Untitled - Faculté de Pharmacie - Aix

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JOURNEE DE LA RECHERCHE
Comme vous le savez, notre Université a développé une très grande activité en faveur de la
recherche, en cohérence avec les orientations définies pars les grands organismes de recherche
et le Ministère de l'Enseignement Supérieur et de la Recherche avec, en particulier, le
programme "Investissements d'Avenir".
S'appuyant sur 73 unités de recherche de qualité, socle essentiel des ambitions de sa politique,
Notre établissement a intensifié ses actions de valorisation à travers des relations fortes
public/privé, mais aussi en s'impliquant dans la plupart des pôles de compétitivité de la
région, participant au développement économique régional.
Dans l'élaboration de notre nouveau contrat "quinquennal", les trois universités de Marseille
ont souhaité développer leur potentiel, accroître leur lisibilité et leur attractivité en définissant
des axes renforcés et en élaborant un contrat commun. Véritable préfiguration de notre
université de demain, ce dispositif nous incite à développer des projets forts, fédérateurs, au
sein d'unités importantes, à lisibilité internationale.
C'est dans cet esprit que nous avons initié la Journée de la Recherche au sein de l'UFR de
Pharmacie, afin d'informer très concrètement nos étudiants sur cette filière et tous les aspects
des métiers qu'elle comporte. Cette manifestation a pleinement rempli les missions qu'elle
s'était fixées, autour de ce rendez-vous annuel désormais institutionnel, touchant un public
diversifié, en renforçant la lisibilité de notre recherche. Consolider le positionnement de notre
recherche, accroître sa compétitivité et répondre efficacement aux enjeux de demain, telles
sont nos ambitions.
Pour cette 8ème édition, nous avons souhaité donner à cette journée un rayonnement un peu
différent, encouragés par l'intégration de la Pharmacie au CHU et la mise en place de la loi
Hôpital, Patients, Santé, Territoire, et avons associé les internes en Pharmacie, qui
s'impliquent tous les jours dans nos équipes et nos thématiques. C'est d'ailleurs avec beaucoup
d'efficacité qu'ils ont pris une large part à l'organisation de la journée. Nous remercions
l'AIPM pour cette participation active.
Dans le domaine de la recherche, comme dans bien d'autres, c'est avec les initiatives
d'aujourd'hui que nous construirons l'avenir de notre composante.
Françoise DIGNAT-GEORGE
Vice-Doyen Chargé de la Recherche
Patrice VANELLE
Doyen
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L’ACTIVATION ENDOTHELIALE DEPENDANTE DU CALCIUM, UN
CATALYSEUR DU PURPURA THROMBOTIQUE THROMBOCYTOPENIQUE
AUTO-IMMUN DE L’ADULTE
Agnès Widemann1, Stéphane Robert1, Philippe Robert3,4, Paul Salers1, Catherine
Farnarier4, Pascale Poullin5, Patrice Lefèvre5, Françoise Dignat-George1,2, Gilles
Kaplanski1,6.
1. UMR-S 608 UFR de Pharmacie Marseille, 2. Laboratoire d’hématologie, hôpital de la
Conception, AP-HM ; 3. UMR 600 ; 4. Laboratoire d’immunologie, hôpital de la
Conception, AP-HM ; 5. Service d’hémaphérèse, hôpital de la Conception, AP-HM ; 6.
Service de médecine interne,hôpital de la Conception, AP-HM
Le purpura thrombotique thrombocytopenique (PTT) est une maladie auto-immune
caractérisée par la présence d’auto-anticorpscirculants anti-ADAMTS-13. Ces anticorps
neutralisent ADAMTS13, une protéase connue pour cliver le facteur vonWillebrand
(fvW) de haut poids moléculaire en monomères de plus faible activité prothrombotique, induisant ainsi une microangiopathie thrombotique.Cependant
l’observation clinique et les modèles animaux suggèrent que le déficit en ADAMTS13
n’est pas le seul mécanisme impliqué dans cette maladie. Nous avons fait l’hypothèse
que l’activation endothéliale pourrait aussi participer à cette pathologie.
Nous avons comparé la capacité de plasma de patients atteints de PTT ou de myasthénie
(MG) obtenus lors d’échanges plasmatiques, à activer les cellules endothéliales(CE) in
vitro.
