IgA titers, which were enhanced by co-administration
of the plasmid with the lipid, monophosphoryl lipid A
(MPL). The intranasal administration resulted in serum IgG
and fecal IgA at titers comparable to those induced by im
injection, and again, co-administration of MPL enhanced
both titers but enhanced the fecal IgA titers to sigificantly
higher levels. Both im and intranasal routes of
administration elicited cellular responses. DTH activity,
which was enhanced by co-administration of MPL, was
observed in all immunized animals, as was specific CTL
activity. Induction of DTH activity was comparable by both
routes, but CTL activity induced by intranasal instillation of
plasmid was higher than that induced by im injection.
There is no doubt that complexing with lipids facilitates the
transfection of DNA and the subsequent induction of
mucosal immunity, though the issue of toxicity of many of
the cationic lipids needs addressing, as does the limited
stability of lipid and liposomal preparations. The successful
induction of mucosal immunity by co-administration of
MPL, a stabilized lipid adjuvant, suggests that the intranasal
immunization with lipid-formulated DNA vaccines may be
a realistic prospect.
PLG entrapped DNA
The po route is a convenient and non-invasive means of
delivering vaccines, and allows access to the largest mucosal
surface in mammals, the gastrointestinal tract (GIT). The GIT
is rich in immune inductive tissues, comprising lymph
nodes, lymphoid follicles and aggregations of lymphoid
follicles known as Peyer’s patches. Collectively these tissues
are called the gut-associated lymphoid tissue (GALT) and as
part of the common mucosal immune system, immunization
of the GALT elicits mucosal immune responses both locally
in the gut as well as in the respiratory and genitourinary
tracts [18].
However, to protect against hydrolysis and proteolysis,
orally delivered antigens should be protected by a barrier.
This is thought to be particularly true for DNA which has a
half-life of minutes when injected im [19] and would be
expected to be even shorter in the gut. This has commonly
been achieved by encapsulation of antigens in poly(lactideco-glycolide) (PLG) microparticles [20]. PLG degrades by
non-enzymic hydrolysis to the normal body metabolites,
lactic and glycolic acids and is therefore biodegradable and
biocompatible. As internal sutures and as human or
veterinary implants, PLG has acquired a documented
history of safe use [20].
Orally administered PLG microparticles ≤10 µm in diameter
are readily phagocytosed from the lumen of the gut by M
cells, the specialized epithelial cells which overlie Peyer’s
patches, and whose function it is to transport particulate
material to antigen presenting cells underlying the dome of
Peyer’s patches. The presentation by dendritic cells within
Peyer’s patches leads to T- and B-cell interactions and the
subsequent initiation of the IgA cell cycle, ie, the induction
of mucosal immunity. In contrast, the uptake of soluble
antigens by Peyer’s patches is less efficient and occurs across
villi with subsequent processing by macrophages in the
lamina propria which may have a suppressive effect on the
subsequent immune responses, ie, induction of tolerance.
Systemic immune responses result from a concomitant
redistribution of microparticles from Peyer’s patches to the
spleen. Thus, po administration of microencapsulated
antigens can elicit both systemic and mucosal immunity [2124]. To pursue the interest in the induction of mucosal
immunity, The Centre for Applied Microbiology and
Research have investigated the oral delivery of PLGmicroencapsulated plasmid DNA vaccines.
The process of microencapsulation involves emulsifying an
aqueous solution of antigen in a solution of PLG in organic
solvent [25]. Shear, which is detrimental to the physical and
biological integrity of DNA, is generated during emulsification, but conditions have been established in our
laboratory which allows plasmid DNA to be encapsulated in
microparticles ≤10 µm diameter with the retention of
biological activity [26].
Figure 1. Serum anti-luciferase IgG
Serum anti-luc IgG
Enc po
Non po
Non im
Enc ip
Non ip
0
500
1000
1500
2000
2500
3000
End point titer
The figure shows the serum anti-luciferase IgG titers 6 weeks after immunizing mice with a single 50 µg dose of encapsulated DNA by ip
injection or gavage (Enc ip and Enc po). Control animals received 50 µg of DNA by ip or im injection (Non ip and Non im) or by gavage (Non
po).
To determine whether encapsulated plasmid DNA would
elicit an immune response, mice were immunized with a
single dose of naked or encapsulated plasmid DNA
containing the gene encoding the insect enzyme, luciferase.
Comparison of the serum IgG responses elicited in mice by
the administration of non-encapsulated plasmid DNA by im
or ip injection or gavage, with that of encapsulated plasmid,
showed that at six weeks post-immunization, mice
immunized with encapsulated DNA, given by both po and
ip routes, showed enhanced titers of luciferase-specific
serum IgG compared to mice given ’naked’ DNA by the
same or im routes (Figure 1). These responses confirmed
that plasmid DNA formulated in microparticles allowed the
expression of the encoded protein and appropriate
presentation of the expressed antigen to lymphoid tissues.
The time course of induction of the immune responses
elicited by a single 50 µg dose of DNA, following ip injection
or gavage of encapsulated DNA, was investigated. Serum
and fecal pellets were collected at various times postimmunization and assayed for elicited antibodies. The ip
injection of PLG-encapsulated DNA elicited good antiluciferase IgG and IgM and only modest IgA titers. The po
administered encapsulated DNA elicited good responses in
all three antibody classes. Over the course of the study, the
immune responses tended to increase with time although
the IgG response following ip injection, peaked at six weeks
post-immunization [27].
The administration of PLG encapsulated DNA also elicited a
local mucosal antibody response. Very high titers of specific
IgA were detected in fecal pellets from animals immunized po
with encapsulated plasmid [27]. These titers peaked at nine
weeks post-immunization but remained high, 16 weeks postimmunization (Figure 2). The lymphoproliferative responses
of pulsed splenocytes from these mice were also measured
and were found to be 50% higher than those measured in
splenocytes from control animals given naked plasmid im.
Thus orally administered encapsulated DNA is capable of
both enhancing the responses elicited by im injection of naked
DNA, and eliciting cellular immune responses.
Preliminary protection experiments have been performed in
a challenge model for measles. Following gavage of infant
mice with an encapsulated plasmid encoding the measles
virus nucleoprotein, immunized mice were challenged with
live virus. At the time when all control animals had died,
half the immunized animals had survived. Thus the vaccine
induced a substantial degree of protection which dose
escalation experiments will hopefully improve.
The Centre for Applied Microbiology and Research’s
collaboration with John Herrmann’s group at the University
of Massachusetts investigated the immune responses and
protection elicited by encapsulated rotavirus DNA vaccines.
Mice were immunized, po, with a single dose of microencapsulated plasmid DNA encoding VP6, which elicited
anti-VP6 serum antibodies and fecal IgA. Immunized
animals were challenged with live homologous virus and
fecal antigen was reduced to approximately 80% that of
controls. Earlier and higher fecal VP6-specific IgA responses
were observed during the period of viral shedding,
suggesting that the protection was mediated by specific
mucosal immune responses [28]. Although gene-gun
immunization of abdominal epidermis or perineal mucosa
Figure 2. Fecal anti-luciferase IgA
Fecal anti-luc IgA
3 weeks
6 weeks
9 weeks
12 weeks
16 weeks
0
500
1000
1500
2000
2500
3000
End point titer
The figure shows the change with time of fecal anti-luciferase IgA titers elicited by gavage of a single 50 µg dose of encapsulated luciferase
pDNA.
with the VP6 plasmid did not elicit fecal IgA, gene-gun
immunized animals were fully protected against challenge.
Conclusions
Over the last few years, DNA vaccines have made a huge
impact on vaccine research and development and the
prospects for human application of DNA vaccines are
bright, with the results of current human trials being eagerly
awaited. Most of the studies to date have delivered DNA
vaccines to animals by im injection, or epidermally with the
gene-gun. The studies discussed in this review bring
another dimension to DNA vaccines, in offering both
alternative means of delivery which may be more acceptable
to patients, and potential for improving the immune
responses elicited.
Injections through the im route elicit vigorous immunity
and protection, even against some mucosally-acquired
pathogens and largely in the absence of detectable mucosal
immune responses. In these cases, the mechanisms of
protection need to be resolved but notwithstanding, the
precision required in the injection of DNA, the availability of
alternative, non-invasive means of delivery, and the lack of
induction of mucosal immunity will probably argue against
this method of delivery in favor of another.
There is a strong case for using the gene-gun in preference
to im injection. With much lower doses of DNA, vigorous
immunity and protection against challenge are elicited,
though in the few instances where protection elicited by
epidermal or mucosal immunization have been directly
compared [9,10], protection was higher in the perineal/anal
or vaginal immunized groups, indicating a more efficient
induction of protection by mucosal routes. These routes,
however, are unlikely to be acceptable to patients.
At present the gene-gun is somewhat cumbersome but
disposable single-shot units are being developed which will
allow, at higher cost per unit, more flexibility and access to
the buccal mucosae. Perhaps the biggest drawback of this
technology concerns the coating of the gold particles. DNA
is precipitated onto the particles which must subsequently
be stored completely dry otherwise DNA disassociates from
the particles. Since the technique is entirely dependent on
cell transfection achieved by the ‘shooting’ of high-density,
DNA-coated particles directly into cells, this will inevitably
limit the stability of gene-gun vaccines.
Direct application of DNA vaccines is feasible, certainly by
the intranasal route, although the efficiency of uptake would
be greatly enhanced by complexing with lipids. It is possible
that, as with subunit vaccines, comparable responses can be
elicited by the administration of small doses of the vaccine
combined with adjuvant (or in the case of a DNA vaccine, a
transfectant) or larger doses of the unformulated vaccine.
This, combined with concerns over possible degradation of
unformulated DNA, suggests that formulation with a
transfectant might offer both some degree of protection to
the DNA and accelerate its internalization. Vigorous
systemic and mucosal immunity can be elicited by lipidcomplexed DNA, especially when given intranasally, but
such formulations tend to be unstable and until this issue is
resolved, such vaccines will remain experimental. Furthermore, many of the cationic lipids possess a degree of toxicity.
Perhaps the most promising and cost effective approach to
inducing mucosal immunity with DNA vaccines is oral
delivery. The simplicity and convenience of administration
will be widely accepted by vaccinees, whilst the induction of
broad spectrum immunity and protection demonstrates the
efficacy of DNA vaccines delivered by this route. The
encapsulation of DNA in PLG microparticles protects the
DNA, ensures uptake by lymphoid tissues, and as a
lyophilized preparation, adds an extra dimension of stability
to the vaccines. These are compelling reasons to favour this
means of delivering DNA vaccines for use in both the
developed and the developing world.
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Review 5-HT receptors in learning and memory 109
Involvement of 5-HT receptors in learning and memory
François S Roman & Evelyne Marchetti
Address
Laboratoire de Neurobiologie des Comportements
UMR 6562 CNRS
Université de Provence
IBHOP
Traverse Charles Susini
13388 Marseille Cedex 13
France
Tel: +33 0491 28 8724
Fax: +33 0491 98 2697
IDrugs 1998 1(1):109-121
Cumulative evidence indicates that the serotonin (5-HT) system
plays a modulatory role in cognitive processes, particularly in
learning and memory. The present review focuses on the effects of
agonists and antagonists on different 5-HT receptors, administered
during pre-learning, post-learning or pre-retention, in different
behavioral tasks. The effects of these pharmacological compounds
were either facilitative or disruptive, or allowed for recovery from
impaired cognitive performance following the activation or blockade
of 5-HT1, 5-HT2, 5-HT3 and 5-HT4 receptors. Since little or no data
on learning and memory are currently available for the 5-ht5, 5-ht6
and 5-HT7 receptors, this article presents an overview of their brain
locations and possible involvement in these highly cognitive
processes.
Introduction
Since Twarog and Page [1] detected the presence of 5-HT in
the cerebral tissues in 1953, intense research has been
directed towards understanding the effect of this
neurotransmitter on CNS functions. Anatomically, the
serotonergic systems are amongst the most diffusely
organized of the brain, and complex axonal systems project
into almost all regions of the CNS, with particularly dense
innervation of the cerebral cortex and limbic structures.
Consequently, it has been suspected that this neurotransmitter is involved in learning and memory processes. However, the initial studies on behavior using pharmacological
tools modified the activity of all serotonergic pathways at
the same time, so the effects on the now well-characterized
5-HT receptors [2] could not be distinguished. To review
these studies of the effect on learning and memory of the
compounds and doses used for these different receptor
classes (ie, mainly agonist or antagonist), the injection
protocol must be taken into account. The pre-learning phase
allows the study of the effects on acquisition, while postlearning administration can be used to determine the effects
on consolidation. When administration is performed during
pre-retention, the effect is ascribed to the retrieval stage of
learning. This approach was recently proposed in a study of
5-HT1A receptors in learning and memory [3].
This review focuses on the effects of serotonergic
compounds, with particular reference to compounds with
selective affinity for a given receptor subtype, in different
tasks in animal models of mnesic processes.
Table 1. Effects of pre-training administration of 5-HT1A receptor
agonists and antagonists (modified from Meneses and Hong,
1997)
BEHAVIORAL TASK
DRUGS
EFFECTS
8-OH-DPAT
buspirone
ipsapirone
MED
(mg/kg)
0.125
16
8
Isolation-induced
social behavioral
deficit
Open-field
8-OH-DPAT
buspirone
ipsapirone
0.25
8
8
-
Passive avoidance
8-OH-DPAT
buspirone
ipsapirone
gepirone
tandospirone
BMY-7378
NAN-190
0.125
1
-1.2
2.5
2.5
1
2
0
-
Active avoidance
8-OH-DPAT
buspirone
ipsapirone
tandospirone
gepirone
0.1
1
20
1
10
0
-
Repeated acquisition
procedure
8-OH-DPAT
buspirone
0.032
0.032
-
Extinction
8-OH-DPAT
0.015
-
Delayed matching
to position
8-OH-DPAT
0.003
0.3
1
1
0.01
2
0.03
0
+
+
0
0
0
ipsapirone
buspirone
NAN-190
WAY100635
-
Delayed nonmatch
to sample task
8-OH-DPAT
0.2
0
Autoshaping
8-OH-DPAT
0.015
+
Water maze
8-OH-DPAT
0.003
0.25
1
0
-
buspirone
Radial maze
8-OH-DPAT
0.1
0.3
+
-
Delayed conditional
flesinoxan
NAN-190
1
0.1
1
3
0
-
+ enhanced; - impaired; 0 no parametric effect; MED Minimum
effective dose
Effects on 5-HT receptor subtypes
Presently, 14 different types of 5-HT receptors have been
cloned in vertebrates. They are divided into seven distinct
receptor classes [4,5], which are discussed below.
Effects on 5-HT1A receptors
5-HT1A receptor agonists are active in certain behavioral tests
in animals [3,4] (Tables 1 - 3).
In mice, the pre-learning injection (Table 1) of the 5-HT1A
receptor agonists, 8-hydroxy-2-(di-n-propylamino)tetralin
(8-OH-DPAT), buspirone (Bristol-Myers Squibb), and
ipsapirone (Bayer), was found to accentuate the deficit on
the isolation-induced social behavioral deficit test [6]. In
addition, the compounds decreased exploratory activity in
the open-field test. Since the minimum active doses were
very similar in the social behavioral deficit and open-field
tests, it was suggested that a common phenomenon of
increased emotionality or reactivity accounted for both of
these reductions in activity.
In the rat, 8-OH-DPAT decreases the acquisition of learning
in behavioral tasks involving passive and active avoidance,
repeated-acquisition procedures, delayed conditional discrimination, and extinction [7-13]. At doses ranging from 0.003
to 0.3 mg/kg, this agonist has either no effect or enhances
learning in delayed matching to position and autoshaping
tasks [4,14-16], whereas at doses of 0.2 to 0.3 mg/kg, it
decreases learning in water and radial maze behavioral tasks
[17-20]. On delayed nonmatch to sample tasks, 8-OH-DPAT
at 0.2 mg/kg has no effect [21]. Buspirone and ipsapirone
decrease learning in passive and active avoidance,
autoshaping, and water maze tasks; buspirone also
decreases learning in repeated-acquisition procedures [1113,22-26]. In delayed matching to position task, buspirone
has no effect while ipsapirone enhances learning [27,28].
Tandospirone (Sumitomo) at 1 mg/kg improves learning in
active avoidance task, but at a higher dose (2.5 mg/kg)
decreases learning of passive avoidance [9,29]. Gepirone
(Bristol-Myers Squibb) decreases passive and active avoidance at 10.0 mg/kg, and flesinoxan (Solvay), at 1.0 mg/kg,
disrupts
the
acquisition
of
delayed
conditional
discrimination [3,12]. Concerning the effects of 5-HT1A
receptor antagonists, it has been reported that the 5-HT1A
receptor partial agonists, BMY-7378 (Bristol-Myers Squibb)
and 1-(2-methoxyphenyl)-4-[4-(2-phthalamido)butyl]-piperazine (NAN-190), either have no effect or decrease learning
in passive avoidance, delayed conditional discrimination,
and delayed matching to position tasks [3,9,11,27], whereas
the silent 5-HT1A receptor antagonist , WAY-100635 (WyethAyerst), has no effect on the delayed matching to position
task [30].
With post-learning injections (Table 2), 8-OH-DPAT
enhances the consolidation of learning in autoshaping and
extinction tasks [7,31], and either impairs or has no effect
on passive avoidance and water maze tasks [10,28],
respectively. Buspirone and tandospirone decrease
learning in autoshaping and active avoidance tasks,
respectively [32]. Tandospirone at 1.0 mg/kg impairs
consolidation in active avoidance task, but at 2.5 mg/kg
has no effect on passive avoidance [9,29]. In contrast, the
partial 5-HT1A receptor agonist, NAN-190, and the silent 5-
Table 2. Effects of post-training administration of 5-HT1A
receptor agonists and antagonists on learning (modified from
Meneses and Hong, 1997)
BEHAVIORAL
TASK
Autoshaping
DRUGS
8-OH-DPAT
buspirone
NAN-190
S-UH-301
WAY-100135
WAY-100635
MED
(MG/KG)
0.015
1
0.5
0.3
5
0.001
EFFECTS
+
0
0
0
0
Extinction
8-OH-DPAT
0.015
+
Passive
avoidance
8-OH-DPAT
tandospirone
0.09
2.5
-
Active
avoidance
tandospirone
1
-
Water maze
8-OH-DPAT
0.1
0
effective dose
Table 3. Effects of pre-retention administration of 5-HT1A
receptor agonists on learning (modified from Meneses and
Hong, 1997)
BEHAVIORAL
TASK
Delayed
nonmatch
to sample task
DRUGS
AGONISTS
8-OH-DPAT
ipsapirone
MED
(mg/kg)
0.2
0.5
2.5
EFFECTS
0
0
Water maze
8-OH-DPAT
buspirone
0.003
0.01
0
Plus maze
8-OH-DPAT
0.01
0
Passive
avoidance
8-OH-DPAT
buspirone
0.01
0.01
0
-
Delayed
matching
to position
ipsapirone
2.5
0
effective dose
HT1A receptor antagonists, 1-(1-naphthyl) piperazine
hydrochloride (1-NP), WAY-100135 (Wyeth-Ayerst), WAY100635, and S-UH-301 (Solvay Duphar), do not alter the
consolidation of learning in autoshaping tasks [3,33-35].
The effects of the preretention administration of 5-HT1A
agonists (Table 3) reveal that 8-OH-DPAT, at 0.01 and 0.3
mg/kg, and buspirone, at 0.1 and 1.0 mg/kg, impair the
retrieval of learning in passive avoidance [10,23,36]. In
contrast, at 0.1 and 0.2 mg/kg, 8-OH-DPAT has no effect in
the water maze, plus maze, or delayed nonmatching to
sample tasks [18,21,36]. Both 8-OH-DPAT (0.5 mg/kg) and
agonist, CGS-12066B (Novartis), does not modify the escape
deficit in the LH paradigm, but it reduces the ability of the 5HT reuptake blocker citalopram (Lundbeck) and
fluvoxamine (Solvay) to reverse the behavioral deficit
resulting from uncontrollable shocks [41], suggesting that 5HT1B receptor agonists, by their inhibition of 5-HT release,
might reduce the stimulation of 5-HT transmission induced
by 5-HT reuptake blockers. In addition, the mixed 5-HT1B/A
agonist, RU-24969, (Roussel-Uclaf) dose-dependently
increases the spontaneous locomotor activity of rats and
mice [42,43], and the selective 5-HT1B agonist, CP-94253
(Pfizer), also produces hyperlocomotion in rats [44], whilst
5-HT1A receptor agonists [45] reduce locomotor activity in
ipsapirone (2.5 mg/kg) decrease learning in the delayed
matching to position, and delayed nonmatching to sample
tasks [21,37].
Effects on 5-HT1B receptors
In learned helplessness (LH) paradigm rats, 5-HT1B receptors
are upregulated in the cortex, hippocampus, and septum,
and downregulated in the hypothalamus [38,39]. These
results suggest that a change in 5-HT1B receptor responsiveness might be related to the escape deficit. 5-HT release
measured in vivo by microdialysis in the cortex of LH rats
decreases [40], which is compatible with 5-HT1B
autoreceptors being upregulated. The 5-HT1B receptor
Figure 1. Drugs acting at 5-HT1A receptors
N
O
N
N
N
O
N
N
N
H
O
S
Cl
O
H
O
Buspirone
Cl
Ipsapirone
(Bayer)
N
N
O
H
N
N
O
N
N
N
N
N
H3C
H 3C
N
N
N
H
O
H
Cl
Gepirone
O
Tandospirone
(Sumitomo)
N
N
N
O
N
O
CH3
H
Cl
H
Cl
WAY-100635
(Wyeth-Ayerst)
H
Cl
N
enedioxyamphetamine (MDA); and dl-methylenedioxymethamphetamine (MDMA) also enhanced the acquisition
of CRS [50,53,54]. Quipazine has a similar effect on the
conditioned avoidance response (CAR) [55]. On the other
hand, in studies that have examined the effects of various 5HT2A/2C receptor antagonists on learning, ritanserin (Janssen),
glemanserin (MDL-11939, Hoechst Marion Roussel),
pizotifen, and cyproheptadine have all been found to have a
negative action in that they produced significant retardation
of learning, whether measured by the conditioned NM
response or the CAR [48,56,57]. In contrast, the 5-HT2A/2C
antagonists, ketanserin (Janssen), mianserin, d-2-bromolysergic acid diethylamide (BOL) and LY-053857 (Eli Lilly),
had no effects on learning [48,55]. However, other authors
have reported that the 5-HT2A/2C antagonist receptors, 2,5dimethoxy-4-iodoamphetamine (DOI), ritanserin, and ketanserin improve learning in aging or animals with induced
learning deficits [3,58]. For instance, ketanserin enhances the
consolidation of learning, attenuates the learning deficits
induced by aging or hypoxia, and improves the facilitative
effects on memory brought about by the acetylcholinesterase
inhibitor, physostigmine, in aged animals. These findings
are contradictory to, but not incompatible with, the
facilitating effects observed with agonists of these receptors
[48].
mg/kg), also reduce locomotor activity in rats [46]. Finally,
recent data indicate that the 5-HT1B/1D receptor antagonist,
GR-127935T (Glaxo Wellcome), enhances the consolidation
of learning [47].
5-HT2 receptor family
Few studies have concentrated on the effects of 5-HT2
receptors on learning and memory processes, and little
information is available (Table 2 and Table 4) [48]. Initial
reports indicated that d-lysergic acid diethylamide (LSD), an
agonist of the 5-HT2A,2C,1A receptors, produced significant
enhancement in the acquisition of the conditioned nictitating
membrane (NM) response in the rabbit [49,50]. LSD (12.9
µg/kg) was shown to enhance acquisition of the
conditioned NM response. More importantly, LSD enhanced
the acquisition of conditioned responses (CRS) during
classical appetitive conditioning of the jaw movement
response as effectively as it enhances the acquisition of the
defensive NM response [51]. The LSD-induced enhancement
of acquisition was found to be due to the enhancement of
associative learning, with no effect on such non-associative
factors as sensitization, pseudoconditioning, baseline rate of
responding, or to changes in the unconditioned responses
[49,51,52]. Later studies employing the conditioned NM
response indicated that other 5-HT2A/2C agonists, including
dl-2,5 dimethoxy-4-methylamphetamine (DOM); dl-methyl-
Figure 2. Drugs acting at 5-HT1B receptors
H
N
H
N
N
H3C
F
N
H3C
O
O
N
N
F
F
N
CH3
RU-24969
CGS-12066B
(Novartis)
O
H3C
N
H
N
H
CP-94253
(Pfizer)
N
N
N
H
N
CH3
N
O
O
GR-127935T
(Glaxo Wellcome)
CH3
CH3
Table 4. Effects of pre-training administration of 5-HT2A/2C receptor agonists and antagonists (modified from Harvey, 1996)
BEHAVIORAL
TASK
Conditioning of rabbits’
nictitating membrane response
DRUGS
AGONISTS
LSD
DOM
MDA
MDMA
MED
(mg/kg)
0.001
0.074
0.54
0.95
0.32
0.2
2.5
> 0.166
> 1.0
1.25
1.0
> 2.0
> 1.0
0.001
ANTAGONISTS
Ritanserin
MDL-11939
Pizotifen
BOL
LY-53857
Quipazine
Active avoidance
Cyproheptadine
Ketanserin
Mianserin
LSD
Classical appetitive
conditioning
EFFECTS
+
+
+
+
0
0
+
0
0
+
+ enhanced; - impaired; 0 no parametric effect; MED Minimum effective dose
Figure 3. Drugs acting at 5-HT2 receptors
OH
O
O
CH3
H3C
CH3
N
F
N
N
H
S
N
H
O
CH3
N
H3C
CH3
F
Ritanserin
(Janssen)
LY-53857
(Eli Lilly)
N
OH
MDL-11939
Involvement of 5-HT3 receptor family
Involvement of the 5-HT3 receptors in learning and memory
was first studied using the antagonist, ondansetron (Glaxo
Wellcome) [59]. With pre-training injections in mice on a
habituation test, ondansetron (10 ng/kg) facilitated
performance in young and aged animals, and inhibited
impairment in habituation induced by scopolamine,
electrolesions, or ibotenic acid lesions of the nucleus basalis
magnocellularis [60]. Similarly, in the T-maze reinforced
alternation task in rats, ondansetron (100 ng/kg)
antagonized a scopolamine-induced impairment [60]. In an
object discrimination and reversal learning task in the
marmoset, pre-training administration of ondansetron, in
doses ranging from of 1 to 10 ng/kg, was shown to improve
learning during the initial discrimination and reversal tests
[59-63]. In aversive experiments using other 5-HT3
antagonist receptors, granisetron (SmithKline Beecham; 1-10
mg/kg) given as a pre-training injection increased the stepdown latency when mice were tested 24 h after footshock on
a passive avoidance task [64]; tropisetron (Novartis)
improved the retrieval of a previously learned aversive habit
in mice at doses of 1, 10, and 100 mg/kg; and pretreatment
with itasetron (DAU-6215, Boehringer Ingelheim;1, 10, 30,
and 100 mg/kg) antagonized a deficit in the acquisition of a
passive-avoidance response caused by scopolamine
administration in rats [65]. In aged animals, itasetron, at 10
mg/kg injected for 3 weeks, significantly improved task
performance compared to that displayed by the old rats
treated with the vehicle in the Morris water maze task [66].
In the water maze also, the effects of the 5-HT3 receptor
antagonists mirisetron maleate (WAY-100579, WyethAyerst) and ondansetron at doses of 0.001, 0.01, and 0.1
mg/kg injected subcutaneously led to recovery of the deficit
created by combined excitotaxic lesions of the nucleus
basalis and medial septal brain regions. Linear dose-related
improvement trends have been found with both 5-HT3
antagonists in acquisition as well as in retention during
spatial learning in the water maze [67]. Given the
importance of the hippocampus in learning and memory,
and the fact that LTP may provide a substrate for certain
forms of memory, ondansetron in the rat induces a reliable
and dose-dependent (100 and 500 µg/kg) increase in
hippocampal theta rhythm, a significant increase in the
magnitude and duration of LTP, and improved retention in
an odor matching problem and a spatial learning task [68].
The action of the 5-HT3 receptor antagonists may be taskdependent, since ondansetron, over a large dose range (1
ng/kg to 1 mg/kg) injected during the pre-session for 10 to
15 days, failed to attenuate a scopolamine-induced impairment in the stone maze, which is considered to be a complex
spatial memory task [69]. Also, repeated treatments may be
required, as suggested by the finding that itasetron was only
effective with chronic treatment [65].
Moreover, post-training intraperitoneal injection of the
5-HT3 agonist, 1-(m-chlorophenyl)-biguanide hydrochloride
(mCPBG; 1 to 10 mg/kg), has been shown to impair
consolidation in the learning of a lever-press response in
autoshaping, tropisetron (0.01 to 0.1 mg/kg) and
ondansetron (0.1 to 1 mg/kg) improved it, and the effect
induced by mCPBG (1.0 mg/kg) was prevented by each of
these 5-HT3 antagonists at the lower dose. Interestingly, the
individual effects of the 5-HT3 receptor agonists and
antagonists are significantly decreased by p-chloroamphetamine (PCA) pre-treatment during the first and
second autoshaping sessions (10 mg/kg x 2), implying that
activation and blockade of presynaptic 5-HT3 receptors may
be involved in the impairment and enhancement of learning,
respectively [70].
Effects on 5-HT4 receptors
Data suggest that the stimulation of 5-HT4 receptors plays a
role in different aspects of the cognitive processes, eg,
improving the acquisition of learning [4,71-73] but impairing
its consolidation [74]. For instance, the 5-HT 4 receptor
agonists BIMU-1 and BIMU-8 (Boehringer Ingelheim)
improve acquisition on various behavioral learning tasks.
Pre-training stimulation of 5-HT4 receptors improve social
learning, prevent amnesia, and reverse deficits in mice
performing a passive avoidance task following exposure to
hypercapnia and hypoxia at 10 to 20 mg/kg [71-73]. When
administered intraperitoneally immediately after the first
presentation in social olfactory recognition tasks in rats,
BIMU-1 (10.0 mg/kg) enhanced short-term memory (ie,
recognition 2 h later). The effect of BIMU-1 was antagonized
by intraperitoneal injection of 1 and 10 mg/kg GR-125487
(Glaxo Wellcome), a specific 5-HT4 antagonist [75]. Similarly,
pre-training injection of BIMU-1 (5 to 20 mg/kg) or BIMU-8
(20 mg/kg) enhanced acquisition in the autoshaping task,
but post-training administration of these drugs impaired
consolidation at 10 to 20 mg/kg and 5 and 20 mg/kg,
respectively [74]. Injection of the 5-HT4 receptor agonist, RS67333 (Roche Bioscience) (0.1, 10 and 1000 µg/kg) reverses
atropine-induced performance deficits in the rat Morris
water maze assay (30 mg/kg); such an effect is blocked by 5HT4 receptor antagonists [58].
Post-training administration of the 5-HT4 receptor, SDZ205557 (Novartis; 10.0 mg/kg) and GR-125487 (0.78 mg/kg),
or PCA (10.0 mg/kg) pretreatment, does not appear to affect
learning itself, but is able to reverse the effect of the 5-HT4
receptor agonist. This observation suggests the participation
of postsynaptic 5-HT4 receptors [74]. It should be noted that
BIMU-1 and BIMU-8 are mixed 5-HT4 agonists/5-HT3
antagonists. Nevertheless, recent data, using an olfactory
association learning task with systemic pre-training
administration of BIMU-1 at 1.0, 5.0, and 10.0 mg/kg,
revealed a substantial improvement in associative memory
[76]. One month later, difficulty rapidly reversing behavioral
responses to previously learned associations, indicated that
the BIMU-1 effect at 10.0 mg/kg was not transient but
correlated to long-term memory. The effects of BIMU-1 are
most likely mediated by 5-HT4 receptors, since they were
blocked by GR-125487. These data suggest that activation of
5-HT4 receptors may modulate cognitive processes like
learning and memory [76]. Such a view could be explained
by the distribution of 5-HT4 receptors in adult rat brain in
various regions belonging to the limbic system, especially in
the hippocampus, olfactory tubercules, and Islands of
Calleja [77-79].
Suspected roles of 5-ht5, 5-ht6 and 5-HT7
receptors
5-ht5 receptors
At the present time there are no selective ligands to evaluate
5-HT5 function [80]. The possible functions of the 5-ht5
receptors can be predicted based on localization studies. For
example, the limbic distribution in the mouse brain indicates
a possible role in learning and in mood [81], while the
distribution of 5-ht5A mRNA in the cerebellum suggests a
role in the coordination of fine motor skills. The distribution
of 5-ht5 messangers in the habenula is interesting. This
region is a relay station between the limbic system and the
midbrain, and it projects into the interpeduncular nucleus,
raphe nuclei, substantia nigra, and ventral tegmental areas.
H3C
N
H3C
H3C
N
N
O
O
N
N
N
N
H
N
CH3
HN
N
O
O
H3C
Ondansetron
(Glaxo Wellcome)
Granisetron
Mirisetron
(American Home Products)
OH
H
N
HO
OH
HN
CH3
O
N
O
O
OH
HO
N
O
O
HN
O
HO
OH
N
H
CH3
CH3
OH
HN
CH3
O
Itasetron
(Hoechst)
The stimulation of GABAergic neurons in the lateral
habenula inhibits both 5-HT neurons in the raphe and
dopamine neurons in the substantia nigra and ventral
tegmentum. Therefore, it is possible that modulation of 5HT and dopamine release through 5-ht5 receptors has
behavioral consequences. Habenular lesions have been
shown to lead to enhanced exploratory behavior. Together,
these data indicate a possible role for the 5-ht5 receptors in
the inhibition of behavior related to emotional states [80].
The first behavioral studies on possible 5-ht6-mediated
functions were conducted using antisense oligonucleotides in
male rats targeted to the 5-ht6 receptor subtype [83]. In these
studies, the rats exhibited a behavioral phenotype consisting
of an increased number of yawns and stretches. This behavior
was blocked by atropine, suggesting the role of the 5-ht6
receptors in the control of cholinergic neuro-transmission. If
so, then a 5-ht6 antagonist might be useful in the treatment of
learning and memory disorders [82,83].
5-ht6 receptors
5-HT7 receptors
The distribution of 5-ht6 mRNA in limbic pathways,
particularly in the dentate gyrus and CA1, CA2 and CA3 of
the hippocampus [82], also suggests that 5-ht6 receptors may
modulate learning and memory processes. The mRNA
localization of the 5-ht6 receptor in the nucleus accumbens
may also be indicative of a role in reinforcement/reward [82].
There are no selective ligands currently available to study the
5-HT7 receptors. Predictions of possible receptor function
based on studies of the localization of 5-HT7 receptor mRNA
and protein in the septum, hypothalamus, centromedial
amygdala, and anterior hippocampal rudiment also include a
role in the so-called limbic processes [84]. Moreover, studies of
the role of 5-HT7 receptors require the development of
selective subtype compounds.
N
CH3
N
H
O
H
NH
O
N
NH
N
O
H
Cl
O
N
H
H3C
BIMU-1
(Boehringer Ingelheim)
CH3
S
N
O
O
CH3
BIMU-9
(Boehringer Ingelheim)
H
N
O
O
Cl
F
H2N
O
N
H
Cl
N
CH3
O
CH3
CH3
GR-125487
(Glaxo Wellcome)
Conclusions
As a whole, it appears that the 5-HT1A, 5-HT2A/2C, 5-HT3, and
5-HT4 receptors are the main subtypes involved in learning
and memory, whilst the role of the 5-ht5, 5-ht6, and 5-HT7
receptors in these cognitive processes can only be
speculated.
The role of 5-HT1A receptors in learning and memory
processes is controversial. There are studies reporting that 5HT1A receptor agonists enhance learning, while some have
failed to show such improvement, and others have observed
impairments (Tables 1 - 3). It should be noted that the use of
different experimental conditions and different behavioral
tasks can help distinguish true cognitive effects from
locomotor, motivational and emotional effects [24,85]. These
inconsistencies may, in fact, be ascribed to differences in the
behavioral tasks, doses, and drug administration schedules.
This is true for all of the effects reported for the different 5HT receptor families in this review. Although the pre- or
post-training and pre-retention stimulation of 5-HT1A
receptors impairs learning in passive and active avoidance
O
CH3
H
N
CH3
Cl
RS-67333
(Roche)
tasks, the passive avoidance task cannot distinguish
memory from motivational and sensory factors [eg,
24,48,86].
The observed difference between 8-OH-DPAT and buspirone has not yet been explained, but it is noteworthy that
early studies found that buspirone does not affect learning
and memory in humans [60,86], while more recent work has
reported that it disrupts such processes [26]. The effects
induced by the pre-training administration of 8-OH-DPAT
seem to be non-specific, whereas those produced by the
post-training injection of the drug are attributed to changes
in learning [16,30,87,88]; however, 8-OH-DPAT enhances
learning at doses of 0.1 mg/kg [21] or 0.015 to 0.062 mg/kg
[15,87]. When administered 15 [87], 30 [14] or 20, but not 40
or 120 min [21,87] before the training session, 8-OH-DPAT
does not alter visual learning or discriminability [16,18]. The
changes observed in these tasks cannot be attributed to
increments in feeding [15,87], as rats trained with the food
magazine and treated 24 h later with 8-OH-DPAT displayed
normal or enhanced learning [15]. In addition, the silent 5-
HT1A receptor antagonist, WAY-100635, blocks the nonspecific effects produced by 8-OH-DPAT in the delayed
matching to position learning task [14,16]. Hence, it is
reasonable to conclude that pre- or post-learning
administration of 8-OH-DPAT enhances learning regardless
of feeding, and that once learning is consolidated, the
stimulation of 5-HT1A receptors does not alter it [3,15].
The learning and memory effects produced by pre- or posttraining injection of 8-OH-DPAT are eliminated by PCA and
parachlorophenylalanine (PCPA) [15,33] and reversed by the
silent 5-HT1A receptor antagonists, WAY-100135, WAY100635 and S-UH-301 [16,30,33]. These findings suggest that
pre- and postsynaptic 5-HT1A receptors participate in the
acquisition and consolidation of learning.
To conclude, it should be noted that 5-HT1A receptors are: (i)
coupled with inhibition of adenylate cyclase, directly
+
activating a K channel [89]; (ii) interact with other 5-HT
receptor classes and subtypes; (iii) are co-expressed in single
neurons with 5-HT2A/2C and 5-HT4 receptors in the hippocampus and cortex, and other neuronal populations [90,91];
and (iv) affect diverse neurotransmission systems [92,93].
Furthermore, from electrophysiological studies, it has been
shown that 5-HT1A receptors in subregions of the hippocampus, and those located pre- and postsynaptically, exhibit
different physiological responses [91,94,95].
Only a few studies have focused on the behavior of the 5HT2A/2C receptors [48]. Some behavioral paradigms (rabbit
nictitating membrane conditioned response, rat conditioned
avoidance response) have demonstrated the enhancement of
learning 5-HT2A/2C receptor agonists, and that this enhancement is only blocked by drugs that are antagonists of these
receptors [48,49,50]. However, it should be noted that the
available 5-HT2A/2C compounds are not selective for these
receptors [96,97], and some of these display negative
intrinsic activity [48].
Various effects, including improvement of learning and
anxiolysis, are observed with 5-HT3 receptor antagonists
administered systemically and centrally [59,67,98-100]. For
instance, granisetron, tropisetron, itasetron and mirisetron
have precognitive effects, and counteract deficits in learning
associated with dysfunction of central cholinergic neurons
[67,100]. Moreover, the 5-HT3 agonist, 1-(m-chlorophenyl)biguanide (mCPBG), impairs the consolidation of learning,
whereas tropisetron and ondansetron improve it; the effect
induced by mCPBG is prevented by both 5-HT3 antagonists
[70]. Interestingly, the individual effect of the 5-HT3 receptor
agonists and antagonists is significantly reduced by PCA
treatment, implying that activation and blockade of
presynaptic 5-HT3 receptors may be involved in the
impairment and enhancement of learning, respectively [70].
Presynaptic 5-HT3 receptors located in the amygdala and
hippocampus may be involved in learning. In addition, 5HT3 receptors may be located presynaptically (in non-5-HT
neurons, but postsynaptically in 5-HT neurons), possibly on
the soma, axon, and/or nerve terminals of the GABAergic
interneurons [67,100-102]. Electrophysiological studies have
shown that 5-HT3 heteroreceptors modulate the activity of
several neurotransmitters, including cholinergic and gluta-
matergic neurotransmitters [92,93,102]. Previous findings
suggest that postsynaptic 5-HT3 receptors are involved in
learning and memory processes [68].
The pre-training stimulation of 5-HT4 receptors improves
social learning and associative olfactory discrimination,
prevents amnesia, and reverses defects in learning and
memory resulting from hypercapnia and hypoxia [7173,75,76]. Injection of the 5-HT4 receptor agonist, RS- 67333,
reverses the performance deficit produced by atropine; the
effect is blocked by 5-HT4 receptor antagonists [103]. The
post-training administration of the 5-HT4 receptor
antagonists, SDZ-205557 and GR-125487, or pretreatment
with PCA does not affect learning itself, but is able to
reverse the effect of 5-HT4 receptor agonists. This observation suggests the participation of postsynaptic 5-HT4
receptors [74]. As autoradiographic studies have revealed
that 5-HT4 receptors are located in the habenula,
hippocampus, and amygdala [27], and electrophysiological
studies have shown that 5-HT4 receptors mediate a slow but
long-lasting excitatory response in the hippocampus
[77,104], stimulation of the 5-HT4 receptors could modulate
learning and memory processes. The development of new
and more selective agonists will determine future studies of
this receptor.
Anatomical data confirm the location of 5-ht5, 5-ht6, and
5-HT7 receptors on cognitive pathways, especially in the
hippocampus. A better understanding of the role played in
cognition by these, and other 5-HT receptors, will be gained
through the discovery of new specific ligands and new
molecular tools, such as gene knockout and transgenic mice
[4,80].
In conclusion, it is clear that 5-HT receptor families
modulate cognitive processes, such as learning and memory.
Nevertheless, more specific ligands with greater selectivity
for a specific subtype of receptor and more elaborate
behavioral tasks are required to enable the discrimination of
the respective effects of these compounds on the different
subcategories of learning and long-term memory. Such new
compounds would be useful as therapeutic agents for
patients with mnesic deficits, such as those observed in
Alzheimer’s disease.
Acknowledgements
We wish to thank Joël Bockaert and Aline Dumuis for the
preparation of the manuscript. We wish to acknowledge the
expert secretarial assistance of Michelle Bauget.