Après 30 secondes de contact avec les CE microvasculaires, les plasma PTT et non les
plasma MG, induisent un flux calcique intracellulaire soutenu. Les plasma PTT et non MG
induisent aussi une exocytose du fvW des CE après 60 min, visible par des techniques
d’immunofluorescence pratiquées dans des conditions statiques ou dynamiques dans
une chambre de flux. De plus, les plasma PTT induisent la libération d’IL-8 et
d’endothéline-1, après 1 h de stimulation des CE. Tous ces effets sont inhibés lorsque les
CE sont cultivées en présence d’un chélateur du calcium.Les plasma PTT épurés en fvW
après passage sur une colonne d’affinité, continuent à induire ces effets, alors que les
mêmes plasma PTT obtenus en phase de rémission, ont perdu leurs propriétés
activatrices endothéliales.
Ces résultats montrent que les plasmas de PTT possèdent des propriétés activatrices de
l’endothelium, de façon calcium-dépendante, conduisant à la dégranulation rapide des
corps de Weibel-Palade et à l’exocytose du fvW de haut poids moléculaire. A côté des
auto-anticorps anti-ADAMST-13, l’activation micro-endothélialecalcium-dépendante
pourrait donc constituer le second « hit » du PTT.
STRATEGIE TDAE :
OUTIL SYNTHETIQUE ET APPLICATIONS PHARMACOCHIMIQUES
Since M., Terme T. et Vanelle P.
Laboratoire Chimie Provence, Universités d’Aix Marseille I, II et III – CNRS UMR 6264, Equipe : PharmacoChimie Radicalaire (PCR), Faculté de Pharmacie, 27 Bd Jean Moulin 13385 Marseille cedex 05, France.
La problématique fondamentale des recherches développées par notre équipe est centrée sur
l’obtention de nouvelles molécules à visée thérapeutique via le développement de nouveaux
outils synthétiques utilisant les réactions par transfert monoélectronique.
Le tétrakis(diméthylamino)éthylène (TDAE) est un puissant donneur d’électrons, qui a la
particularité d’activer la liaison Carbone-Halogène pour conduire à la formation d’un radical
électrophile et d’un anion nucléophile stables.
CH3
CH3
H3C
N
N
CH3
H3C
N
N
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CH3
CH3
Depuis 2002, notre équipe associe ces deux réactivités dans un programme sur les réactions par
transfert monoélectronique initiées par le TDAE d’agents alkylants bioréductibles
nitroaromatiques, nitrohétérocycliques et quinoniques dans le but de synthétiser de nouvelles
molécules à visée thérapeutique.
Après avoir introduit le TDAE et ces applications synthétiques développées au sein de notre
équipe, nous présenterons 2 exemples de mise au point de nouvelles réactivités initiées par le
TDAE intégrées dans un travail de pharmacomodulation antiparasitaire ou dans le
développement d’un nouveau programme concernant la préparation de nouvelles molécules à
visée antinociceptive.
Giuglio-Tonolo, G.; Terme, T.; Médebielle, M.; Vanelle, P. Tetrahedron Lett. 2003, 44, 6433.
Giuglio-Tonolo, G.; Terme, T.; Médebielle, M.; Vanelle, P. Tetrahedron Lett. 2004, 45, 5121.
Giuglio-Tonolo, G.; Terme, T.; Vanelle, P. Synlett 2005, 251.
Amiri-Attou, O.; Terme, T.; Vanelle, P. Synlett 2005, 3047.
Amiri-Attou, O.; Terme, T.; Vanelle, P. Molecules 2005, 10, 545.
Montana, M.; Terme, T.; Vanelle, P. Tetrahedron Lett. 2005, 46, 8373.
Montana, M.; Terme, T.; Vanelle, P. Tetrahedron Lett. 2006, 47, 6573.
Amiri-Attou, O.; Terme, T.; Médebielle, M.; Vanelle, P. Tetrahedron Lett. 2008, 49, 1016.
Khoumeri, O.; Montana, M.; Terme, T.; Vanelle, P. Tetrahedron. 2008, 64, 11237.
Montana, M.; Crozet, M.D.; Casteras-Ducros, C.; Terme, T.; Vanelle, P. Heterocycles 2008, 75, 925.