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GS-4104 Gilead Sciences Inc
Andreas Billich
Address
Novartis Research Institute
General Dermatology
PO Box 80
Brunner Strasse 59
A-1235
Vienna
Austria
IDrugs 1998 1(1):122-128
Gilead and Roche are codeveloping GS-4104, a
neuraminidase inhibitor, for the potential treatment and
prevention of influenza virus infections, type A and type B.
The companies began international phase II/III trials in
December 1997 [272638].
The phase II/III double-blind, placebo-controlled study
involves centers in the US, Canada, Europe and Hong Kong
[272638]. The program will consist of two treatment studies
and two prophylaxis studies, each of which will recruit 750
patients [273331]. The trials involve over 1500 patients, who
will receive either GS-4104 or placebo for 6 weeks, as a
once- or twice-daily dosing schedule [276650], [276895]. The
company has also registered 73 centers for the two
treatment studies, and 55 of these had been activated by the
end of January 1998. Patients in the treatment arm will
receive either GS-4104 or placebo for 5 days. Roche is
conducting similar trials in Europe, Canada and Hong Kong
[276895].
In September 1997, results were announced from two phase
II studies, initiated in May 1997, in which volunteers were
exposed intranasally to influenza A. The patients received
oral capsules of GS-4104, or placebo, once-daily or twicedaily. In the first study, GS-4104 decreased the duration of
symptoms by nearly 50%. In the second study, all patients
who were given GS-4104 prophylactically did not have
detectable levels of virus, as compared to 50% in the placebo
group [264362]. In phase I trials, GS-4104 was welltolerated with good oral absorption and distribution in
blood and tissue [248180].
Introduction
Influenza viruses cause respiratory illness with significant
morbidity and mortality. At present, influenza is managed
mainly by preventative vaccination or by symptomatic
treatment. The success of vaccinations is limited by the
continuous mutation of the influenza virus. Symptomatic
treatment with amantadine and rimantadine (Du Pont Merck)
shortens the duration of symptoms caused by type A virus
strains, but is inactive against type B strains, which account
for about one third of infections. Also, side-effects have been
associated with these drugs and the emergence of resistant
influenza strains has been observed.
The viral enzyme, neuraminidase (sialidase), is a surface
glycoprotein that cleaves sialic acids from glycoproteins and
Originator Gilead Sciences Inc
Status Phase III clinical
Indication Influenza virus infection
Action Viral replication inhibitor, Sialidase inhibitor
Biotechnology Oral formulation
CAS 1-Cyclohexene-1-carboxylic acid, 4-(acetylamino)-5amino-3-(1-ethylpropoxy)-, [3R-(3.α.,4.β.,5.α.)]Registry No: 187227-45-8
Note: GS-4071 - free acid of GS-4104.
Synonyms GS-4109, Ro-64-0796, GS-4071, GS-4116, GS4104 analogs, Gilead
O
O
O
O
N
H
NH 2
glycolipids. The enzyme is responsible for the release of new
particles from infected cells and may also assist in the
spreading of the virus through the mucus within the
respiratory tract. Therefore, neuraminidase is thought to
represent a suitable target for antiviral therapy. Based on the
initial discovery of 2-deoxy-2,3-dehydro-N-acetyl-neuraminic
acid (Neu5Ac2en) as a transition state analog inhibitor of
neuraminidases, a large range of more specific and more
potent derivatives have been synthesized. The present
evaluation decribes the status of one of these, GS-4104, which,
based on data from recent clinical trials [266214], holds
promise for future therapy of influenza infections.
Synthesis and SAR
Derivatives of the transition state inhibitor, Neu5Ac2en,
featuring an amino or guanidino function in the 4-position
have been identified as more potent inhibitors of influenza
A and B virus sialidase than the original lead compound
[254841,158324]. 4-Guanidino-Neu5Ac2en (zanamivir, Biota/
Glaxo) is a highly effective inhibitor of both the sialidase and
the growth of a wide range of influenza A and B strains in
vitro [158324]. Zanamivir has shown efficacy in phase II
challenge studies for the prophylaxis and treatment of
influenza virus infection [280306]. The drug has to be
administered by either intranasal or inhaled routes since it
exhibits poor oral absorption. In addition, the compound is
rapidly excreted [165582]. The search for a molecule with
similar potency, but improved pharmacokinetic properties
lead researchers at Gilead to three major changes of the
zanamivir structure [280310]: (i) the labile dihydropyran
Drug evaluation GS-4104 123
ring was converted to a cyclohexene ring; (ii) the polar side
chain in the 6-position of zanamivir was replaced by a more
lipophilic ether moiety which is accommodated in a
hydrophobic pocket in the active site of the enzyme; (iii) the
4-guanidino group was changed to an amino group. The
resulting compound, GS-4071, was equipotent with
zanamivir in an in vitro plaque reduction assay; the ester
prodrug of GS-4071, designated GS-4104, showed good oral
bioavailability in several species [280310].
The synthesis of multi-kilogram amounts of GS-4104 has
been achieved by a lengthy linear sequence (about 16 steps,
with no chromatography needed) starting from (-)-quinic
acid [280310]. It is reasonable to expect that a more direct
route can be achieved for process development.
Clinical Development
Phase I
The phase I program demonstrated that GS-4104 was well
tolerated with good oral absorption and distribution in
blood and tissue [248180].
Phase II
Eighty volunteers received doses (20 mg, bid, to 200 mg,
qid) of GS-4104 for 5 days beginning 28 h after intranasal
exposure to influenza A. The viral load was reduced by
more than 100-fold in those receiving GS-4104 compared
with placebo; the median time to cessation of symptoms in
GS-4104-treated patients was 53 h versus 95 h in the placebo
group [266214].
Synthesis of several series of derivatives has not yet led to
further improvement of the activity of GS-4071, with the
exception of the guanidino analog, GS-4116. However, this
compound and its ethyl ester showed poor oral
bioavailability [258727,258728,258052].
In a separate prophylaxis study, 37 patients received 100 mg
GS-4104 once or twice daily, or placebo, for 5 days,
beginning 26 h before intranasal exposure to influenza A.
None of the treated patients had detectable virus in nasal
passages compared with 50% of those receiving placebo
[266214].
Pharmacology
Although GS-4104 was generally well tolerated in both
studies, transient, mild to moderate post-dosing nausea was
reported in some individuals [266214].
GS-4071 is a potent inhibitor of influenza neuraminidase (Ki
< 1 nM) [280313]. In a plaque reduction assay in cell culture,
the compound was active against an influenza A strain
(H1N1) (ED50 = 16 nM) [5]. In a more detailed study, GS-4071
showed potent inhibitory activity against a range of
different isolates of influenza H1N1, H3N2, H2N2 and B
strains (ED50 from 0.2 to 14 nM) [271744]. In addition, it was
active against clinical isolates grown on cells as well as in
eggs and in adenoid explants; GS-4071 had a potency and
antiviral spectrum similar to zanamivir and was also active
against certain viruses with reduced susceptibility to
zanamivir [247132].
In a mouse infection model, on oral dosing at 1 mg/kg/day,
the prodrug, GS-4104, exhibited excellent efficacy (87%
survival versus 6% in the placebo group) [271744]. Delaying
po administration with GS-4104 as late as 60 h post-virus
exposure was still inhibitory to a lethal influenza A virus
infection [254764]. In ferrets infected with influenza A virus,
GS-4104 showed efficacy in reducing flu symptoms, such as
pyrexia, nasal cell infiltrate, nasal signs, at an oral dose of 5
mg/kg [256493].
Metabolism
GS-4071 has low (< 5%) oral bioavailability in animals, but
the ethyl ester prodrug, GS-4104, has exhibited good oral
bioavailability in rats (36%), mice and dogs (65 to 100%)
[254763,247131,280313]. Following absorption from the
gastrointestinal tract, GS-4104 undergoes rapid enzymatic
conversion to the active form, GS-4071 [280314].
Furthermore, the parent drug, GS-4071, was delivered into
rat bronchoalveolar fluid following oral administration of
the prodrug [280313].
Toxicity
In a 5-day repeat dose toxicology study in rats, GS-4104 did
not demonstrate any toxic effects with a dose of 300
mg/kg/day, po [271744].
Current Opinion
Currently available modalities for therapy of influenza
infection are suboptimal. Therefore, novel drugs, such as the
neuraminidase inhibitors, are needed, especially for therapy
in elderly patients and in small children. GS-4104 is an orally
bioavailable prodrug which is readily converted in the
gastrointestinal tract to GS-4071, a potent and highly
selective inhibitor of the viral sialidase. Following absorption, GS-4071 is distributed to the bronchoalveolar lavage
fluid where, at least in rats, it is encountered at substantial
and sustained concentrations. GS-4071 is approximately
equipotent to zanamivir against influenza A and B strains.
However, GS-4071 can be delivered orally by its prodrug,
whilst zanamivir can only be used topically. Although
arguments in favor of both routes of application can be
sought, such as the convenience of oral dosage versus more
direct and fast interference at the target site by the topical
route, only extended phase III trials and finally, acceptance
by the market, will determine which of the two approaches
is superior with respect to efficacy, side-effects, and patient
compliance.
A general concern in antiviral therapy is the occurrence of
resistance. Currently, it is not known how important an
issue this will be in the case of the neuraminidase inhibitors.
Any speculations about the superiority of this class of
compound over the M2 protein inhibitors with respect to
development of resistance, appear not to be based on
experimental data. Combination therapy might be the future
strategy for the treatment of influenza infection to potentiate
the efficacy of the drugs and to minimize the risk for
spreading of resistant virus.
A concern voiced by epidemiologists, is the possible
reluctance of people to be vaccinated, once effective drugs
for treatment and prophylaxis are available [256487]. This
argument cannot be used against the introduction of
neuraminidase inhibitors, especially for use in high risk
groups; rather, the issue should be considered as a challenge
for the medical community and public health authorities to
ensure thoughtful use of these drugs.
To conclude from the currently available data, GS-4104
appears to be an effective drug that is based on a
scientifically sound concept and is likely to be successful on
the market.
Licensing
Hoffmann-La Roche AG
Exclusive worldwide rights to Gilead’s proprietary neuraminidase inhibitors, including GS 4104. Roche will make an initial
cash payment to Gilead of $10 million and up to an additional $40 million in cash upon achievement of developmental and
regulatory milestones. In addition, Roche will fund all research and development costs and pay Gilead undisclosed royalties
on the net sales of any products developed under the collaboration [220572].
Development history
DEVELOPER
COUNTRY
STATUS
INDICATION
DATE
REF
F Hoffmann-La Roche Ltd
UK
C2
Influenza virus infection
30-MAY-97
248180
Gilead Sciences Inc
US
C3
17-DEC-97
272638
Hoffmann-La Roche Inc
US
C3
17-DEC-97
272638
Gilead Sciences Inc
Western Europe
C3
17-DEC-97
272638
Gilead Sciences Inc
Canada
C3
17-DEC-97
272638
Gilead Sciences Inc
Hong Kong
C3
17-DEC-97
272638
Canada
C3
17-DEC-97
272638
Western Europe
C3
17-DEC-97
272638
Hong Kong
C3
17-DEC-97
272638
Literature classifications
Key references relating to the drug are classified according to a set of standard headings to provide a quick guide to the
bibliography. These headings are as follows:
Chemistry: References which discuss synthesis and structure-activity relationships.
Biology: References which disclose aspects of the drug’s pharmacology in animal models.
Clinical: Reports of clinical phase studies in human volunteers providing, where available, data on the following: whether
the experiment is placebo-controlled or double or single blind; number of patients; dosage.
Metabolism: References that discuss metabolism, pharmacokinetics and toxicity.
Chemistry
STUDY TYPE
RESULT
REF
Synthesis and SAR
Design, synthesis and structural analysis of GS-4104.
280310
SAR
Synthesis of C3-thia and C3-carba analogs of GS-4071.
258727
SAR
Synthesis of C2-substituted analogs of GS-4071.
258728
SAR
Systemic variation in positions C3, C4 and C5 of GS-4071.
258052
Biology
STUDY TYPE
EFFECT STUDIED
EXPERIMENTAL MODEL
RESULT
REF
In vitro
Activity against influenza
virus.
Plaque reduction assay.
GS-4071 is active against a series
of A and B strains (ED 50 = 0.1 to 14
nM).
271744
In vitro
Activity against influenza
virus.
Assays using cell-, egg-, or
tissue-grown viruses.
GS-4071 shows similar potency and
antiviral spectrum as zanamivir.
247132
In vivo
Survival of mice after
influenza infection.
Oral dosing at 1 mg/kg/day
after infection.
87% survival versus 6% in placebo
group.
271744
In vivo
Survival of mice after
influenza infection.
Oral dosing at 10 and 1
mg/kg/day after infection.
GS-4104 protects from lethal
infection with strain H1N1, even
when administered 60 h exposure to
the virus.
254764
Biology (continued)
STUDY TYPE
EFFECT STUDIED
EXPERIMENTAL MODEL
RESULT
REF
In vivo
Flu symptoms in ferrets.
Oral dosing to ferrets infected
with influenza A virus.
Efficacy in reducing pyrexia, nasal
cell infiltrate and nasal signs at a
dose of 5 mg/kg, bid.
256493
STUDY TYPE
EFFECT STUDIED
MODEL USED
RESULT
REF
In vivo
Penetration of GS-4071 into
rat bronchoalveolar lavage
fluid.
Oral administration of prodrug
to rats. Measurement of
parent drug in plasma and
bronchoalveolar lavage fluid.
Oral bioavailability based on plasma
level is 36%. Significant delivery of
GS-4071 to bronchoalveolar lavage
fluid, longer persistence than in
plasma.
280313
In vivo
Oral bioavailability.
Oral dosage to rats.
GS-4104 undergoes rapid cleavage
to GS-4071 after absorption from the
GI tract; bioavailability in rats is
36%.
254763
In vivo
Oral bioavailability.
Oral dosage to dogs.
Oral bioavailability in dogs is 100%.
247131
Metabolism
Clinical
EFFECT STUDIED
MODEL USED
RESULT
REF
Safety, tolerability and
absorption in phase I study.
Oral dosing in healthy volunteers.
GS-4104 was well tolerated with good oral absorption
and distribution.
248180
Phase II study examining the
effect on flu symptoms and
virus load.
Exposure of volunteers to influenza
A; dosing of GS-4104 either postexposure or prophylactically.
GS-4104 showed efficacy by reducing the time to
cessation of symptoms (therapeutic treatment) and
preventing virus replication (prophylaxis).
266214
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M, Beadle D, Brindle S, Black J, ABN AMRO Hoare Govett
ANALYST REPORT 1998 January
277127 Development of antivirals against influenza. Cianci C,
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277501 DRUG DEV PIPELINE 1998 3 1 1 - 4
277572 Flu patients in US may now have access to
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synthesis, and structural analysis of carbocyclic sialic acid
analogs with potent anti-influenza activity. Kim CU, Lew W,
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analogs, culminating GS-4071 and its prodrug, GS-4104.
280313 Penetration of GS-4071, a novel influenza
neuraminidase inhibitor, into rat bronchoalveolar lining fluid,
following oral administration of the prodrug, GS-4104.
Eisenberg EJ, Bidgood A, Cundy KC ANTIMICROB AGENTS
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• Demonstration of bioavailability and delivery into the
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272638 Gilead and Roche begin phase II/III studies of an
experimental product to treat or prevent influenza. Gilead
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280314 New potent, orally active neuraminidase inhibitors as
anti-influenza agent: in vitro and in vivo activity of GS-4071 and
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Lynch ANALYST REPORT 1998 February 18
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282057 Mutations in a conserved residue in the influenza virus
neuraminidase active site decreases sensitivity to Neu5Ac2enderived inhibitors. McKimm BRESCHKINJL, Sahasrabudhe A,
Blick TJ, McDonald M, Colman PM, Hart GJ, Bethell RC, Varghese
JN J VIROL 1998 72 3 2456 - 2462
273508 Gilead begins oral flu drug phase II/III. BIOWORLD
WEEK 1997 5 51 3
275686 Gilead’s oral drug blocks, relieves flu. BIOWORLD
WEEK 1997 5 40 3
282059 ’Flu’ and structure-based drug design. Wade RC
STRUCTURE 1997 5 9 1139 - 1145
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276077 Gilead announces fourth quarter and 1997 year-end
financial results. Gilead Sciences Inc PRESS RELEASE 1998
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276650 Gilead/Roche flu vaccine went from phase I to phase III
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Swaminathan S, Mendel DB, Tai CY, Escarpe PA, Laver WG,
Stevens RC, Bischofberger N ICAAC 1997 37 Toronto 388
282075 The novel influenza neuraminidase inhibitor prodrug,
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KLINISCHER PRUFUNG. Jungmayr P DTSCH APOTH ZTG 1997
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282084 Tolerability and pharmacokinetics of the influenza
neuraminidase inhibitor, Ro-64-0802 (GS-4071), following oral
administration of the prodrug, Ro-64-0796 (GS-4104) to healthy
male volunteers. Wood ND, Aitken M, Sharp S, Evison H ICAAC
1997 37 Toronto 25
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Drug evaluation Cystemustine 129
Cystemustine INSERM
Brian Palmer
Address
Auckland University
Cancer Research Laboratory
School of Medicine
Private Bag 92019
Auckland
New Zealand
IDrugs 1998 1(1):129-135
Cystemustine is a chloroethyl-nitrosourea under development by INSERM as a potential anticancer drug. It is in
phase II trials in France for malignant melanoma [207163],
advanced neck and head cancer [207164], advanced renal
cancer [207165], and colorectal cancer [207187].
Cystemustine appears to have moderate activity against
2
malignant gliomas using a 60 mg/m dosing regimen as a
second line therapy, following surgery, chemotherapy or
radiotherapy.
Cystemustine has potent antitumor activity in mice. It has
a short plasma half-life which makes it potentially
amenable to circadian scheduling in order to enhance
tolerability and dose intensity. The influence of circadian
dosing time on toxicity was first investigated in a total of
368 synchronized male B6D21F1 mice. Leukopenia was the
6
main hematological effect encountered. The activity of O alkylguanine-DNA alkyltransferase (AGT) was studied in
the liver of 31 additional mice obtained at six different
circadian times. A large amplitude circadian rhythm
characterizes both cystemustine toxicity and liver AGT
activity in mice. This suggests that the AGT rhythm is an
important mechanism of the chronopharmacology of
cystemustine [203827].
Introduction
The [2-chloroethyl]nitrosoureas, such as BCNU (carmustine), CCNU (lomustine) and MeCCNU, are a wellestablished class of DNA-alkylating antitumor agents, which
are particularly useful for the treatment of gliomas and
lymphomas [273753]. As a result of their lipophilicity, they
readily cross the blood-brain barrier and display good
bioavailability. Under physiological conditions, they are
extensively decomposed, liberating small electrophilic
species which are capable of alkylating DNA at nucleotide
base and phosphate sites. Alkylation at O-6 of guanine has
been suggested as an important cytotoxic event for this class
of agent. Recently, a series of second generation nitrosoureabased agents, such as fotemustine (Servier), nimustine
(ACNU; Sankyo) and chlorozotocin (DCNU; NIH), have
been developed, which display an improved spectrum of
activity, as well as better activity against drug-resistant
tumors. The development of clinical resistance to the
nitrosourea class of agents is frequently associated with
6
overexpression of the enzyme, O -alkylguanine DNA
alkyltransferase (AGT), which acts to repair the cytotoxic
Originator INSERM
Status Phase II clinical
Indication Neoplasm, Melanoma, Head & neck tumor,
Renal tumor, Colorectal tumor, Glioma, Carcinoma,
Sarcoma
Action Anticancer, Alkylating agent
CAS Urea, N-(2-chloroethyl)-N’-[2-(methylsulfonyl)ethyl]-NnitrosoRegistry No: 79955-36-5
Synonyms CENU, chloroethylnitrosourea, CYST,
CMSO2EN2
O
O
O
S
Cl
N
H
N
N
O
lesion formed upon DNA alkylation. The co-administration
of inhibitors of this enzyme improves the antitumor effect of
nitrosoureas in experimental systems, and many of the
newer agents of this class are under evaluation in
conjunction with newly synthesized alkyltransferase
inhibitors.
Cystemustine is a second generation nitrosourea which is
derived from the amino thiol cysteamine, and which has
recently completed a series of phase II clinical trials against a
variety of tumor types.
Synthesis and SAR
Cystemustine (N-[2-chloroethyl]-N’-[(2-methylsulfonyl)ethyl]-N-nitrosourea, CENU, CMSO2EN2, CYST) was originally
identified as a metabolite of the experimental agent CNCC
[N,N’-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl]cystamine],
where it occurred with the corresponding sulfoxide
[273755]. Both metabolites result from the in vivo reduction
of the disulfide linkage in CNCC, followed by S-methylation
and oxidation. Subsequent studies established that both
cystemustine and the sulfoxide, perrimustine, were as active
as CNCC against the L1210 leukemia in mice, and led to the
development of cystemustine as an experimental antitumor
agent in its own right [273755], [207185]. Cystemustine is
prepared in four steps from 4-nitrophenol and 2-chloroethyl
isocyanate and is obtained as light yellow crystals (melting
°
point 94 to 96 C). It has good water solubility, enabling its
direct formulation in aqueous solutions for in vivo studies.
14
[ C]cystemustine has been synthesized for pharmacokinetic
studies with the label in complementary portions of the
14
14
molecule, from either [ C]ethanolamine [273757] or [ C]2chloroethyl isocyanate [273762].
Pharmacology
The in vivo activity of cystemustine was evaluated in a panel
of 12 murine tumors, and was compared with that of CNCC
[273765]. It was at least as active as CNCC against eight
lymphomas and solid tumors, and gave a higher number of
long term survivors. Cystemustine was clearly better than
CNCC against B16 melanoma, 26 glioma and grafted L1210
leukemia, with an optimum iv dose of 15 mg/kg, half that of
CNCC. The activity of cystemustine was significantly
improved following the co-administration of an
experimental, water soluble inhibitor of AGT to nude mice
bearing the M4Beu melanoma xenograft [207169].
Metabolism
14
The disposition of [ C]cystemustine was evaluated in nontumor bearing rats following an iv dose of 60 µmol/kg
[273766]. Elimination of cystemustine and its metabolites
was predominantly via the urine, with 70% of the total
radioactivity collected in this fraction, 72 h following drug
administration. At this time, the feces contained only 5% of
the radioactivity, with 10% excreted as carbon dioxide in the
lung. An analysis of the tissue distribution of unchanged
cystemustine at 5 min and 30 min, following iv
administration, showed a particularly high level of drug in
the kidney, together with significant levels of the drug in the
brain, lung and liver. In this study, cystemustine was
cleared with first order kinetics, with a plasma half-life of
30.5 min, and a volume of distribution of 1.05 l/kg. The
AUC was 42.8 nmol/h/ml. The human pharmacokinetics of
cystemustine have been reported as part of a phase II clinical
trial [207184]. HPLC analysis was used to determine plasma
levels up to 3 h following iv administration of cystemustine,
2
at doses in the range 40 to 90 mg/m . The drug was
eliminated with a half-life of 50 min, and had a distribution
half-life of less than 10 min. The rapid metabolism and
clearance of cystemustine is similar to that observed for the
clinically used nitrosoureas, CCNU and BCNU.
Cystemustine is extensively metabolized in the rat, with the
main urinary metabolites being thiodiacetic acid and its
sulfoxide (sulfonylthiodiacetic acid), which together
comprise about half of the urinary radioactivity. N-acetyl-Shydroxyethylcysteine and N-acetyl-S-carboxymethylcysteine
together account for 7% and 2-(methylsulfonyl)ethylamine
16% of the urinary radioactivity. About 10% of urinary
radioactivity is associated with four unidentified metabolites.
The main plasma metabolite is 2-chloroethanol [273766,
273768].
Toxicity
As with other nitrosourea-based antitumor agents,
cystemustine showed mainly hematological toxicity in
preclinical murine studies [273765]. Cystemustine caused
dose-related leukopenia in mice, peaking on days 8 to 12
following administration of multiple iv doses of the drug up
to day 5, which recovered to control levels by day 19.
Cystemustine had little effect on platelets or red blood cells,
in contrast to CNCC.
In murine studies, the toxicity of cystemustine displayed a
surprising dependence on the point in the circadian cycle of
the mice at which drug administration occurred [268219].
This effect is pronounced for cystemustine due to its short
plasma half-life. Following administration of a single dose of
cystemustine, the nadir of leukocyte count (day 7) was lower
for those mice injected with drug 7 h after light onset, as
compared with 13 or 19 h afterwards, and bone marrow
necrotic lesions were also more pronounced in the former
case. Furthermore, recovery from leukopenia was faster,
following administration of cystemustine 19 h after light
onset. The 60-day survival rate following a dose of 35
mg/kg was 4% when injected 7 h after light onset, rising to
90% when given at 19 h. Overall, cystemustine displayed the
lowest toxicity when administered in the middle of the
active phase of the rest-active circadian cycle. Further
studies established a significant 5-fold circadian variation in
the liver AGT activity of mice which correlated with
cystemustine toxicity, suggesting a basis for the observed
chronotoxicity of cystemustine [203827].
The dose-limiting toxicity of cystemustine in the phase I
clinical trial was dose-dependent and hematological. Mild
2
toxicity began, following a cumulative dose of 55 mg/m of
cystemustine, and the maximum tolerated dose was
2
established as 90 mg/m [207188]. In the phase II studies,
toxicity per cycle of treatment was mild and well-tolerated
2
2
at the 60 mg/m dose level, while at 90 mg/m , the toxicity
was also hematological, grade 3 to 4 anemia (19%), grade 3
to 4 neutropenia (23%), grade 3 to 4 thrombopenia (27%) and
grade 3 to 4 nausea and vomiting (11%) [156265]. All
toxicities were reversible.
A phase I study of cystemustine was carried out in 34
patients with a variety of advanced metastatic cancers
[207188]. The drug was administered iv in seven or eight
cycles over a period of up to 190 days, in escalating doses
2
ranging from 0.9 to 180 mg/m . The recommended dose for
2
2
phase II evaluation was 60 mg/m , increasing to 90 mg/m .
Three partial responses were reported in this phase I study.
The phase II evaluation of cystemustine began in 1990 in a
range of tumor types, under the auspices of the EORTC.
More than 160 patients with advanced or recurrent tumors
were given cystemustine q2 weeks as a 15 min iv infusion at
2
2
a dose of either 60 mg/m or a dose of 90 mg/m for the first
2
three cycles, with subsequent doses of 60 mg/m [156265],
[207165]. Although the drug had been observed to
concentrate to some extent in the kidney in preclinical rat
studies, when cystemustine was evaluated in 56 patients
with advanced renal cancer, it had only minimal activity
[207165]. Cystemustine also displayed only marginal activity
against metastatic colorectal carcinoma (27 patients)
[207187], soft tissue sarcoma (32 patients) [273774], head and
neck cancer (28 patients) [207164] and non-small cell lung
cancer (19 patients) [156265]. Against advanced malignant
melanoma in 25 patients, cystemustine displayed limited
activity at the lower dose level [268218], but a smaller study
in 17 pre-treated patients using the higher dosing regimen of
2
90 mg/m gave an overall response rate of 23% [273770]; this
is significant for this tumor type, but remains to be verified
in a larger number of patients. Cystemustine was evaluated
2
at the 60 mg/m dose in 38 patients with malignant gliomas,
comprising 14 glioblastomas, 20 grade 3 to 4 astrocytomas
and four grade 3 to 4 oligodendrogliomas [156265], [273771].
The majority of these patients had received prior
radiotherapy. Of the 32 evaluable patients, three had partial
remissions (giving an overall response rate of 9.4%), while
18 patients had stable disease for at least 8 weeks. Several of
the responding patients had received high doses of BCNU in
previous adjuvant chemotherapy, suggesting that
cystemustine might be cross-resistant to conventional
nitrosourea-based agents. There have been no specific
reports of the clinical evaluation of cystemustine against
either Hodgkin’s or non-Hodgkin’s lymphoma.
Current Opinion
The clinical utility of the first generation of nitrosoureas is
limited mainly to the treatment of lymphomas and the
palliative treatment of gliomas (the latter usually in
combination with radiotherapy and/or surgery). As
expected, the second generation nitrosourea-based agents
are also active against lymphoproliferative diseases and
gliomas, but in addition, have low to moderate activity
against small cell lung carcinoma, gastrointestinal cancers,
head and neck cancer and melanoma [273753]. The
published phase II clinical data for cystemustine suggest
that it is no more active than other second generation agents
against these latter tumor types, and cystemustine is
unlikely to be developed further for these indications.
Cystemustine appears to have moderate activity against
2
malignant gliomas, using the 60 mg/m dosing regimen,
when used as a second line therapy following surgery,
chemotherapy or radiotherapy. The response rate observed
with cystemustine against this tumor type appears to be
broadly comparable with response rates obtained with
currently used nitrosoureas, such as CCNU and BCNU
[23593], [273776], although strict comparisons await a
detailed analysis of the median survival times (rather than
response rates) obtained with cystemustine, in conjunction
with the adjuvant therapy used and tumor staging at the
start of treatment. Any further advancement of cystemustine
for gliomas will depend on a clear demonstration of its
superior activity to currently used agents at the higher
2
dosing regimen of 90 mg/m , and/or a demonstration of
clinical activity in tumors which have become resistant to
conventional nitrosourea-based antitumor agents.
Development History
DEVELOPER
COUNTRY
STATUS
INDICATION
DATE
REF
INSERM
France
C2
Melanoma
08-MAY-96
207163
INSERM
France
C2
Head & neck tumor
08-MAY-96
207164
INSERM
France
C2
Renal tumor
08-MAY-96
207165
INSERM
France
C2
Neoplasm
08-MAY-96
207184
INSERM
France
C2
Colorectal tumor
08-MAY-96
207187
INSERM
France
C2
Glioma
21-JAN-98
273771
INSERM
France
C2
Carcinoma
21-JAN-98
156265
INSERM
France
C2
Sarcoma
21-JAN-98
273774
Chemistry
STUDY TYPE
RESULT
REF
Synthesis and antitumor activity
Synthesis of four metabolites of the experimental agent, CNCC, one of which is
cystemustine.
273755
Synthesis
Synthesis of [ 14C]cystemustine from labeled ethanolamine.
273757
Synthesis
Synthesis of [ 14C]cystemustine from labeled 2-chloroethyl isocyanate.
273762
Biology
STUDY TYPE
In vivo
EFFECT STUDIED
Toxicity.
EXPERIMENTAL MODEL
Cystemustine was administered at 7,
13 or 19 h after light onset (HALO) and
toxicity was evaluated in B6D2F1
mice.
RESULT
Leukopenia recovery was faster and
bone marrow necrotic lesion was not
severe after cystemustine at 19 HALO.
This was dependant on the rhythm of
AGT activity.
REF
203827
In vivo
Antitumor activity.
L1210 tumors grown ip in mice.
Activity of cystemustine against murine
L1210 tumors was found to be greater
than that of CNCC.
273755
In vivo
Antitumor activity.
12 murine and xenograft tumors in
mice.
Cystemustine demonstrated greater
overall activity than CNCC, at half the
dose.
273765
In vivo
Antitumor activity in
the presence of
AGT inhibitors.
M4Beu melanoma xenografts in mice.
In vitro and in vivo activity of
cystemustine evaluated in the presence
of a series of inhibitors of the enzyme,
AGT. Enzyme inhibitors containing
benzyl groups augmented the antitumor
activity.
207169
In vivo
Circadian toxicity.
368 synchronized B6D2F1 mice.
Cystemustine hematological toxicity
markedly lower when administered in the
middle of the active portion of the activerest cycle.
268219
In vivo
Circadian toxicity.
Radiochemical detection of liver AGT
activity at six circadian timepoints in
31 mice.
5-fold variation in AGT activity found,
correlating with the chronotoxicity of
cystemustine.
203827
Metabolism
STUDY TYPE
EFFECT STUDIED
MODEL USED
RESULT
REF
In vivo
Drug disposition and
pharmacokinetics.
Non-tumor bearing rats, dose of 60
mmol/kg of [ 14C]cystemustine.
70% of radioactivity in the urine after 72 h,
with 5% in feces, 10% in lung. Plasma halflife 30.5 min, first order. Volume of
distribution = 1.05 l/kg, AUC = 42.8
nmol/h/ml.
273766
In vivo
Pharmacokinetics.
Phase II trial. HPLC analysis of
plasma levels.
Distribution half-life less than 10 min.
Plasma half-life = 50 min.
207184
In vivo
Metabolism.
[14C]cystemustine in the rat.
Extensive metabolism, with metabolites
appearing mainly in the urine. Major
metabolites identified.
273768
Clinical
EFFECT STUDIED
MODEL USED
RESULT
REF
2
Phase I trial.
Phase I evaluation in 34 pretreated cancer
patients.
Dose for phase II trials set at 60 mg/m ,
increasing to 90 mg/m2. Three responses seen.
207188
Antitumor activity.
Phase II evaluation in 27 patients with metastatic
colorectal carcinoma.
No activity.
207187
Antitumor activity.
Phase II evaluation in 56 patients with advanced
renal cancer.
Minimal activity.
207165
Antitumor activity.
Phase II evaluation in 32 patients with soft tissue
sarcoma.
Minimal activity.
273774
Antitumor activity.
head and neck cancer.
Minimal activity.
207164
Antitumor activity.
Phase II evaluation in 19 patients with non small
cell lung carcinoma.
Minimal activity.
156265
Clinical (continued)
EFFECT STUDIED
MODEL USED
RESULT
REF
Antitumor activity.
malignant melanoma.
Limited activity at a dose of 60 mg/m 2.
268218
Antitumor activity.
malignant melanoma.
Limited activity at a dose of 60 mg/m 2.
268218
Antitumor activity.
malignant melanoma.
Overall response rate of 23% at a dose of 90
mg/m2.
273770
Antitumor activity.
Phase II evaluation in 38 patients with mostly preirradiated gliomas, comprising 14 glioblastomas,
20 high-grade astrocytomas and four
oligodendrogliomas.
Three partial remissions, and a total of 18
patients with stable disease for at least 8 weeks.
An observation that cystemustine could have
activity against BCNU-refractory gliomas.
156265
Antitumor activity.
Phase II evaluation in 38 patients with mostly preirradiated gliomas, comprising 14 glioblastomas,
20 high-grade astrocytomas and four
oligodendrogliomas.
Three partial remissions, and a total of 18
patients with stable disease for at least eight
weeks. An observation that cystemustine could
have activity against BCNU-refractory gliomas.
273771
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Krakowski I, Lentz MA, Fumoleau P EUR J CANCER PART A GEN
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Mathe G, Misset JL, Triana BK, Goden‘ eche D, Madelmont JC,
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Veyre A J LABEL COMPOUNDS RADIOPHARM 1988 25 1135 1142
• A report of the use of 14C labeled 2-chloroethyl isocyanate for the
preparation of labeled cystemustine for use in pharmacokinetic and
metabolism studies.
273765 Cytostatic action of two nitrosoureas derived from
cysteamine. Bourut C, Chenu E, Godeneche D, Madelmont JC,
Maral R, Mathe G, Meyniel G BR J PHARMACOL 1986 89 539 546
• An in vivo evaluation of the antitumor activity of cystemustine (and
perrimustine) in 12 murine tumor models, in comparison with
another nitrosourea, CNCC. Hematological toxicity studies are
reported.
273766 Disposition of new sulfur-containing 2-(chloroethyl)nitrosoureas in rats. Godeneche D, Madelmont JC, Labarre P,
Plagne R, Meyniel G XENOBIOTICA 1987 17 59 - 70
• A distrubution study of 14C radiolabeled cystemustine in rats.
273768 Main urinary metabolites of two cysteamine containing
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Glabbeke M, Urosevic V, Chevallier B, Fumoleau P NCI EORTC
SYMP NEW DRUGS CANCER THER 1994 Abs 290
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273771 Phase II trial of cystemustine, a new nitrosourea, as a
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• A report from the early studies group of the EORTC on the activity
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M, Chollet PH, Chevallier B, Fumoleau P ANN ONCOL 1992 3
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• A report of a phase II trial of cystemustine against advanced soft
tissue sarcoma, establishing only marginal activity.
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• A short note evaluating the usefulness of nitrosoureas as adjuvant
therapy for gliomas.
282039 O6-methylguanine-DNA methyltransferase (MGMT)
transfectants of a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)sensitive colon cancer cell line selectively repopulate
heterogenous MGMT+/MGMT- xenografts after BCNU and O6benzylguanine plus BCNU. Phillips WPJR, Willson JK, Markowitz
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Debiton E, Cussac Buchdhal C, Mounetou E, Rapp M, Dupuy JM,
Maurizis JC, Veyre A, Madelmont JC BR J CANCER 1997 76 9 1157
- 1162
282049 Evidence for nucleotide excision repair as a modifying
factor of O6-methylguanine-DNA methyltrans-ferase-mediated
innate chloroethylnitrosourea resistance in human tumor cell
lines. Chen ZP, Malapetsa A, McQuillan A, Marcantonio D, Bello V,
Mohr G, Remack J, Brent TP, Panasci LC MOL PHARMACOL 1997
52 5 815 - 820
282053
Application
of
antisense
ribonucleic
acid
complementary to O6- methylguanine-deoxyribonucleic acid
methyltransferase messenger ribonucleic acid for therapy of
malignant gliomas. Nagane M, Asai A, Shibui S, Nomura K,
Kuchino Y, Ali Osman F, Greenberg HS, Raffel C, Rutka JT
NEUROSURGERY 1997 41 2 434 - 441
KA-672 Dr Wilmar Schwabe GmbH & Co
Hermann Mucke
Address
Sanochemia
Enenkelstrasse 28/32
A-1160 Wien
Austria
IDrugs 1998 1(1):136-140
KA-672 is a multifunctional antidementia agent under
development by Schwabe as a potential agent to combat
cognitive and other symptoms of Alzheimer’s disease (AD),
via modulation of the interplay between serotonergic,
adrenergic, dopaminergic and cholinergic systems [260590].
It has also been categorized as a neuronal activator.
Various studies support the notion that this compound
could indeed have a broad range of nootropic properties.
The compound is in phase II clinical trials [260592].
KA-672 selectively protects against NMDA-induced
neurotoxicity but not against other chemoconvulsants or
electroshock [260590]. At 0.3 mg/kg it inhibits the reduction
in 5-HT brain levels following NMDA administration, and
exacerbates the reduction in dopamine levels [260593]. It has
been shown to bind to 5-HT1A, 5-HT7, α1, D2 and D3 receptors
with nanomolar affinities, acting as an antagonist at α1
and D2 receptors [260592]. It also has nerve growth factor
(NGF)-like activity [260591].
Introduction
Most drugs that are being developed for the symptomatic
treatment of AD are directed exclusively towards one of the
several neurotransmitter deficits that are known to occur in
primary degenerative dementia; in most cases the target is
the cholinergic system. A more balanced approach would be
desirable, especially with respect to the treatment of noncognitive symptoms which are not directly mediated
through acetylcholine (ACh) deficits. Whether such an
approach requires combination therapy, or can be achieved
in a single molecule, remains an open question.
Originator Dr Wilmar Schwabe GmbH & Co
Status Phase 2 Clinical
Indication Alzheimer’s disease
Action Nootropic agent
CAS 2H-Benzopyran-2-one, 7-methoxy-6-[3-[4-(2methoxyphenyl)-1-piperazinyl]propoxy]-3,4-dimethylRegistry No: 155773-59-4
O
N
O
O
O
N
H
Cl
O
with 2-methyl-3-oxobutanoic acid ethyl ester in 75% sulfuric
acid. This intermediate is reacted with 1-[3-chloropropyl]-4[2-methoxyphenyl]piperazine in the presence of K2CO3 and
KI in ethanol, resulting in the target compound, KA-672, as
free base [281905], [258435].
Pharmacology
KA-672 binds to 5-HT1a, 5-HT7, α1, D2 and D3 receptors in
nanomolar concentrations, but does not interact with other
subtypes of monoaminergic receptors or ion channels.
Functional studies suggest that KA-672 is an α1 and D2
antagonist, but the nature of receptor interaction remains to
be defined [260592]. Given to rats at a dose of 0.3 mg/kg, po,
the compound elevated serotonin levels and decreased the
levels of dopamine and its metabolite, 3-methoxytyramine.
The parent structure for KA-672 is scoparone (6,7dimethoxycoumarin), a naturally-occuring compound with
vasodilatory,
anti-atherogenic,
antiproliferative
and
immunosuppressive properties. It is isolated from the
flower, Artemisia scoparia, an agent in Chinese herbal
medicine.
In rat brain homogenates and in commercial Torpedo
preparations, the potency of KA-672 as an acetylcholinesterase inhibitor (AChEI) is reported to be as equal to
that of tacrine and 10-fold higher than that of galanthamine,
corresponding to an IC50 value in the submicromolar range
[281915], [258435]. However, no such effect was observed in
vivo, and Schwabe no longer classifies KA-672 as an AChEI
[281908]. The compound does not inhibit plasma butyrylcholinesterase, and is also without effect on rat brain
arylacylamidase. At moderately high doses of 3 to 5 mg/kg
po it does not produce tremor, salivation or other peripheral
cholinergic effects. Furthermore, KA-672 does not bind to
nicotinic or muscarinic ACh receptors [281915].
KA-672 is synthesized via 6-hydroxy-7-methoxy-3,4dimethyl-2H-1-benzopyran-2-one, which is accessible
through a Pechmann reaction of 2-methoxy-1,4-benzenediol
Most importantly, however, KA-672 appears to be a
functional NMDA antagonist. In doses that are effective in
cognitive rodent models [177791], it protects mice against
Schwabe has dropped its provisional synonym for KA-672,
anseculin [281908]. No international non-proprietary name
has been assigned so far.
Synthesis and SAR
Drug evaluation KA-672 137
convulsions and mortality induced by NMDA or
homocystein, but protection against chemoconvulsants or
electroshock is not achieved even at doses several-fold
higher. The compound does not compete with glutamic or
kainic acid, glycine, AMPA or MK-801 for binding sites at
the NMDA receptor complex. In in vitro electrophysiological studies, KA-672 abrogated currents triggered by
NMDA, but not those activated by AMPA or GABA. If
administered prior to an NMDA challenge, it acted
synergistically with NMDA in lowering brain dopamine by
enhancing its metabolism along the homovanillic acid
pathway, antagonized the serotonin depletion induced by
NMDA, and also increased the brain norepinephrine
content. [260593].
Finally, in an in vitro system of rat embryonic septal cells,
KA-672 has been shown to have NGF-like activity
(comparable to that of NGF in terms of potency and quality).
No synergy between the compound and NGF was observed,
suggesting that both might stimulate neurite outgrowth
using essentially the same mechanisms [260591].