Juspin, T.; Terme, T.; Vanelle, P. Synlett 2009, 1485.
Since, M.; Terme, T.; Vanelle, P. Tetrahedron 2009, 65, 6128.
Khoumeri, O.; Terme, T.; Vanelle, P. Synthesis 2009, 3677.
Khoumeri, O.; Crozet, M. D.; Terme, T.; Vanelle, P. Tetrahedron Lett. 2009, 50, 6372.
Juspin, T.; Laget, M.; Terme, T.; Azas, N.; Vanelle, P. Eur. J. Med. Chem. 2010, 45, 840.
Juspin, T.; Giuglio-Tonolo, G.; Terme, T.; Vanelle, P. Synthesis 2010, 844.
Montana, M.; Terme, T.; Vanelle, P. Lett. Org. Chem. 2010, 7, 453.
Nadji-Boukrouche, A. R.; Khoumeri, O.; Terme, T.; Liacha, M.; Vanelle, P. ARKIVOC, 2010, X, 358.
Microtubule dynamics in chemotherapy-induced neurotoxicity: A protective role of
olesoxime (TRO19622)
Rovini A.1, Carré M.1, Bordet T.2, McKay N.1, Pruss R.2 and Braguer D.1
1
INSERM UMR911, Centre de Recherche en Oncologie biologique et Oncopharmacologie
(CRO2) ; Université de la Méditerranée, Marseille, France
2
Trophos, Parc Scientifique de Luminy, Marseille, France.
The microtubule cytoskeleton plays a central role in cell processes, such as mitosis
completion, cell spreading, morphology and organelle trafficking. Microtubules constitute the
primary target of Microtubule-Targeting Agents (MTAs) used as cancer chemotherapeutics.
However, peripheral neuropathy is often a dose-limiting side effect of MTAs, and the
mechanism for this neurotoxicity is still poorly understood. To date, there are no approved
therapies for prevention or treatment of neuropathies triggered by MTAs. We addressed this
challenge by investigating the neurotoxic effect of MTAs in vitro and the protective effect of
olesoxime (TRO19622), a new drug candidate that has promising neuroprotective properties
in vivo.
We first showed that MTAs induced loss of neurite outgrowth in rat and human neuronal cells
in vitro and that olesoxime prevented this effect. In parallel, olesoxime did not alter cytotoxic
properties of MTAs in human tumor cell lines, a necessary prerequisite to further study such a
combination. Since regulation of microtubule dynamics is critical for neuron organization, we
analysed the distribution of proteins (+TIPs) that specifically promote microtubule plus-ends
growth. Interestingly, MTAs triggered EB1 and EB3 dissociation from microtubules to
cytosol in human neuroblastoma cells grown either under proliferative conditions or under
neuronal differentiation conditions. Concomitant treatment with olesoxime preserved normal
EB1 distribution while maintaining neurite outgrowth, specifically under neuronal
differentiation conditions suggesting a tight link between microtubule dynamics disruption
and neurite architecture loss. Time-lapse videomicroscopy analysis showed that olesoxime
significantly increased the growing rate of microtubules and decreased the attenuation mean
duration in neuronally differentiated cells. All these data strongly suggest that olesoxime
neuroprotection could result from its ability to specifically protect the microtubule network
against MTAs in differentiated neuronal cells.
To conclude, the present work provides fundamental data supporting the correlation between
microtubule dynamics and the maintenance of neurite outgrowth. It also reveals a mechanism
underlying the neuroprotective effects of olesoxime, a new drug candidate that could address
a huge unmet medical need by providing a treatment for side-effects associated with
chemotherapy with MTAs.
Présenté au congrès FEBs Prague 2009
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The interaction of CD146/MCAM with galectin-1 is involved in the control of endothelial cell
apoptosis
Nathalie JOUVE1, Aurélie S. LEROYER1, Nicolas DESPOIX1, Marion ESPELI2, Karim FALLAGUE1, Nathalie BARDIN1, Marcel BLOT-CHABAUD1,
Laurent GAUTHIER3, Frédéric VELY1, Claudine SCHIFF2 and Françoise DIGNAT-GEORGE1
- 1 UMR-S608, INSERM-Université de la Méditerranée, Marseille, France
- 2 U631-UMR6102, INSERM-CNRS-Université de la Méditerranée, CIML, Marseille, France.