In neuropsychological standard screening tests, KA-672 is
either inactive or behaves as a weak antidepressant. In
cognitive paradigms, such as passive or conditioned
avoidance, oral doses of 0.1 to 1.0 mg/kg strongly facilitate
acquisition and retention in rats and mice, and antagonize
the amnestic effect of scopolamine [177791], [258435].
Metabolism
No data have yet been reported concerning the metabolic
fate of KA-672; however, educated guesses can be made
based on what is known from coumarin derivatives used as
anticoagulants. These compounds show almost complete
plasma protein binding, and a metabolic rate that might in
part be genetically determined. The aromatic ring system is
extensively hydroxylated. This results in glucuronidation or
sulfatation products being excreted in urine, although
hardly any unchanged parent compound is lost in this way.
The metabolism of scoparone, the parent compound of KA672, has been thoroughly investigated because it can be used
as a substrate to differentiate cytochrome P450 monooxygenase induction stages. Scoparone is regio-selectively
demethylated at different rates for the methyl groups at
positions 6 and 7, yielding scopoletin (7-hydroxy-6methoxycoumarin)
and
isoscopoletin
(6-hydroxy-7methoxycoumarin). Rat hepatocytes metabolize scoparone 7to 10-fold slower than those of hamsters and monkeys.
Human hepatocytes do not secrete scopoletin in detectable
amounts, suggesting that scoparone metabolism in humans
may be qualitatively different from that in other mammals
[281907].
Toxicity
In mice and rats, the only dose-dependent toxicological
symptoms observed after oral doses of 10 mg/kg or more
were sedation, hypothermia and anorexia, with doses up to
50 mg/kg not causing mortality. In 26-week chronic
exposure studies, daily oral doses of up to 13 mg/kg in rats
and 1.5 mg/kg in dogs were well-tolerated, and side-effects
were reversible. Oral doses up to 40 mg/kg were not
clastogenic in the mouse bone marrow micronucleus test
[258435].
Phase I
Single and repeat oral dose tolerability of KA-672 was
determined in adult and elderly healthy male and female
subjects in the non-fasting state. The compound was welltolerated and linear pharmacokinetics, in terms of Cmax and
AUC, were observed after repeat doses. Half-life was in
excess of 10 h, suggesting that therapeutic trials with 10 or
20 mg once-daily dosing could be feasible [260594].
Phase II
KA-672 has been reported to have entered phase II in 1997
[260592], [258423]; however, no data are currently available.
Side-effects and Contraindications
In phase I studies, the side-effect profile of 40 mg, po, single
doses was indistinguishable from placebo; at 60 mg, about
half the subjects experienced a moderate drug-related
orthostatic syndrome. Repeated dosing of up to 20 mg
po/per day, for 14 days, resulted in only mild adrenergic
side-effects (decrease in semisupine and standing blood
pressure, but no orthostasis) without a systematic pattern
[260594].
Current Opinion
The current lack of peer-reviewed original papers on KA-672,
and the multitude of pharmacological activities reported in
patents and congress abstracts, makes it difficult to form a
solid opinion on this compound. The all-in-one concept of a
single molecule combining several activities that are relevant
to the therapy of AD is certainly intriguing, and would indeed
put KA-672 in a class of its own if these claims can be fully
substantiated. If this were case, it would add dramatic support
to the notion that a strategy consisting of targeted synthesis
and pharmacological screening could, at this stage of our
knowledge, still be superior to the onslaught of combinatorial
chemistry, or purely rational drug design through molecular
modeling. It is, however, difficult to imagine that so many
different functional elements should actually reside in a single
chemical structure. In particular, it should be investigated
whether some of the claimed effects might be conferred by
metabolites of KA-672.
Regardless of what explanation can be found for this broad
activity spectrum, an inseparable and fixed combination of
an NMDA receptor antagonist with an α-adrenergic,
dopaminergic and NGF agonist might not be without
problems in clinical practice. While functional NMDA
antagonism could be invaluable in combating neurodegeneration, and a measure of stimulatory activity in the
aminergic systems might allow better therapeutic access to
the non-cognitive disturbances of AD, NGF-like- and D2antagonistic-activity might not be desirable for all patients,
or in all stages of the syndrome. It is also conceivable that
interactions with other drugs could be difficult to control.
There could be significant additional potential for KA-672 as
an anticonvulsant, in ischemic stroke, and in traumatic brain
injury.
Patent Commentary
Based on the German patent, DE-04111861 (April 11, 1991),
Schwabe has secured a family of patents (Benzopyranones, a
method for producing them and uses thereof) covering the
synthesis of the series of coumarin derivatives to which KA672 belongs, and their use as neuroprotectants and antiallergics. This family includes, among others, WO-09218493
(October 29, 1992), US-05428038 (June 27, 1995), and JP94506922. This family is extended by another series under
the same title (DE-04233963, October 8, 1992; WO-09408985,
April 28, 1995; US 05550129, August 27, 1996).
Development History
DEVELOPER
COUNTRY
STATUS
INDICATION
DATE
REF
Dr Wilmar Schwabe GmbH & Co
Germany
C2
Alzheimer’s disease
08-AUG-97
258423
Dr Wilmar Schwabe GmbH & Co
Germany
C1
Alzheimer’s disease
23-MAY-95
177791
Chemistry
STUDY TYPE
RESULT
REF
Synthesis and SAR
110 novel 2H-1-benzopyran-2-ones (coumarin derivatives) prepared. Neuroprotective and analgesic
properties described.
281905
Biology
STUDY TYPE
In vitro
EFFECT STUDIED
NGF-like activity.
EXPERIMENTAL MODEL
Embryonic rat septal cells.
RESULT
Addition of 100 ng/ml KA-672 to the culture
medium induced outgrowth of predominantly short
(5 to 25 mm) neurites, comparable to NGF at the
same concentration.
REF
260591
In vitro
Spasmogenic activity.
Isolate guinea pig ileum.
No interference with the effects of acetylcholine,
bradykinin, serotonin, substance P and nicotine.
Dose-dependent inhibition of histamine effects.
177791
In vitro
Cholinesterase
inhibition.
Rat brain homogenate.
Electric eel
acetylcholinesterase.
KA-672 inhibited acetylcholinesterase as potently
as tacrine and was approximately 10-fold stronger
than galanthamine. Butyrylcholinesterase and
arylacylamidase were not inhibited.
281915
In vivo
Cognitive effect (passive
and conditioned
avoidance).
Rat and mouse.
KA-672 (0.1 to 1.0 mg/kg, po) dose-dependently
enhanced memory acquisition and retrieval. 0.01
to 0.3 mg/kg, po, shortened conditioned
avoidance latencies from day 1 to 2 of training,
and increased the number of correct responses
up to 15 days after discontinuation of treatment.
177791
In vivo
Cholinergic activity.
Rat and mouse.
KA-672 (0.1 to 0.3 mg/kg, po) protected against
scopolamine-induced amnesia. No tremor or
salivation at 3 to 5 mg/kg.
177791
In vivo
Adrenergic activity.
Rat and dog
(anesthetized).
KA-672 (10 mg/kg, iv) inhibited the cardiovascular
actions of phenylephrine, and lowered systolic
and diastolic blood pressure 15 to 20%, without
altering heart rate.
258435
Drug evaluation KA-672 139
Biology (continued)
STUDY TYPE
In vivo
EFFECT STUDIED
Serotonergic activity.
EXPERIMENTAL MODEL
Rat and mouse.
RESULT
KA-672 dose-dependently inhibited 5-HT-induced
head twitch in mice, and potentiated the 8-OHDPAT-induced inhibition of spontaneous cageleaving behavior in rats.
REF
258435
In vivo
Functional NMDA
receptor antagonism.
Mouse.
KA-672 (0.1 to 0.5 mg/kg, po) protects against
convulsions induced by NMDA, but not against
those triggered by pentetrazol, picrotoxin,
strychnine, or electroshock. No binding interference
with glutamic or kainic acid, AMPA, glycine, MK-801
or TCP. Electrophysiological currents activated by
NMDA (but not those activated by AMPA or GABA)
were inhibited.
260590
In vivo
Receptor binding.
Rat.
KA-672 drastically reduced 5-HT in the striatum, and
elevated its levels in the hippocampus. Binds to 5-
260592
HT1a, 5-HT7, α1 and D2 receptors in nanomolar
concentrations. No binding to other monoaminergic
receptor subtypes or ion channels.
In vivo
Serotonin and dopamine
metabolism.
Mouse.
KA-672 (0.3 mg, po, 1 h prior to injection of 50 mg/kg
NMDA) potentiated the reduction of dopamine levels,
and antagonized the reduction of 5-HT levels.
260593
In vivo
Nootropic activity.
Passive avoidance test in
rats and mice.
KA-672 (0.1 to 1 mg/kg po) dose-dependently
enhanced learning ability and improved
consolidation and retrieval of memory.
177791
In vivo
Onset and duration of
action.
Onset and duration of
action in conditioned
avoidance response tests
in rats.
Rapid onset of action following administration of
0.01 to 0.3 mg/kg, po, resulting in correct avoidance
response from first to second training day; long
duration with nootropic activity 15 days after
discontinuation of treatment.
177791
RESULT
Maximal side-effect (free single) dose is 40 mg, po; 50% of
individuals experienced orthostatic syndrome after single
doses of 60 mg, po. Mild decrease in supine and standing
blood pressure seen as the only side-effect with repeat
dosing of 10 or 20 mg, po. Linear pharmacokinetics;
terminal-phase half-life values after 20 mg repeat doses and
single doses were 11.1 h and 13.7 h, respectively
REF
260594
Clinical
EFFECT STUDIED
Tolerability, safety and
pharmacokinetics.
EXPERIMENTAL MODEL
Double-blind, randomized, placebocontrolled study in healthy males and
females aged 52 to 74 years.
Associated patent WO-09218493 (Dr Willmar Schwabe GmbH Co)
Abstract Heterocyclic substituted coumarins have been
synthesized and are claimed as specific NMDA agonists. They
act as anti-allergic and neuroprotective agents and may be
useful in the treatment of hayfever, asthma and urticaria.
Compounds were tested in vivo in mice for NMDA agonist
activity, the most potent having efficiency at doses of 5 to 25
mg/kg, iv. Sixty-six compounds are exemplified by synthesis
via 44 intermediates, and using standard techniques. Yields
and melting points are provided. Twenty-six compounds are
specifically claimed, one of which is 6-ethoxy-7-(3-(4-phenyl-1piperazinyl)propoxy)-2H-1-benzopyran-2-one.
Priority DE-00111861 11-APR-91
Inventors Chatterjee,S; Noeldner,M; Hauer,H; Koch,E.
Designated States AT AU BE CA CH DE DK ES FR GB GR IT JP LU MC NL
SE US
Title Benzopyranones, methods of manufacture and use thereof.
Action NMDA agonist
Indication Allergy, Neurodegenerative disease, Urticaria
Publication WO-09218493-A1 29-OCT-92
Title Benzopyranones, methods of manufacture and use thereof.
Action NMDA agonist
Indication Allergy, Neurodegenerative disease, Urticaria
Publication WO-09218493-A1 29-OCT-92
Priority DE-00111861 11-APR-91
Inventors Chatterjee,S; Noeldner,M; Hauer,H; Koch,E.
Designated States AT AU BE CA CH DE DK ES FR GB GR IT JP LU MC NL
SE US
Bibliography
177482 Neurologic Drugs. DRUG NEWS PERSPECT 1995 8 1
47-48
177791 KA-672.HCL: A new orally active antidementia agent.
Klusa V, Germane S, Chatterjee SS, Noeldner M EUR J PHARM
SCI 1994 2 1-2
• The seminal presentation on KA-672, describing cognitive effects
in mice and rats.
258423 Willmar Schwabe GmbH COMPANY COMMUNICATION
1997 August 08
• Confirmation of KA-672 being in phase II trials for Alzheimer’s
disease.
258435 Anseculin hydrochloride. DRUGS FUTURE 1996 21 8
779-781
• A review on KA-672.
260590 Effects of anseculin on some excitatory amino acidinduced phenomena. Chatterjee SS, Noldner M PHARMACOL
TOXICOL 1997 80 Suppl 1 abs16
• The interactions of KA-672 with the NMDA receptor complex and
its neuroprotective and anticonvulsant effect is discussed.
260591 Neurotrophic effects of KA-672 HCl on cultured
embryonic septal cells. Klimaviciusa L, Chatterjee SS, Noldner M,
Svirskis S, Klusa V PHARMACOL TOXICOL 1997 80 suppl 1 abs28
• KA-672 and nerve growth factor act in a comparable way in vitro.
260592 Modulation of neurotransmitter by Anseculin. Noldner
M, Chatterjee SS PHARMACOL TOXICOL 1997 80 suppl 1 abs38
• An overview of the interaction of KA-672 with neurotransmitter
receptors, and its influence on serotonin and dopamine levels and
metabolism.
260593 Influence of KA-672.HCl on the NMDA-induced
neurotoxicity in mice: the contents of the brain monoamines.
Skujins A, Svirskis M, Noldner M, Chatterjee SS, Klusa V
PHARMACOL TOXICOL 1997 80 suppl 1 abs48
• This abstract describes the separate and combined actions of
KA-672 and NMDA, as reflected in 5-HT and dopamine levels. The
effect of KA-672 on NMDA-induced convulsions and mortality might
be mediated through central monoaminergic mechanisms.
260594 Anseculin, a neuronal activator against dementia:
tolerability, safety, preliminary pharmacokinetics in male and
female volunteers. Sourgens H, Horr R, Steinbrede H, Derendorf
H PHARMACOL TOXICOL 1997 80 suppl 1 abs50
• A presentation of the phase I studies of KA-672.
270790 Therapeutic approach in dementia. Trabucchi M,
Bianchetti A RIV PSICHIATR 1997 32 4 SUPPL. 20-26
275467 [Treatment of dementia with Tacrine]. THERAPIE
DEMENTIELLER ERKRANKUNGEN MIT TACRIN. Gastpar M
FORTSCHR FORTBILD MED 1997 21 - 451-458+495
281905 Benzopyranones, a method for producing them and
uses thereof. Chatterjee SS, Noeldner M, Hauer H, Kock E US
PATENT 1995, June 27 Dr W Schwabe GmbH & Co, Germany.
281907 Biotransformation of scoparone used to monitor
changes in cytochrome P450 activities in primary hepatocyte
cultures derived from rats, hamsters and monkeys. Mennes
WC, van Holsteijn CW, Timmerman A, Noordhoek J, Blaauboer BJ
BIOCHEM PHARMACOL 1991 41 8 1203-1208
• The demethylation of scoparone (the parent structure of KA-672)
in hepatocytes of various species is described.
281908 Dr Wilmar Schwabe GmbH & Co COMPANY COMMUNICATION 1998 March 6
•Schwabe has dropped its provisional synonym for KA-672 HCl,
anseculin. No international non-proprietary name has been assigned
to date.
281915 KA-672 HCl: A novel anticholinesterase inhibitor with
no effects on arylacylamidase or butyrylcholinesterase.
Noeldner M, Chatterjee SS 9TH INT SYMPOSIUM CHOLINERGIC
MECHANISMS 19957-10 June
• The cholinesterase inhibitor properties of KA-672 are discussed.
Drug evaluation Tranilast 141
Tranilast Kissei Pharmaceutical
Matthew Konneh
Address
Institut des Vaisseaux et du Sang
University VII of Paris
Paris
France
IDrugs 1998 1(1):141-146
Kissei, in collaboration with SmithKline Beecham, is
developing tranilast (Rizaben) as a prophylactic agent for
restenosis, following percutaneous transluminal coronary
angioplasty (PTCA). As of May 1997, tranilast was
awaiting approval in Japan as an oral formulation given
for three months following PTCA [246273], [248281]. The
agent has been launched for use in allergic rhinitis, asthma
and atopic dermatitis. Analysts had expected the early
approval of Rizaben for PTCA-associated restenosis.
However, due to delays by the Ministry of Health and
Welfare, it is unlikely that Rizaben will be approved until
the end of 1998. Sales of Rizaben for the treatment of its
launched indications did not reach company expectations.
The companies are also developing tranilast, in a nasal
spray formulation, as a potential treatment for allergic
rhinitis. The drug is in phase II trials for this indication
[262810].
EP-00588518 from Kissei discloses the use of tranilast for
the treatment of restenosis. Kissei has also filed patents
disclosing the use of tranilast for arteriosclerosis (JP09227371) and diabetic retinopathy (WO-09729744).
Introduction
Tranilast (N-3,4-dimethoxycinnamoyl anthranilic acid) has
been in clinical use in Japan since 1982 for the treatment of
allergic diseases, such as allergic rhinitis, bronchial asthma
and atopic dermatitis. Its mechanism of action is based on
the inhibition of antigen-induced release of chemical
mediators from mast cells and basophils [275344]. Tranilast
is also effective against keloids, the formation of which is
thought to be associated with excessive proliferation of, and
excessive collagen synthesis by, fibroblasts. From these lines
of evidence, researchers have investigated the therapeutic
benefit of tranilast against the development of fibroproliferative restenotic lesions, following PTCA.
Coronary heart disease (CHD) may be treated surgically by
PTCA, coronary artery bypass graft or cardiac
transplantation. The method of intervention depends on the
severity of the disease. These invasive surgical interventions
are associated with an early complication of increased
vascular smooth muscle cell growth and extracellular matrix
expression. Coronary angioplasty uses a balloon catheter to
dilate coronary stenotic lesions. Restenosis at the site of
dilation of these lesions occurs in 30 to 40% of cases [275349],
usually over a time course of three to six months. Acute
failure of angioplasty in 30% of cases is due to ’elastic recoil’
and can be prevented, to a certain extent, by stenting. The
Originator Kissei Pharmaceutical Co Ltd
Status/Indication Launched (Allergic rhinitis, Asthma, Atopic
dermatitis); Pre-registration (Restenosis)
Synonyms MK-341, Tramelas, Rizaben
O
OH
O
O
N
H
O
late failure of PTCA is normally due to fibrous intimal
proliferation; the excessive overgrowth of vascular smooth
muscle cells (VSMCs) together with the excess production of
extracellular matrix (ECM), including collagen, elastin and
glycoproteins (fibronectin, vitronectin and proteoglycans)
lead to the narrowing of the vessel lumen.
Pharmacology
The pharmacological approaches under consideration for
reducing restenosis include anti-inflammatory drugs,
antiplatelet agents, antithrombotics, anticoagulants, antiproliferatives, vasodilators, antioxidants, lipid-lowering
drugs, chimeric toxins, antisense oligonucleotides and gene
therapies. Several medical devices, such as atherectomy
systems that use a rapidly rotating device to reduce plaques,
intravascular stents and laser technology have come into
clinical testing and are used for the types of lesions for
which they are suited. Although some show benefit with
regard to acute complications, most do not seem to offer
distinct advantages in avoiding restenosis.
Using VSMC in culture, Tanaka et al [167010] showed that
tranilast could inhibit DNA synthesis, and hence cell
growth, as well as their migration and their ability to
synthesize collagen. These inhibitory effects occurred in a
dose-dependent manner; tranilast is also able to modulate
the effects of contractile agents. In isolated rat aortic rings,
which were made to contract in an organ bath by the
addition of endothelin (ET-1), exposure of these rings to
tranilast (0 to 500 µM) dose-dependently inhibited ET-1induced increases in tension and intracellular calcium
[258570]. Chronic recoil, following PTCA, may account for
more than 60% of the late lumen loss in restenosis. This
recoil may be due to a chronic vasoconstrictor component
such as endothelin release [275350] or loss of nitric oxide
(NO) vasodilatory activity. The impairment of NO release
by platelet-derived growth factor (PDGF) production can be
abrogated in the presence of tranilast [258582]. Therefore,
the ability of tranilast to inhibit vasoconstrictor activity may
be an important mechanism in its anti-restenotic profile.
Mechanical injury causes endothelial denudation and
platelet accumulation, which results in the release of several
growth factors [275350]. These growth factors may stimulate
the migration and proliferation of smooth muscle cells
(SMCs) in the dilated artery and some of the growth factors
may be acting through intracellular calcium release, [Ca2+]i.
Nie et al [266805] have shown that in rat VSMCs, 1 nM of
PDGF induced a biphasic elevation of cytoplasmic free
calcium concentration, however, tranilast abolished the
sustained phase of [Ca2+]i in a dose-dependent manner.
Tranilast also abolished the concomitant DNA synthesis of
these cells with a similar IC50 to that of the inhibition of
calcium flux. In a model of rat-vessel denudation, in which
insult to the femoral artery is induced by a photochemical
reaction between light and a photosensitive dye (Rose
Bengal), the generation of singlet molecular oxygen leads to
endothelial injury with subsequent platelet adhesion and
aggregation, and growth factor release. This initiates the
cascade responsible for recruiting SMCs and eventually
neointimal lesion formation.
In rats which received tranilast (10, 100, 300 mg/kg) two
days before surgery and continued for three weeks after,
lesional changes, measured by morphometric analysis,
showed that there was a dose-dependent decrease in the
cross-sectional area of the intimal layer (28.8, 62.4, 86.9%,
respectively), a trend which was also observed in
intimal/medial ratios. In SMCs cultured from the same rat
species, incubation with tranilast (3 to 300 µM) 24 h
previously caused a dose-dependent inhibition in
[3H]thymidine uptake [266806].