- 3 INNATE-PHARMA, Marseille, France
Background : CD146, also known as Melanoma Cell Adhesion Molecule (MCAM), MUC18,
A32 antigen or Sendo1, is an adhesion molecule, localized at the inter-endothelial junctions,
playing a key role in the control of vessel permeability, transendothelial migration and
angiogenesis. However, nothing is described about its ligands. Interestingly, galectin-1 shares
structural and functional similarities with CD146. Because galectin-1 was known to promote
apoptosis on other cells types, we hypothesized that galectin-1 could be one CD146 ligand and
could play a role in endothelial cell apoptosis.
Results : We demonstrated a direct and specific binding of galectin-1 to recombinant CD146,
dependent of glycosylations, either in ELISA and Biacore (Kd=3.10-7 M) whereas galectin-2 was
not able to bind CD146. Moreover, galectin-1 was able to interact with endothelial cells
harbouring CD146 as shown by either co-immunoprecipitation of these two molecules or
immunofluorescence stainings of Human Umbilical Vein Endothelial Cells (HUVECs) in culture.
We observed that galectin-1 induced a time- and dose-dependent apoptosis of endothelial cells,
demonstrated by annexin-V/PI staining and by the presence of cytoplasmic histone-associatedDNA-fragments in HUVECs exposed to galectin-1. Moreover, deletion of endothelial CD146 by
the use of siRNA or blocking CD146 with a neutralizing antibody increased this galectin-1
induced-endothelial apoptosis. Consequently, our data suggest a protective role of CD146 in
galectin-1-induced apoptosis.
Conclusion : Taken together, our data demonstrated that galectin-1 is a ligand for CD146 and
this interaction modulated apoptosis of endothelial cells with a protective role attributed to
CD146. Thus, the association of soluble galectin-1 with anti-CD146 should be a new therapeutic
strategy targeting the endothelial cell apoptosis in diseases such as cancer.
Plasmatic Leukocyte-derived microparticles : the first biological marker for predicting unstable
plaque in asymptomatic patients with high-grade carotid stenosis
1,2
2
3
1
4
5
G. Sarlon-Bartoli , Y. Bennis , MD Piercecchi-Marti , MA. Bartoli , L. Arnaud , J. Mancini , R.
2,4
1
6
1
2
7
7
Lacroix , A. Boudes , E Sarlon , B. Thevenin , A. Leroyer , C. Squarcioni , F. Nicoli , PE.
1
2,4
2,8
Magnan , F. Dignat-George and F. Sabatier , for the RISC Study Group
1
Service de Chirurgie Vasculaire, Faculté de Médecine de Marseille, Université de la Méditerranée,
Assistance Publique Hôpitaux de Marseille - Hôpital de la Timone, Marseille, France.
2
INSERM UMR-S608, Faculté de Pharmacie, Université de la Méditerranée, Marseille, France
3
Service d’Anatomo-pathologie, Faculté de Médecine de Marseille, Université de la Méditerranée,
Assistance Publique Hôpitaux de Marseille - Hôpital de la Timone, Marseille, France.
4
Laboratoire d’hématologie, Assistance Publique Hôpitaux de Marseille - Hôpital de la Conception,
Marseille, France.
5
Service de santé publique (SSPIM), Assistance Publique Hôpitaux de Marseille - Hôpital de la
Timone, Marseille, France.
6
INSERM U669, Faculté de médecine Paris Sud, Paris, France.
7
Service de Neurologie Vasculaire, Assistance Publique Hôpitaux de Marseille - Hôpital de la Timone,
Marseille, France.
8
Laboratoire de culture et thérapie cellulaire, INSERM CIC BT 510, Assistance Publique Hôpitaux de
Marseille - Hôpital de la Conception, Marseille, France.
ABSTRACT (249 words)
Objectives: To analyse whether testing plasmatic levels of leukocyte-derived microparticles (LMP) in
patients with high-grade carotid stenosis could be useful to identify those with unstable plaque
Background: Preventive carotid surgery in asymptomatic patients is actually debated given the
improvement of the best medical therapy. LMP are present in human carotid plaques, and their
circulating levels have been proposed as biomarker of cardiovascular risk.