Although investigations employing injury to animal carotid
artery have provided invaluable insight into neointimal
development, the response to injury in these models may
not be entirely similar to the situation following PTCA of
atherosclerotic lesions in man. Leukocytes, which are
minimal in rat vessels, comprise a major but variable cellular
component of human and hypercholesterolemic rabbit
experimental models [275351], [275358], [275359].
In white male rabbits fed 1% cholesterol and 5% lard,
followed by balloon catheter injury in the right common
carotid artery, there was increased medial thickening,
intimal hyperplasia, and lipid deposits, compared to rabbits
on a normal diet. Tranilast was given orally (100 to 300
mg/kg/day) one day after the balloon injury. At 28 days
after injury, morphometric analysis showed that treatment
with tranilast (300 mg/kg) decreased the intimal thickening
as measured by intimal/medial ratios, and also increased
the lumenal/total area ratio, which was decreased in the
hypercholesterolemic rabbits compared with normocholesterolemic rabbits [258580].
Tranilast (600 mg/day) given to patients post-angioplasty for
three months, demonstrated a reduced rate of post-PTCA
restenosis [275420]. In an enlarged multicenter, double-blind,
placebo-controlled study of patients with native coronary
lesions and other predisposing factors, tranilast (300 mg/day
or 600 mg/day) or placebo was given for three months
following PTCA. Patients receiving tranilast (600 mg/day)
had a reduced rate of restenosis; 14.7% in comparison to 46.5
% for the placebo group (p < 0.001) [266811].
In another study, the efficacy of tranilast was investigated in
patients with restenosis undergoing directional atherectomy of coronary lesion. Patients were randomized into
two groups; 40 patients receiving tranilast (600 mg/kg) and
152 patients received placebo. In comparing angiographic
and clinical variables, the minimal lumen diameter was
significantly larger in the tranilast group at both the threeand six-month follow-up times than in the placebo group.
The restenosis rates (loss of > 50% of the initial gain) were
lower in the tranilast treated patients (11% versus 26%, p =
0.03). The number of clinical events over the 12-month
period after the procedure was significantly reduced by
tranilast administration (p = 0.0013) [275361].
Current Opinion
Hospital registries show that acute complications, including
hospital deaths, myocardial infarctions, and emergency
bypass surgery, have greatly declined. Much of this
improvement has been attributed to the aggressive use of
agents to prevent elastic recoil (antispasmodics), anticoagulants, thrombolysis and new mechanical devices including
perfusion balloons and intravascular stents. There are
300,000 procedures performed annually in the US. Although
the acute occurrence of complications has been greatly
reduced, the marked prevalence of restenosis clearly
compromises the long-term benefits associated with PTCA.
The advantage of the lower cost of performing PTCA
relative to coronary artery bypass surgery has been greatly
diminished by the expense of repeat diagnostic and
therapeutic catheterization as a result of restenosis [275362],
currently estimated to be US$2 billion dollars per year in the
United States alone [275423].
Tranilast has a potent inhibitory effect on smooth muscle cell
proliferation, migration and extracellular matrix production.
It also reduces the neointimal response to balloon injury in
the various animal models used to simulate this condition.
Of the few trials in which tranilast has been used in the
clinical setting, it is encouraging to see that all have shown a
beneficial outcome with respect to tranilast-induced
decreases in restenosis rates, following PTCA and coronary
atherectomy procedures. It remains for tranilast to undergo
the ’gold standard’ in clinical trials in which a large cohort of
patients (2000 to 3000) are enrolled to evaluate clinical events
following PTCA with a further subanalysis of patients using
angiography to determine the restenosis rate in doubleblind randomized, placebo-controlled conditions. With the
huge increase in healthcare costs as a result of restenosis and
its resultant complications (angina and repeat procedures), a
drug which limits these events would provide a powerful
pharmacological therapy for interventional cardiologist and
reduce the cost of coronary heart disease management.
Licensing
SmithKline Beecham plc
In May 1997, SmithKline Beecham acquired exclusive development and marketing rights for tranilast in areas outside of
Japan, Taiwan, South Korea, China and Russia [248281].
Development History
DEVELOPER
COUNTRY
STATUS
INDICATION
DATE
REF
Kissei Pharmaceutical Co Ltd
Japan
C1
Restenosis
12-MAR-97
275361
Japan
PR
Restenosis
14-MAY-97
246273
UK
PR
Restenosis
14-MAY-97
246273
UK
C2
Allergic rhinitis
15-SEP-97
262810
Japan
L
Allergic rhinitis
18-MAR-98
Japan
L
Asthma
18-MAR-98
Japan
L
Atopic dermatitis
18-MAR-98
South Korea
L
Allergic rhinitis
18-MAR-98
South Korea
L
Asthma
18-MAR-98
South Korea
L
Atopic dermatitis
18-MAR-98
Biology
STUDY TYPE
In vitro
EFFECT STUDIED
Vasorelaxation.
EXPERIMENTAL MODEL
Isolated rat aortic strips in an organ
bath were induced to contract by
endothelium ET-1 and high potassium
+
(K ) addition. Changes in calcium
2+
(Ca ) levels were measured by fura-2
loading.
RESULT
Prior exposure of these rings to tranilast
(0 to 500 µM), dose-dependently inhibited
ET-1-induced increases in tension and
2+
Ca elevation in the muscle strips.
REF
258570
In vitro
Vasorelaxation.
Transverse strips of porcine coronary
artery in an organ bath prior to
incubation with tranilast for 30 min,
followed by challenge with histamine
and KCl.
Histamine-induced contractions were
decreased by 25% and 65% on incubation
with 100 µM and 500 µM tranilast,
respectively. Tranilast inhibited the
+
contraction induced by K (30 mM) in a
dose-dependent manner.
258569
In vitro
Smooth muscle cell
(SMC) proliferation.
Extracellular matrix
production and
migration.
Stimulated [ H]thymidine and proline
uptake by SMC. Induced SMC
migration on culture plates.
Tranilast inhibited thymidine uptake dosedependently, with maximal inhibitory
activity being reached at 50 µg/ml. The
calcium antagonist, nilvadipine and the
PDE antagonists, E-1020, and elastase, all
showed concentration-dependent inhibitory
effects on vascular smooth muscle cell
proliferation, with maximal effects that
were approximately half that of tranilast.
167010
In vivo
Neointima formation.
Intimal thickening in femoral artery of
rats initiated by photochemical
reaction between green light directed
at the artery by an optic fiber and iv
infusion of Rose Bengal (10 mg/kg).
Tranilast at doses of 30, 100, 300 mg/kg,
po, given two days prior to endothelial
injury, decreased intimal area by 29%,
62% and 87%, respectively, in comparison
to vehicle control-treated animals.
266806
3
Biology (continued)
STUDY TYPE
In vivo
EFFECT STUDIED
Neointima formation.
EXPERIMENTAL MODEL
Intimal thickening in common carotid
artery induced by balloon
catheterization in rabbits which had
been fed a 1% cholesterol diet.
RESULT
28 Days after balloon angioplasty, marked
intimal thickening was observed in control
rabbits. Treatment with tranilast (300
mg/kg, po) decreased the intimal
thickening as measured by intimal/medial
ratios.
REF
258580
Clinical
EFFECT STUDIED
Restenosis after
percutaneous
transluminal coronary
angioplasty (PTCA).
MODEL USED
Double-blind, placebo-controlled study of
patients with native coronary lesions and
other predisposing factors. Patients were
given either 600 mg/day, 300 mg/day, or
placebo for three months following PTCA.
RESULT
Patients receiving tranilast 600 mg/day showed a
restenosis rate of 14.7% in comparison to 46.5%
for the placebo group (p < 0.001).
REF
266811
Restenosis following
successful directional
atherectomy.
Patients who underwent directional
atherectomy of coronary lesions were
randomized into groups. 40 Patients received
tranilast (600 mg/kg) and 152 patients
received placebo.
Comparing angiographic and clinical variables;
the minimal lumen diameter was significantly
larger in the tranilast group at both the threemonth and six-month follow-up times.
The restenosis rates (loss of > 50% of the initial
gain) were lower in the tranilast treated patients
than those on placebo (11% versus 26%; p =
0.03). The number of clinical events over the 12month period after procedure was significantly
reduced by tranilast administration (p = 0.0013).
275361
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275359 Morphological observations late (greater than 30 days)
after clinically successful balloon coronary angioplasty. Waller
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coronary atherectomy. Kosuga K, Tamai H, Ueda K, Hsu YS,
Ono S, Tanaka S, Doi T, Myou UW, Motohara S, Uehata H AM
HEART J 1997
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than bypass surgery? Reeder GS, Krishnan I, Nobrega FT , et al
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Murakami T, Yoshioka M, Yumoto R, Higashi Y, Shigeki S, Ikuta Y,
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281057 Pharmaceutical Industry: Global Model Book
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282064 Suppression by tranilast of fetal myosin heavy chains
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282065 Effectiveness of tranilast on restenosis after directional
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Drug evaluation Iralukast 147
Iralukast Novartis AG
Andrew Bramley
Address
Pulmonary Research Laboratory
123-3440 West Broadway
Vancouver
V6R 4R2
Canada
IDrugs 1998 1(1):147-151
Iralukast is an LTD4 and LTE4 antagonist under
development by Novartis and in phase II clinical trials as a
potential treatment for asthma [244117], [177071].
In a double-blind, placebo-controlled trial in 16 patients
with mild to moderate asthma, a single 1.5 mg inhaled dose
of iralukast reduced the incidence of exercise-induced
bronchiospasm and was well-tolerated [272161].
Originator Novartis AG
Status Phase II clinical
Indication Asthma
Action LTD4 antagonist, LTE 4 antagonist
CAS 4H-1-Benzopyran-2-carboxylic acid, 7-[[9-(4-acetyl-3hydroxy-2-propylphenoxy)-1-[hydroxy[3(trifluoromethyl)phenyl]methyl]-2,4-nonadienyl]thio]-4-oxo-,
monosodium salt, [S-[R*,S*-(E,Z)]]Registry No: 125617-94-9
Note: CGP-45715A - Irakulast sodium salt.
CAS 4H-1-Benzopyran-2-carboxylic acid, 7-[[9-(4-acetyl-3hydroxy-2-propylphenoxy)-1-[hydroxy[3(trifluoromethyl)phenyl]methyl]-2,4-nonadienyl]thio]-4-oxo-,
[S-[R*,S*-(E,Z)]]Registry No: 151581-24-7
Synonyms CGP-45715A
Novartis has an agreement with Rhone-Poulenc Rorer to
use its Ultrahaler delivery system for iralukast and phase II
trials are underway [220836]. The agreement also involves
the development of further related compounds and delivery
systems.
O
S
O
OH
O
OH
O
Introduction
Leukotrienes (LT) are potent LTD4 antagonists which are
synthesized from arachidonic acid and play a major role in
the inflammatory response in asthma. LT C4, D4 and E4 are
some of the most potent bronchoconstrictors known and
have recently been shown to play an important role in the
complex interactions of inflammatory cells in asthma.
Iralukast (CGP-45715A) is an LTD4/LTE4 antagonist which is
under development by Novartis (formerly Ciba-Giegy) and
is currently in phase II trials for the treatment of asthma
[244117], [177071].
Synthesis and SAR
The structure of iralukast is based on LTD4 [194318]. The
carboxylic acid group in the eicosanoid chain of LTD4 is
replaced by a trifluoromethyl group that is important for the
antagonist properties [274603], and the dipeptide sulfur side
chain is replaced by a chromone carboxylic acid [274604].
The polar region of the eicosanoid backbone is stabilized by
phenyl binding [274605]. In addition, the lipophilic region of
the molecule is stabilized by a substituted acetophenone
which serves as a lipophilic anchor [274605]. Iralukast has
the same chain length as LTD4, ie, 20 carbon atoms [194318].
During the development of LT antagonists, the introduction
of a methyl group at carbon 1 led to the LTD4 antagonist
activity with no sign of agonist activity [274603]. The
development towards a highly stable and potent LTD4/E4
antagonist involved the introduction of a chromone group to
replace the cysteinyl-glycine residue, which gave the
compound better chemical stability. This was then enhanced
O
OH
F
F
F
by the integration of the double bonds into a phenyl group,
or as in the case of other LT antagonists, by reducing the
number of double bonds [274604]. Introduction of a phenyl
group at carbons 2, 3 and 4 was found to stabilize the polar
region of the eicosanoid structure [274605], and further
stability was confirmed by replacing a methyl group at
carbon 1 with a trifluoromethyl group [194318].
The 1R,2S configuration of the iralukast structure is
absolutely crucial for LTD4 antagonist potency. Various
other stereoisomers were at least 20-fold less potent. The
number and geometry of double bonds in the eicosanoid
backbone are also essential for the activity of the agonist
both in vitro and in vivo. Analogs having the same number
and geometry of double bonds as synthetic LTD4 were less
active in vitro [194318].
Pharmacology
3
Iralukast displaces [ H]LTD4 from its receptor in guinea pig
lung membrane preparations; it also displaces 1 nM LTD4 in
a dose-dependent fashion (IC50 = 60 nM, Ki = 26 nM) when
added at the same time as the ligand. In studies where the
preparation was pre-incubated with the iralukast at 15, 30
and 45 min prior to the ligand, the IC50 values were 7.35, 10
and 15.3 nM, respectively (Ki values = 2.2, 2.9 and 4.0 nM)
[194318]. The increased potency when pre-incubated with
the antagonist is also reflected in the increased potency of
the compound when pre-incubated in bioassays.
Iralukast is an extremely potent antagonist of both LTD4 and
LTE4 receptors on guinea pig ileum, with IC50 values of 2.7
nM and 0.4 nM at 2 min and 15 min incubation, respectively,
and PA2 of 10.1. For LTE4 antagonism, the IC50 values were
7.9 nM and 0.36 nM at 2 min and 15 min incubation,
respectively. Although the IC50 values are similar for both
LTD4 and LTE4, there were differences in the agonist
concentration used in this assay, ie, for LTD4, 1.8 nM and
LTE4, 22.7 nM. This suggests that iralukast is more potent as
an LTE4 antagonist on guinea pig ileum [194318]. Iralukast
also inhibits histamine, bradykinin, PGE1, ACh, SP and
barium chloride, with IC50 values in the µM range when
added 2 min prior to the agonist. For all of these agonists,
other than BK and SP, the potency of iralukast was increased
when incubation was increased to 15 min.
and 240 min and 24 h pre-treatment. The iv ED50 value in
this model was 0.11 mg/kg at 60 min prior to challenge.
Iralukast also has a non-specific action against LTC4 and
LTD4-induced responses in the guinea pig isolated heart
[26731].
Aerosolized iralukast was found to be a potent LTD4
antagonist on spontaneously breathing guinea pigs
stimulated with LTD4. IC50 values of 0.09, 0.03, 0.2, 0.1 and 10
µg/kg for 1 min aerosol at incubation times of 15, 60, 120,
240 and 480 min. Iralukast is also a potent antagonist of LTE4
receptors in the same model. For animals treated with
iralukast 15 min prior to LTE4 challenge, the IC50 value was
0.3 µg/kg. When administered 60 min prior to challenge,
iralukast was even more potent, with an IC50 value of 0.01
µg/kg. Iralukast has also been shown to be orally active
against LT-induced bronchospasm in this in vivo model. ED50
values range from 4.3 mg/kg for a 1 h pre-treatment to 0.32
mg/kg after a pre-treatment period of 2 h. In addition,
activity was well maintained at a pre-treatment period of 4 h
(ED50 = 1.1 mg/kg). This declined at 8 and 24 h pretreatment to 2.9 and 10.9 mg/kg, respectively.
Toxicity
Iralukast administered iv, was also active, with ED50 values
of 0.16, 0.046 and 0.024 mg/kg at 1, 15 and 60 min prior to
aerosol LTD4 challenge. Similar activity has been shown at
120, 180 and 240 min pre-treatment, with ED50 values less
than 0.1 mg/kg.
In addition, iralukast showed potent antagonism of antigeninduced leukotriene-mediated bronchospasm in sensitized
guinea pigs [194318]. IC50 values were approximately 2, 3,
2.5 and 0.8 µg/kg at 15, 60 and 480 min, respectively. Oral
and iv activities have also been demonstrated in this antigen
model, with oral ED50 values of 2.8, 3.8 and 3.9 mg/kg at 120
In comparison with the other more established and clinically
advanced LT antagonist, such as zafirlukast (Zeneca) and
montelukast (Merck), iralukast has been shown to be
considerably more potent in inhibiting LTD 4-induced
bronchospasm in the guinea pig model. However, both
zafirlukast and montelukast are far more advanced in
clinical trials than iralukast, and it is not yet known whether
iralukast is more potent than its rivals in clinical trials.
No toxic effects have been reported.
Phase I
No data are currently available.
Phase II
To date, results of only one phase II trial on iralukast have
been published [272161]. In this double blind, placebocontrolled crossover trial, the ability of 1500 µg/kg of
inhaled iralukast, administered 60 min prior to evaluation,
was assessed for its ability to prevent exercise-induced
bronchospasm in asthmatics. In 16 patients with mild to
moderate asthma, iralukast significantly prevented exerciseinduced bronchospasm as assessed by a fall in FEV1 and
was shown to be well tolerated.
Current Opinion
Iralukast is a potent LTD4/LTE4 antagonist which blocks
both leukotriene and antigen-mediated bronchoconstriction
in guinea pigs. In animal studies, its activity compares well
with the other competing LT antagonists and, in fact, was
shown to be more potent than pranlukast (Ono), montelukast and zafirlukast. However, as yet there are very little
data demonstrating its efficacy and toxicity in human
subjects.
Licensing
Rhone-Poulenc Rorer SA
Co-development and co-marketing agreement with Ultrahaler technology.
Development History
DEVELOPER
COUNTRY
STATUS
INDICATION
DATE
Ciba-Geigy AG
Switzerland
C1
Asthma
01-OCT-92
Ciba-Geigy AG
Switzerland
C2
Asthma
01-NOV-94
Chemistry
STUDY TYPE
SAR
RESULT
Evaluation and structure-activity relationships of various synthetic analogs of LTD 4 found to be potent
leukotriene antagonists.
REF
194318
Biology
STUDY TYPE
In vitro
EFFECT STUDIED
Leukotriene antagonistic
effects.
EXPERIMENTAL MODEL
Guinea pig; isolated heart;
leukotriene-induced vascular
contraction; inhibitory effects.
RESULT
Leukotriene-induced vasculature
contraction inhibited non-specifically.
REF
26731
In vitro
LTD4 binding.
Ability of iralukast to displace LTD 4
from its receptor, using guinea pig
lung membrane preparations.
Iralukast displaced 1 nM LTD4 with IC50
of 60 nM (Ki = 26 nM).
194318
In vitro
Smooth muscle relaxant.
Antagonist properties of iralukast on
LT contractions of guinea pig ileum.
IC50 value for iralukast on LTD4 (2.7 nM)
LTE4 (7.9 nM) at 2 min incubation and at
15 min incubation LTD4 (0.4 nM) LTE4
(0.36 nM).
194318
In vivo
LTD4-induced
bronchoconstriction.
Effect of aerosol administration of
iralukast on LTD4 challenge.
Iralukast is an effective and potent LTD 4
antagonist.
194318
Associated patent EP-00419411 (Ciba-Geigy AG)
Abstract Novel alkanophenone derivatives possessing
leukotriene antagonistic and phospholipase A2 inhibitory
activities are disclosed. The compounds are useful for
treating various inflammatory diseases, including contact
dermatitis, asthma and allergies. Protocols for the in vitro
evaluation of the compound's leukotriene D4 antagonist
activity and phospholipase A2 and phospholipase C
inhibitory activity are disclosed. Thirty two compounds
are specifically claimed, but no specific data are given.
Title Additional alkanophenones.
Indication Allergy, Asthma, Dermatitis, Inflammation
Action LT antagonist, LTD4 antagonist, PLA2 inhibitor
Publication EP-00419411-A2 27-MAR-91
Priority CH-00003402 19-SEP-89
Inventors Von Sprecher,A; Schaub,B; Lang,RW.
Designated States AT BE CH DE DK ES FR GB GR IT LI LU NL SE
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1991 41 395-406
• Reports on effects of iralukasts non-specific actions on LTC4
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• Report on Ciba (Novartis) pipeline as of 1995.
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of leukotriene D4 with special emphasis on CGP 45715A.
Sprecher A Von, Beck A, Sallmann A, Breitenstein W, Wiestner
H, Kimmel S, Anderson GP, Subramanian N, Bray MA DRUGS
FUTURE 1991 16 9 827-843
• Report on synthesis and structure-activity relationships of
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203429 Novartis - Backgrounder.
BROCHURE 1996 March 27
Novartis
COMPANY
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272161 Information update DRUGS FUTURE 1997 22 9 10271061
• Reports on the only phase II trial using iralukast.
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Les anti-leucotrienes: leur positionnement dans l’asthme. Devillier P, Millart H, Advenier C REV MED BRUX 1997 18 4 279-285
274603 Samuelsson B, Paoletti R, Ramwell PW ADV PROSTAGLANDIN THROMBOXANE LEUKOT RES 1987 17 519-525
• Reports on the importance of methyl substitution of the
carboxylic group in the eicosanoid backbone of LTD4 to produce
leukotriene antagonist properties.
274604 Von Sprecher et al ANN NY ACAD SCI 1988 524 438-441
• Shows the importance of replacing the cysteinyl-glycine
dipeptide sulfur side chain with a chromone carboxylic acid to
confer chemical stability.
274605 Samuelsson B, Wong PYK, Sun FF ADV PROSTAGLANDIN THROMBOXANE LEUKOT RES 1988 19 647-650
• Shows the importance of stabilizing the polar region of the
eicosanoid backbone of an LTD4 antagonist by phenyl binding.
207193 Novartis’ "new skills". SCRIP 1996 2111 7
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1990-1995. Mlodzik H EXP OPIN THER PAT 1996 6 1 57-60
• Reports on possible commercial viability and market potential
of agents used for the treatment of asthma.
215839 Modulators of leukotriene biosynthesis and receptor
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257897 The action of the peptidoleukotriene, LTD4, on
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257899 Protective effect of inhaled iralukast (a new LTD4
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1994 19 9 872.
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long lasting peptido-LT PLT antagonist activity in vivo. Bray
MA, Anderson GP, Erard E, Kuhn M, Niederhauser U, Reisbrodt
P, Rordorf C, Sills M, Stalder R, et al AM REV RESPIR DIS
1992 145 4 PART 2 A284
281987 Peptidoleukotrine antagonists: structural analogs of
leukotriene D4 with special emphasis on CGP-45715A. Von
Sprecher A, Beck A, Sallmann A, Breitenstein W, Wiestner H,
Kimmel S, Anderson GP, Subramanian N, Bray MA DRUGS
FUTURE 1991 16 9 827-843
281989 Pharmacological and molecular evidence for the
expression of the two steroid 5-α-reductase isozymes in
normal and hyperplastic human prostatic cells in culture.
Berthaut I, Mestayer C, Portois MC, Cussenot O, Mowszowicz I
PROSTATE 1997 32 3 155-163
Epristeride SmithKline Beecham
Sharath S Hegde
Address
Roche Bioscience
Center for Biological Research
Department of Urogenital Pharmacology
3401 Hillview Avenue
Palo Alto
CA 94304
USA
IDrugs 1998 1(1):152-157
 Current Drugs Ltd ISSN 1369-7056
Epristeride is a transition-state, noncompetitive steroid 5-αreductase inhibitor under development by SmithKline
Beecham for the treatment of benign prostatic hyperplasia
and acne. Phase III trials for prostatic hypertrophy with an
oral formulation, have been initiated in the UK, the US and
Japan [188478], [219166].
Drug name Epristeride
Originator SmithKline Beecham plc
Status Phase III clinical
Indication Acne, Prostatic hypertrophy, Prostate disease
Action α-reductase inhibitor
CAS Androsta-3,5-diene-3-carboxylic acid, 17-[[(1,1dimethylethyl)amino]carbonyl]-, (17β)Registry No: 119169-78-7
Synonyms SKF-105657, Zariflo, ONO-9302, SKB-105657
O
In healthy male volunteers, epristeride caused a reduction in
serum dihydrotestosterone levels but serum testos-terone
levels remained stable. In animal studies, it showed a
similar potency to finasteride for the inhibition of 5-αreductase [164200].
Epristeride is licensed to Ono which has exclusive rights in
Japan, South Korea and Taiwan. Recordati has comarketing rights for epristeride [162547], [162519].
Analysts at Yamaichi estimate that epristeride will be
launched in Japan between 1999 and 2000 and peak annual
sales have been predicted to be over ten billion Yen [216018].
Introduction
Epristeride is a potent and selective non-competitive
inhibitor of the Type 2 steroid, 5-α-reductase, the enzyme
responsible for the conversion of testosterone to
dihydrotestosterone. By suppressing the biosynthesis of
dihydrotestosterone, epristeride has the ability to retard
prostate growth and relieve bladder outlet obstruction in
patients with benign prostatic hyperplasia. Other potential
therapeutic uses of epristeride include the treatment of acne,
prostate cancer and male pattern baldness.
Synthesis and SAR
Epristeride (17-β-(N-tert-butylcarbomyl)androsta-3,5-diene3-carboxylic acid) was identified from a series of steroid 5-αreductase inhibitors that belong to the 3-androstene-3carboxylic acid class. Structure-activity studies showed that
2
the high affinity is dependent on sp hybridization at C-2, C3 and C-4 [275886]. Furthermore, the negative charge
supplied by the carboxylate function at C-3 is essential for
activity in as much as reduction to the aldehyde or alcohol
leads to a pronounced drop in inhibitory potency.
Two synthetic routes of epristeride have been reported
[199657]. The first route starts from methyl 3-oxoandrost-4ene-17-β-carboxylate, which is converted to epristeride in
H
N
H
H
HO
H
O
four steps, with a 44% overall yield. The second route starts
from the commercially available 3-oxoandrost-4-en-17-betacarboxylic acid, which is converted to epristeride in two
synthetic steps, with a 63% overall yield. Both routes have
been employed to produce kilogram quantities of epristeride
of high purity.
Pharmacology
Epristeride is a potent inhibitor of the human Type 2 steroid
5-α-reductase (Ki = 0.7 to 2 nM) but is a much weaker
inhibitor of the human Type 1 isoform of steroid 5-αreductase (Ki = 400 to 450 nM) [164200]. Epristeride also
inhibits the native form of the enzyme in human prostate (Ki
= 2 to 15 nM), rat prostate (Ki = 10 to 20 nM), rat liver (Ki = 5
to 10 nM), rat epididymis (Ki = 2 to 4 nM) and monkey
(cynomologus) prostate (Ki < 1 nM) [164200], [275888].
Epristeride is a non-competitive inhibitor of both isoforms of
steroid 5-α-reductase by the formation of a ternary complex
with NADP+ and the enzyme [164200]. It is a highly specific
inhibitor of steroid 5-α-reductase since it has at least a 1000fold lower affinity for seven other steroid processing
enzymes and five steroid hormone receptors [164200].
In rats and cynomologus monkeys, orally-administered
single doses of epristeride produce significant and longlasting (>24 h after a single dose) reductions in plasma 5-αdihydrotestosterone levels and also prostatic 5-αdihydrotestosterone content, consistent with inhibition of
Drug evaluation Epristeride 153
steroid 5-α-reductase [164198]. Chronic oral adminis-tration of
epristeride (0.1, 1.0 or 10 mg/kg, bid, for six weeks) to mature
rats resulted in significant decreases in prostatic 5-α-dihydrotestosterone content, which were associated with dose- and
time-dependent decreases in mass of ventral prostate [164198].
Toxicology
In castrated rats, epristeride (25 mg/kg, bid, po) reduced
testosterone, but not 5-α-dihydrotestosterone. It induced
increases in ventral prostate weight, prostatic secretion and
glandular proliferation, which is consistent with the notion
that the compound operates by inhibiting the conversion of
testosterone to 5-α-dihydrotestosterone and is not an
androgen receptor antagonist [199669]. Epristeride (25 or 50
mg/kg, bid, po) produced significant decreases in 5-αdihydrotestosterone content and inhibited tumor growth in
rodents transplanted with androgen-responsive prostatic
cancer cells (Dunning R-3327 G and PC-82) [199674].
Phase I
In cultured stromal cells from benign, hyperplastic adult
prostates, epristeride dose-dependently inhibited the
proliferative response to testosterone, but had no effect on 5-adihydrotestosterone-induced growth or growth of androgenunresponsive, testosterone-treated cells [275889]. In these
studies, upregulation of prostate-specific antigen secretion
from epithelial cells by androgens was downregulated by
epristeride in testosterone-treated cells. Furthermore, transforming growth factor-β-1 secretion was downregulated by
testosterone treatment and this was reversed by epristeride.
Metabolism
The phamacokinetics of epristeride have been investigated
in young and elderly healthy male subjects [275890],
[275891]. Following oral dosing, peak plasma levels were
usually attained around 1.5 to 4 h. Epristeride plasma
concentrations decline in a biexponential fashion with
secondary peaks usually evident around 24 h, following
intravenous and oral dosing. The mean apparent
termination half-life estimates were similar after intravenous
and oral dosing and in the range of 24 to 27 h, which allows
once-daily dosing. The mean plasma clearance and steadystate volume of distribution were 0.33 ml/min/kg and 0.54
l/kg, respectively. The mean absolute bioavailability was
93%. Epristeride was highly bound to plasma proteins
(98.9%) and binding was concentration-dependent over the
therapeutic range and was reduced in patients with liver
disease [206422].
The pharmacokinetics of epristeride have been studied in
benign prostatic hyperplasia patients after daily dosing (5, 20
and 80 mg) for 8 weeks [199659]. Mean trough concentrations
were dose-proportional and steady-state levels were attained
at the end of the first week. No evidence of drug accumulation
were noted over the 8-week period. A good correlation was
observed between steady-state drug levels and the reduction
of 5-α-dihydro-testosterone levels in serum and prostate.
14
Preclinical studies in rats and dogs with [ C]epristeride
have shown that the majority of radioactivity can be
recovered in the bile, predominantly as the acyl glucoronide
conjugate, whereas less than 0.5% was excreted in the urine
[164198]. Enterohepatic circulation of the drug has also been
demonstrated in animals.
The data from toxicological studies with epristeride have not
yet been reported.
In single dose phase I studies, epristeride (median dose of 0.25
mg/kg), decreased serum 5-α-dihydrotestosterone levels by
more than 50% in 86% of the patients. This effect persisted for
more than 24 h [276064]. Two subsequent multi-dose phase I
studies in healthy male subjects confirmed the biochemical
efficacy of epristeride [275897]. In the first open label study, 40
subjects received ascending doses of 0.4, 2.0, 20, 80 or 160
mg/day. In the second placebo-controlled and double-blind
study, 51 subjects were randomized to receive doses of 4, 10,
20 or 40 mg/day of epristeride or placebo. In both studies,
epristeride produced suppression of serum 5-α-dihydrotestosterone levels at all doses, with the maximal effect being
noted at about 12 h post-dose.
Phase II
Two double-blind, placebo-controlled phase II trials with a
total of 96 men have evaluated the effects of epristeride (0.4
to 80 mg once daily for 10 to 14 days) in patients with benign
prostatic hyperplasia prior to undergoing trans-urethral
resection of the prostate [276075], [275898]. Epristeride at 80
mg daily produced significant lowering (74 to 78%) of
prostatic 5-α-dihydrotestosterone levels when compared to
placebo. In another study, 56 patients with benign prostatic
hyperplasia were treated with placebo or epristeride (2, 10
or 80 mg) for 10 days. Levels of 5-α-dihydrotestosterone in
serum and prostate decreased with increasing serum
epristeride levels.
Phase III
No phase III data are currently available.
Side-effects and Contraindications
No side-effects and contraindications have yet been reported.
Current Opinion
Previous clinical studies with finasteride (Merck), the first
selective steroid 5-α-reductase inhibitor, have shown that this
therapeutic approach can confer symptomatic benefit, albeit
modestly, to patients with benign prostatic hyperplasia. Like
finasteride, epristeride is also a selective inhibitor of steroid 5α-reductase and reduces serum and prostatic levels of
dihydrotestosterone in benign prostatic hyperplasia patients.
Epristeride differs from finasteride in its mode of inhibition of
steroid 5-α-reductase, ie, non-competitive as opposed to
competitive. From a theoretical standpoint, the noncompetitive behavior of epristeride may be advantageous,
since this would diminish the potential of excessive substrate
(testosterone) surmounting the inhibitory effects of
epristeride. From the clinical view, it is unclear whether the
non-competitive effects of epristeride would augment the
therapeutic efficacy of the drug. The data from large ongoing
phase III trials are awaited to ascertain the therapeutic value
and superiority of epristeride in comparison to other
inhibitors of steroid 5-α-reductase.
Licensing
Ono Pharmaceutical Co Ltd
A cross-licensing agreement exists between SmithKline Beecham and Ono Pharmaceuticals for the products, epristeride and
pranlukast, respectively [167442]. Ono has the rights in Japan, South Korea and Taiwan.
Recordati SpA
Recordati has co-marketing rights for SmithKline Beecham’s epristeride and ropinirole in Italy and Spain. This is in exchange
for rights given to SmithKline Beecham to develop and market Recordati’s highly-selective α-receptor antagonist compounds
indicated for benign prostatic hypertrophy. This includes the lead compound, REC-15-2739 [162547], [162519].
Development history
DEVELOPER
COUNTRY
STATUS
INDICATION
Taiwan
C2
Prostatic hypertrophy
DATE
REFERENCE
167442
South Korea
C2
167442
Recordati SpA
Italy
C3
162519
Recordati SpA
Spain
C3
162519
UK
C3
188421
UK
C?
Acne
UK
C3
Prostate disease
US
C3
Prostate disease
Japan
C3
203772
203772
11-SEP-96
219166
Chemistry
STUDY TYPE
RESULT
REF
SAR
Studies provide data for identification of key pharmacophores for activity.
275886
Synthesis
Describes two commercially-viable synthetic routes for epristeride.
199657
Biology
STUDY TYPE
EFFECT STUDIED
EXPERIMENTAL MODEL
RESULT
REF
In vitro
Inhibitory potency against
steroid 5-α-reductase.
Recombinant human type I and II
steroid 5-α-reductase; native steroid
5-α-reductase in human, monkey
and rat tissues.
Ki (Type I) = 0.7 to 15 nM; K i
(Type II) = 400 to 450 nM.
164200
In vitro
Proliferative response and
prostate-specific antigen
secretion and TGFβ-1
secretion.
Cultured stromal and epithelial cells
from benign hyperplastic adult
prostates; 1 nM to 0.3 mM
epristeride.
275889
In vivo
Prostatic 5-α- dihydrotestosterone content, ventral
prostate weight, prostate
secretion and glandular
proliferation.
Castrated rats 25 mg/kg bid, po
Inhibition of proliferative
response to testosterone and
upregulation of prostate-specific
antigen secretion and TGFβ-1
secretion.
Reduces testosterone, but not 5α- dihydrotestosterone-induced
increase in ventral prostate
weight, prostatic secretion and
glandular proliferation.
In vivo
5-α-dihydrotestosterone
content and tumor growth.
Rats transplanted with androgenresponsive prostate cancer cells
(Dunning R-3327G and PC-82);
epristeride 25 or 50 mg/kg bid, po.
Decreases in 5-αdihydrotestosterone content and
inhibition of tumor growth.
199669
199669
Metabolism
STUDY TYPE
EFFECT STUDIED
MODEL USED
RESULT
REF
In vivo
Clearance of [14C]epristeride.
Rats and dogs.
Majority of radioactivity recovered
in bile, principally as acyl
glucoronide, < 0.5% excreted in
urine.
164198
Clinical
EFFECT STUDIED
Pharmacokinetics.
MODEL USED
Young and elderly male subjects.
RESULT
Pharmacokinetics.
Patients with benign prostatic hyperplasia; 5,
20, 80 mg, qid, for 8 weeks.
Dose-dependent increase in mean trough
concentrations. Steady-state achieved in 1 week. No
evidence of drug accumulation.
199659
Biochemical
efficacy.
Healthy male subjects (n = 40) in open label
study. Ascending doses of 0.4, 2.0, 20, 80 or
160 mg/day.
Decreases in serum 5-α-dihydrotestosterone levels.
275897
Biochemical
efficacy.
Healthy male subjects (n = 51) in double-blind,
placebo-controlled study. 4, 10, 20 or 40
mg/day.
Decreases in serum 5-α-dihydrotestosterone levels.
275897
Biochemical
efficacy.
Benign prostatic hyperplasia patients (n = 56)
in double-blind, placebo-controlled study; 2, 10
or 80 mg/day for 10 days.
Reduction in prostatic and serum 5-α-dihydrotestosterone levels.
275898
Biochemical
efficacy.
Benign prostatic hyperplasia patients (n = 96)
in double-blind, placebo-controlled study; 0.4
to 80 mg/day for 10 to 14 days.
Reduction in prostatic 5-α-dihydrotestosterone
levels.
276075
Termination t1/2 = 24 to 27 h; plasma clearance =
0.33 ml/min/kg; steady state volume of distribution =
0.54 l/kg; bioavailability = 93%.
REF
275890
Associated patent EP-00427434 (SmithKline Beecham)
Abstract 11-Keto or 11-hydroxy 3,5-diene steroids are
claimed as inhibitors of steroid 5-α-reductase. The
compounds are useful in the treatment of androgenrelated diseases. The treatment of benign prostatic
hypertrophy is indicated in one claim. Inhibition of
human prostatic steroid 5-α-reductase is described for two
compounds. 17β-(N,N-diisopropylcarboxamide)-11-oxoandrosta-3,5-diene-3-carboxylic acid had a Ki value of 30
nM while the corresponding 11β-hydroxy compound had a
Ki value of 20 nM. Eight examples with details are given.
Four compounds are claimed, including N,N-diisopropylcarboxamide-11-oxo-acid.
Title 11-Keto or hydroxy 3,5-diene steroids as inhibitors of steroid 5-αreductase.
Indication Prostatic hypertrophy
Action Testosterone 5-α-reductase inhibitor, Androgen synthesis inhibition
Publication EP-00427434-A2 15-MAY-91
Priority US-00430152 01-NOV-89
Inventors Holt,DA; Metcalf,BW; Levy,MA.
Designated states AT BE CH DE DK ES FR GB GR IT LI LU NL SE
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Ping L J, Fox M, Mewshaw RE, Tickner AM, Kowalski CJ J ORG
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• Describes the synthetic route for epristeride.
167442 Ono Pharmaceutical Co Ltd annual report 1993
171208 SB claims top R&D performance. SCRIP 1995 1992 12 13
• Comments by SB management on better cost/toxicity profile of
SKF 107647 compared with CSFs.
172957 SmithKline Beecham plc. PHARMA BUSINESS 1995 1
123 - 129
176815 Sanofi information
BROCHURE 1995 Spring
meeting.
SANOFI
199655 Benzophenone- and indolecarboxylic acids: Potent
type-2 specific inhibitors of human steroid 5-α-reductase. Holt
DA, Yamashita DS, Konialian-Beck AL, Luengo JI, Abell AD,
Bergsma DJ, Brandt M, Levy MA J MED CHEM 1995 38 1 13 - 15
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199659 Pharmacodynamic analysis of plasma epristeride
concentrations and dihydrotestosterone levels in patients with
benign prostatic hyperplasia. Benincosa LJ, Miller A, Knox S,
Rappoport E, Morris R, Lamb Y PHARM RES 1993 10 10 Suppl
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COMPANY
188421 Research and development review - SB Pharmaceuticals
advanced development portfolio: Products in phase III clinical
trials. SMITHKLINE BEECHAM COMPANY BROCHURE 1995
October 02
188478 Research and development review - SB Pharmaceuticals:
Development portfolio by therapeutic area. SMITHKLINE
BEECHAM COMPANY BROCHURE 1995 October 02
189127 SmithKline Beecham to file 13 new chemical entities
and 8 vaccines over the next 3 years. SmithKline Beecham plc
PRESS RELEASE 1995 October 06
189136 New drugs in the R&D Pipeline : Ono. PHARMA JPN
1995 1467 22
• Disclosure of ONO-1603 having reached phase II.
189187 Smithkline Beecham upbeat on R&D. SCRIP 1995 2066 14
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androgenic response. Lamb JC, English H, Levandoski PL,
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the conversion of testosterone to 5-α-dihydrotestosterone and not by
antagonism of androgen receptors.
199671 Hormonal balance and the risk of prostatic cancer.
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192905 Recent progress in the pharmacotherapy of diseases of
the lower urinary tract. Hieble JP, McCafferty GP, Naselsky DP,
Bergsma DJ, Ruffolo RR Jr EUR J MED CHEM 1995 30 Suppl 269s
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type 1 and type 2 steroid 5-α-reductases from Cynomolgus
monkey: comparisons with human and rat isoenzymes. levy
MA, Brandt M, Sheedy KM, Holt DA, Heaslip JI, Trill JJ, Ryan PJ,
Morris RA, Gasrrison LM, Bergsma DJ J STEROID BIOCHEM MOL
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novel 5-α-reductase inhibitor, SK&F 105657. lamb JC, Levy MA,
Johnson RK, Isaacs JT PROSTATE 1992 21 1 15 - 34
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for treatment of BPH. DRUGS FUTURE 1991 16 11 996 - 1000
203772 Striving to make peoples lives better. SmithKline
Beecham plc ANNUAL REPORT 1995 March
206422 Pharmacokinetic (PK) and protein binding (PB) of
epristeride in liver disease patients LDP and healthy male
subjects. Benincosa LJ, Thompson KA, Everitt DE, Oldham H,
Patterson S, Jorkasky D, Bay MK, Schenkler S AM SOCIETY CLIN
PHARMACOL THER 1996 97 Annu Meet March 20 - 22 PIII-52
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Pharm Co COMPANY BROCHURE 1996 March 30
219166 New drugs in the R&D pipeline.
1515 18
PHARMA JPN 1996
255734 5-α-Reductase (5α-R) and androgenic responses in
HOS-TE85 (TE85) human osteosarcoma cells. Leibovitch IY,
Gygi CM, Goode RL, Dixon EP, Little SP, Sutkowski DM, Neubauer
BL PROC AM ASSOC CANCER RES 1997 38 Abs 3863
261150 Effects of steroid 5-α-reductase inhibitor, ONO-9302,
and anti-androgen allylestrenol on the prostatic growth, and
plasma and prostatic hormone levels in rats. Yasuda N, Fujin K,
Shiraji T, Nambu F, Kondo K JPN J PHARMACOL 1997 74 3 243 252
263452 Epristeride. DRUGS FUTURE 1997 22 7 789
275886 Potent inhibition of human steroid 5-α-reductase (EC
1.3..30) by 3-androstene-3-carboxylic acids. Metcalf BW, Holt
DA, Levy MA, Erb JM, Heaslip JI, Brandt M, Oh HJ BIOORG CHEM
1989 17 372 - 376
275888 3-Phosphonic acid and 3-phosphonic acid steroids as
inhibitors of steroid 5-α-reductase: Species comparison and
mechanistic studies. Levy MA, Metcalf BW, Brandt M, Erb JM, Oh
HJ, Heaslip JI, Yen HK, Razamus LW, Holt DA BIOORG CHEM
1991 19 245 - 260
275889 Effects of a new 5-α-reductase inhibitor (epristeride) on
human prostate cell cultures. Robinson EJ, Collins AT, Robson
CN, Neal DE PROSTATE 1997 32 4 259 - 265
275890 Pharmacokinetics and pharmacodynamics of SK&F
105657 in healthy elderly male subjects. Chapelsky MC, Nichols
A, Jorkasky DK, Pue MA, Lundberg BS, Knox MS, Audet PR CLIN
PHARMACOL THER 1992 51 154
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epristeride in healthy male subjects. Benincosa LJ, Audet PR,
Lundberg D, Zariffa N, Jorkasky DK BIOPHARM DRUG DISPOS
1996 17 3 249 - 258
275897 Effect of multiple doses of epristeride, a steroid 5-αreductase inhibitor on serum dihydrotestosterone (DHT) in
older male subjects. Audet P, Nurcombe H, Lamb D, Jorkasky K,
Lloyd-Davies A, Morris R J CLIN PHARMACOL 1993 53 231
275898 Effect of fourteen days treatment with epristeride, an
uncompetitive 5-α-reductase inhibitor on serum and prostatic
testosterone and dihydrotestosterone in men with benign
prostate hyperplasia. Johnsonbaugh RE, Cohen BR, McCormack
EM, George FW, Wilson JD J UROL 1993 149 432A
• Provides preliminary evidence for the biochemical efficacy of
epristeride in patients with benign prostatic hyperplasia.
276064 Hormonal effect of SK&F 105657, a 5-α-reductase
inhibitor in normal healthy male subjects. Audet P, Ilson B,
Jorkasky D 9TH INT CONGRESS ENDOCRINOLOGY 1992 30
Aug - 5 Sept Nice Abst 13.03.033
276075 Double-blind, placebo-controlled study to evaluate the
pharmacodynamic effect of SK&F 105657 in patients with
benign prostatic hypertrophy. Peeling WB, Abrams P, Ramsey
JWA, et al 10TH CONGRESS EUROPEAN ASSOCIATION
UROLOGY 1992 240 22 July - 25 July, Genoa 148
• Provides preliminary evidence for the biochemical efficacy of
epristeride in patients with benign prostatic hyperplasia.
Patent News
Peter Steele
Address
Current Patents Limited
Middlesex House
London
W1P 6LB
UK
IDrugs 1998 1(1):158-161
Schering-Plough
If there was a patent award, then one of the winners might
well be the nine-part invention from Schering-Plough Corp,
published as WO-09811091 to WO-09811093, WO-09811096
to WO-09811100 and WO-09811106. Dr Karl Thomae GmbH,
however, is also a strong contender, with 75 pages of claims
in WO-09811128 to libraries of neurotransmitter modulators,
the complete specification extending to almost 500 pages.
Schering-Plough’s invention relates to farnesyl protein
transferase (FPTase) inhibitors, and the tricyclic compounds
belong to a series which the company has been investigating
for some time. Several code-numbered compounds of this
type, including Sch-54419, have already been the subject of
preclinical studies. FPTase inhibitors have become a very
popular target for the industry, and more than 20 such
agents are currently undergoing preclinical investigations.
Schering-Plough’s major commit-ment to this field is clearly
yielding a rich supply of promising compounds, although
both Merck & Co Inc and Warner-Lambert Co are also
known to have located several interesting leads.
ICE/CED-3 from IDUN
Three substantial applications from IDUN Pharmaceuticals
Inc, WO-09810778, WO-09811109 and WO-09811129, relate to
inhibitors of the interleukin-1β-converting enzyme CED-3
family. There are two distinct strands to this work,
involving indoles on the one hand and peri-fused tricyclics
on the other. The indoles are shown to act as apoptosis
inhibitors, with potential in transplantation as a result of
their effectiveness in expanding and prolonging survival of
hematopoietic cell populations. IDUN’s work on apoptosis
inhibition has attracted long-term support from Novartis
AG, and these compounds may well fall within the terms of
that agreement. The tricyclics too are linked to the work of
Novartis, since there is a very close structural similarity with
CGS-28106, an azepino[3,2,1-hi]indole in which dual
ACE/NEP inhibitory activity is being studied.
NIH
NIH is commonly understood to denote the National
Institutes of Health, but in management-speak stands for
"not invented here", a syndrome afflicting chauvinistic R&D
personnel. There are several examples this month of
inventions which appear to build on other companies’
earlier discoveries, which is precisely the way in which the
patent system is intended to work. Servier, for example,
patenting as ADIR & Co, claims PDE-IV inhibitors in EP00831090 which are clearly derived from rolipram, Schering
AG’s phase III antidepressant. Likewise Astra AB, a
company with no background in protein kinase C inhibitors,
is now seen to be following the lead of Eli Lilly & Co, a
company with several such compounds in active
development. The bis-indolyl compounds claimed by Astra
in WO-09811102, WO-09811103 and WO-09811105 are
structurally similar to LY-333351; the latter, though still only
at the preclinical stage, has been the subject of several Lilly
process and use cases in recent months, which seems to
indicate that intensive development activity is in progress.
The antihyperproliferatives claimed by Celltech Group plc
in WO-09811095 also act on PKC, but in addition are
described as inhibitors of the p56lck and ZAP-70 kinases.
The 2-pyrimidineamine template adopted by Celltech for
this project has already been used by Novartis as the basis
for such candidates as CGS-53716 and CGS-57148, which act
as inhibitors of tyrosine kinase.
Cross-site developments
A sure sign of precommercial activity is the patenting of
secondary inventions by inventors located at two or more
corporate sites. An example this month is WO-09810762, in
which workers at SmithKline Beecham plc’s (SB) Collegeville, Pennsylvania site join forces with those in the UK to
claim a controlled release form of the azabicyclooctyl
acetonitrile SB-202026; this muscarinic agonist, in phase III
trials in Alzheimer’s disease, has now been assigned the
approved name sabcomeline, and seems destined to become
an important new product for SB within a year or two.
Another Pennsylvania-based team, this time Rhone-Poulenc
Rorer Inc (RPR), is collaborating with colleagues in France
on the development of chiral syntheses of ethylamine
derivatives. From an inspection of RPR’s pipeline, it
becomes clear that the optically active intermediates claimed
in WO-09811064 are intended for adenosine receptor
modulators such as RPR-100579, a cardiovascular agent
which is already progressing well in clinical trials. Sanofi
Recherche SA’s WO-09811053 is also concerned with chiral
synthesis, though without input from abroad; the claimed
benzyl alcohol derivatives could be used in the synthesis of
SR-58611A, a spasmolytic atypical β3 agonist which is in
phase II trials. Another SB invention, WO-09811067, relates
to the use of SB-207266A in irritable bowel syndrome; the 5HT4 antagonist is in phase III trials for this indication. In
WO-09810782, Merck & Co claims various combinations of
pneumocandins with other antifungals, naming such
compounds as ER-30346, Sch-56592, CAN-296 and XMP-97.
Most of Merck’s antifungal development effort seems to be
devoted to echinocandins rather than pneumocandins, and
it is not clear which, if any, of the latter are in active
development. Several of the other antifungals, on the other
hand, are progressing well; Eisai Co Ltd’s ER-30346, for
example, is reported to be licensed to Bristol Myers Squibb
Co, and the Schering-Plough compound, already the subject
of prodrug patenting, has reached phase II trials. There can
be no doubt that secondary patenting of this type, though
Patent News 159
not necessarily of great scientific interest, gives advanced
warning of future commercial activity, if placed in context. It
is no coincidence that several of the candidates mentioned
above are regarded as leading compounds in their
respective classes.
York, describes how as many as 108 compounds might be
prepared by light-directed synthesis on the surface of a
single 5½" disc, and then assayed in a similar fashion. Not
the least interesting aspect of this specification is the prior
art review, covering more than a dozen seminal references
in this emerging field.
Se and SAM
The rise of selenium as a therapeutic element continues with
the publication of WO-09811122, in which Nutramax
Laboratories Inc of Maryland claims nutritive compositions
consisting of selenium and S-adenosylmethionine; optional
additional ingredients in this concoction include vitamin B 12
and, rather improbably, cyanide. The efficacious properties
of this nutritional supplement are associated with leukotriene B4 antagonist activity.
Crystal forms
Eli Lilly & Co’s antipsychotic, olanzapine, is the subject of
WO-09811893, in which a novel physical form known as
dihydrate D is claimed. Patenting of this type is now
common, but the timing of this particular invention is
interesting. Registration submissions were deposited
towards the end of 1995, and were followed a year later by
the first product launches. The present application was filed
initially in September 1996, which seems to indicate that this
work on crystal forms was taking place as registration was
pending, and that possibly the active ingredient was
switched to the dihydrate D form immediately prior to
launch. Olanzapine, acting by a combination of 5-HT2 and
dopamine antagonist mechanisms, is regarded as a leading
product in its class, and would normally enjoy patent
protection until the year 2011, based on claims to the novel
chemical entity. These claims to the new physical form
should in effect extend that protection until 2017. Meiji Seika
Kaisha Ltd is seen to be adopting a similar tactic in WO09812200, although in this instance the need for extended
protection is more pressing, The subject is a crystalline form
of the antibacterial cefditoren pivoxil, which was launched
in Japan in 1994 as Meiact, but which is still in clinical trials
elsewhere with licensees such as Grüenenthal GmbH and
Abbott Laboratories. Basic patent protection for cefditoren is
due to begin expiring in 2005, so that these licensees will be
heavily dependent on the reinforced protection which Meiji
Seika is now securing.
CD-ROM chemistry and signatures
One of the more intriguing aspects of pharmaceutical
patenting is the occurrence of interdisciplinary inventions,
relating to subjects which cannot be fitted neatly into a
single location in the International Patent Classification
(IPC). One such case is WO-09812354, classed under nucleic
acids (C07h), microbiological processes (C12q) and
computers (G06f). The invention claimed by Affymetrix Inc
is a technique for identifying molecular sequence signatures,
clearly of relevance to the discovery of gene therapy
products. In contrast, WO-09812559 is assigned to only a
single IPC, G01n-33, which is concerned with analysis of
biological materials. However, the invention is unmistakably multidisciplinary, since it relates to the use of a CDROM format for spatially addressable combinatorial
chemical arrays. The inventor, James P Demers of New
Alcohol manufacture
Two companies are claiming processes leading to alcohols,
and though the inventions are themselves relatively unspectacular, they indicate that vigorous development activity is
under way. Merck & Co Inc's WO-09812340 claims the
reduction of bisaryl ketones to the corresponding alcohols,
the aim being a chiral product useful in the synthesis of
PDE-IV inhibitors. This is a class of compound not strongly
represented in the company's extensive development
portfolio, although some previous patenting of bisphenyl
ethanes has been noted. This lack of correlation is explained
by the fact that Merck had licensed-in CDP-840 from
Celltech Group plc; that collaboration ceased early in 1996,
when the candidate was in phase II trials, but the present
process patenting seems to indicate that Merck retains an
interest in this class of compound. Further intelligence on
this subject appears in WO-09812178, in which a different
Merck team claims bisarylethanes bearing a 4-pyridyl
substituent, as in CDP-840. The accumulated evidence
suggests that Merck is now actively developing an antiasthmatic acting by PDE-IV inhibition, either CDP-840 itself
or a close analog. Chiral alcohols are also the subject of WO09812155, in which Shionogi & Co Ltd claims a reduction
process employing asymmetric boranes. This process seems
to have broad applicability, although one of the suggested
target structures is saphingol, Lilly's phase II PKC inhibitor.
A similar ambiguity exists in WO-09812197, since the target
molecule, duocarmycin SA, is associated with Kyowa Hakko
Co Ltd, rather than with the joint applicants, Kyorin
Pharmaceutical Co Ltd and the Sagami Institute.
HIP hedgehog and tub genes
Hedgehog proteins are known to have therapeutic potential
in a range of CNS and metabolic diseases, and are being
studied in several institutions. Proteins from the Indian,
Desert and Sonic hedgehogs, for example, form part of a
project at Ontogeny Inc, under which both Biogen Inc and
Boehringer Mannheim GmbH are now licensees. Much of
the work which underpins this progress has however been
carried out in academic institutions, including Columbia
University and Johns Hopkins University. Harvard College
has been involved too, and now in WO-09812326 claims
HIP-1, the hedgehog-interacting protein-1; HIPs show high
affinity for hedgehog binding protein. This again is a
conspicuously eclectic invention in terms of the technologies
upon which it draws, being placed in no fewer than six
distinct subclasses of the IPC. A similarly broadly-based
invention, Millennium Pharmaceuticals Inc's WO-09812302,
is concerned with tub and fat, loci on the ob gene implicated
in the development of obesity. Genetic approaches to obesity
control are now being widely studied, by companies such as
Lilly, Amgen Inc, Amylin Pharmaceuticals Inc and Ligand
Pharmaceuticals Inc. Millennium's own involvement has
attracted the attention of Roche Holding AG, now
collaborating on the development of targeted gene products.
Another company to have studied the tub gene is AxyS
Pharmaceuticals Inc, newly formed by the merger of Arris
Pharmaceutical Corp and Sequana Therapeutics.
β2-Agonist in labor
β2-Agonists might be thought of as a rather unfashionable
research field, since the pioneering products with this
selectivity, salbutamol and ritodrine, were first synthesized
more than 30 years ago. Much of the development work
currently being carried out on these compounds is concerned
with formulations and compositions, principally in the context
of asthma therapy. However, the primary indication for
ritodrine has always been control of premature labor, and
from WO-09813035 it is clear that, in Japan at least, improved
agents for this indication are still being sought. The applicant,
Hokuriku Seiyaku Co Ltd, has enlisted the help of Nitto
Denko Corp, a specialist manu-facturer of transdermal tape,
and is claiming a percutaneous formulation of HSR-81. This
compound, a close analog of the anti-asthma agent
tulobuterol, is in phase III trials for control of labor, and has
been licensed to Tokyo Tanabe Co Ltd. Another Japanese
company, Kissei Pharmaceutical Co Ltd, is also reported to be
developing a novel β2-agonist for this indication, although it is
apparently still at the preclinical stage. Kissei is one of several
companies to have licensed ritodrine from the originator,
Solvay SA (originally Philips).
Subepithelial turbidity
Kissei attracts attention also in the context of subepithelial
turbidity, a condition associated with corneal trauma. The
active agent in WO-09813038 is an anthranilic acid derivative
identifiable as tranilast, an anti-allergic agent first synthesized in the early 1970s, which has now begun to lose its
basic patent protection. An ophthalmic solution for use in
allergic conjunctivitis is already marketed, but Kissei clearly
needs the strongest possible protection for any additional
indications. SmithKline Beecham plc is reportedly studying
the use of tranilast in stenosis, but for the ophthalmic
indication Kissei’s partner is Nihon Tengan-yaku, a
company with no previous history of international
patenting, presumably an ophthalmics specialist; one of the
inventors is known to have worked in this field previously,
apparently with Fujisawa Pharmaceutical Co Ltd.
Beecham, collaborating with the National Institutes of
Health and the Virus Research Institute Inc, respectively.
Now, according to WO-09813065, Merck & Co Inc has also
adopted this target. One of the workers assigned to this
project was previously working on fibroblast growth factor
formulations, a topic now apparently abandoned in favor of
rotavirus.
NNRTIs from Du Pont Merck
The antiviral research at Du Pont Merck Pharm Co’s
Wilmington site is directed mainly towards HIV protease
inhibitors. However, from WO-09814436, it is clear that there
is now also interest in non-nucleoside type reverse
transcriptase inhibitors, or NNRTIs. The company’s own
research into such agents in the late 1980s yielded a lead
which was designated DuP-925, but which seems not to have
progressed beyond the preclinical stage. The present
benzoxazinones resemble DMP-266, now known as efavirenz,
in clinical development jointly with the originator, Merck &
Co Inc. This candidate has progressed rapidly through the
early stages of development, having first appeared in June
1994 when WO-09403440 was published.
Demeter’s MIMs
A rather younger company, Demeter Biotechnologies Ltd,
had two applications published early in 1996 describing
membrane interacting molecules (MIMs), WO-09603519 and
WO-09603522. Now, in WO-09814201, further work on these
MIMs is described, in the context of HIV therapy. Demeter’s
entire product development pipeline is oriented around the
concept of membrane permeability enhancement, but there
is no evidence to date that even this ultra-specialized
approach has attracted any licensing partners to support
product development. The relatively small scale of
Demeter’s operations is further emphasized by the naming
of the company’s R&D VP as the sole inventor. From the
previous inventions of this individual, however, it is
possible to trace Demeter’s lytic peptides back to work on
plant pathogens carried out in the mid 1980s in the
Agricultural and Mechanical College of Louisiana State
University. This rather unconventional background is
confirmed by the naming of the US Department of
Agriculture as joint applicant on one of the earlier Demeter
cases mentioned previously.
PGI2 agonists
Oddly, although several dozen PGI2 agonists have been
under development during the past decade or so, and a
handful have matured into successful products, there seem
to be virtually no candidates under investigation currently.
Nevertheless, Takeda Chemical Industries Ltd is now
entering the field, with claims in WO-09813356 to agents
with high affinity for the receptor and useful platelet
aggregation inhibitory action. The tricyclic templates on
which these compounds are based include naphtho[1,2d]thiazole, a fragment seen previously in certain naturallyoccurring rifamycins.
Merck’s rotavirus vaccine
Rotavirus has attracted the attention of several companies,
including American Home Products Corp and SmithKline
NAALADase inhibitors from Guilford
Baltimore-based Guilford Pharmaceuticals Inc filed no fewer
than nine patent applications at the US Patent Office
between September 1996 and June 1997 relating to inhibitors
of NAALADase. This enzyme, N-acetylated-α-linked acid
dipeptidase, is involved in the conversion of N-acetylaspartyl glutamate into glutamate, which results in presynaptic glutamate release. Compositions useful in the
treatment of neurological disease, including compulsive
disorders, are claimed in WO-09813044 and WO-09813046,
following on from the publication of two related international applications at the end of 1997. This complex family
of patents began to appear, however, in September 1997,
when US-05672592 was granted with claims to novel
phosphonomethylpentanedioic acid derivatives. At about
the same time, Guilford announced that these NAALADase
Patent News 161
inhibitors were showing promise in neurodegenerative
disease and amyotrophic lateral sclerosis, naming GPI-5000
as the lead compound. Guilford has until now filed very few
patent applications, and has focused on the enzyme
rotamase; the inventor team working on NAALADase is
relatively inexperienced too, the only possible exception
being a chemist who may previously have been associated
with a neurological project at Zeneca Group plc’s US site. A
puzzling aspect of Guilford’s earlier patenting was the use of
an agent in Turin; for the current substantial project,
however, the services of a Washington-based attorney are
being used.
Merrell NMDA antagonists
With only a brief diversion into IL-1 antagonists, a small
team at the Merrell Pharmaceuticals Inc site in Cincinnati
has been steadily researching NMDA antagonists for more
than a decade. The latest compounds, claimed in WO09814427, are indole derivatives, and from the sheer
consistency of Hoechst Marion Roussel Inc’s effort in this
particular field, it seems likely that by now some promising
compounds must have been identified. Astra AB’s remacemide, now in phase III trials, is among the more advanced
NMDA modulators in development.
ImmuLogic collaborations
Vomeronasal organ targeted
Pherin Pharmaceuticals of California is a small company
specializing in vomeropherins, synthetic compounds acting
on the vomeronasal organ, or VNO. Such agents are
described in WO-09814194, a document of well over 500
pages, almost half of which are devoted to figures and
diagrams. Vomeropherins have potential in the therapy of a
range of conditions, including anxiety and prostate cancer;
there is a suggestion that two distinct classes of compound
are involved. Although the present patenting forms part of a
series of cases going back to the early 1990s, it is only within
the past year that a licensee, Organon NV, has been
identified to support Pherin in the development of these
pheromone-related compounds. A key inventor, associated
with the company throughout this period, has also appeared
on inventions from companies such as BioSource
International Inc and Erox Corp.
Unlike some of the smaller biotech companies, ImmuLogic
Pharmaceutical Corp has always adopted a policy of
vigorous patenting. Several dozen inventions have
accumulated during the decade since the Massachusettsbased company was founded, and the high level of joint
patenting suggests a very outgoing attitude towards
collaboration, especially with non-industrial partners.
Within the US there have been joint filings with North
Carolina University, Leland Stanford University, and the
New England Medical Center. However, there has also been
collaboration with academics elsewhere, including teams
based in Perth, Western Australia, and London. The latest
addition to this interesting portfolio is WO-09814216, in
which hapten-carrier conjugates for drug-abuse therapy are
claimed. On this occasion there is no joint applicant, and the
company seems not to have identified a development
partner so far. Schering AG is known to be cooperating in
the development of a potential multiple sclerosis therapy,
and at one time Merck, ImmuLogic’s only industrial copatentee, was reported to have an interest in a candidate for
use in transplant rejection.
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