Methods Forty-two patients with greater than 70% carotid stenosis were enrolled. Circulating LMP
were measured by flow cytometry before thromboendarterectomy. Histopathological analysis of the
removed carotid plaque was performed to classify it as stable or unstable. LMP levels were compared
to plaque morphology.
Results: Unstable plaque was determined in 28 patients (66.7%). Median level of LMP was
significantly higher in patients with unstable plaque (CD11b66b+ MP: 240±83 MP/µl, and CD15+
MP:147±48 MP/µl) as compared to those with stable plaque (17±36 MP/µl and 55 ±22 MP/µl, p<0.001
and p<0.01 respectively). This difference remained significant when only asymptomatic patients were
considered (CD11b66b+ MP: 199 ±665 vs 20 ±137, p<0.05; CD15+ MP: 78 ±178 vs 55 ±66, p<0.05).
After logistic regression, only the presence of neurologic symptoms (OR 32.6, 95% CI 3.0-358.5,
p<0.05) and the level of LMP (OR 2.4, 95% CI 1.1-4.9, p<0.05) independently predicted plaque
instability.
Conclusions: Increased circulating levels of LMP are associated to plaque instability in patients with
high grade carotid stenosis. LMP may constitute promising biomarker for prediction of carotid plaque
vulnerability and identification of asymptomatic subjects who are mostly benefit from carotid surgery.
Keywords: Leukocyte-derived microparticles, high grade carotid stenosis, atherosclerosis, unstable
plaque
Ex vivo priming of late outgrowth endothelial progenitor cells with erythropoietin before
transplantation enhances their angiogenic potential and requires the CD131 receptor subunit
(1)*
(1)(2)*
(1)
Bennis Youssef , Sarlon-Bartoli Gabrielle
, Guillet Benjamin , Marcel Blot-Chabot
(1)(3)
(1)(4)
(1)
George Françoise
, Sabatier Florence
, and Pisano Pascale
(1)
, Dignat-
(1) INSERM UMR 608, Université de la Méditerranée, Faculté de Pharmacie, Marseille, France
(2) Chirurgie vasculaire, CHU Timone, Faculté de Médecine, Marseille, France
(3) Département d’Hématologie, CHU Conception, Marseille, France
(4) Laboratoire de Culture et Thérapie Cellulaire, INSERM CIC-BT 510, CHU Conception, Marseille,
France
* Both equally contributed to this work
ABSTRACT (323 words)
Background and purpose: Endothelial colony-forming cells (ECFC) are promising candidate for cell
therapy of ischemic tissues. Erythropoietin (EPO) is a cytokine known to promote angiogenesis after
ischemic injury. The aim of this study was to postulate that priming of ECFC with EPO might increase
their angiogenic properties in vitro and in vivo on a hindlimb ischemia model. We explored also the
role of the CD131 subunit in EPO-priming ECFC.
Materials: EPOR and CD131 expression were assessed by immunoprecipitation, Western blotting
and immunocytochemistry on cord blood ECFC. Priming of ECFC, transfected or not with CD131
siRNA, was done by adding 1.5-10 IU/ml αEPO for 24 hours in EBM2 culture medium prior
experiments. In vitro, we assessed proliferation, migration, wound-healing and tube formation tests on
primed ECFC. In vivo, 5UI/ml EPO-primed ECFC were IV injected 24 h after hindlimb ischemia in
athymic nude mice. On day 14, hindlimb blood perfusion was performed by laser-Doppler and capillary
density quantified on gastrocnemius PECAM-stained frozen slices.
Results: EPOR and CD131 expression on ECFC lysates increased significantly after EPO treatment
compared to non primed ECFC (respectively 147±20% and 219±18%, n=3, P<0.05). These proteins
coimmunoprecipitated and colocalized, suggesting that they are covalently bound in ECFC. In vitro
assays showed that 5UI/ml EPO stimulated significantly proliferation, migration, wound healing and
tube formation of ECFC compared to non primed ECFC (respectively: 143±22%; 179±60%; 191±73%
and 175±46%; n=4, P<0.01-0.05). These EPO stimulatory effects were prevented by CD131 siRNA
transfection (respectively 105±10%; 124±11%; 105±10% and 112±28%; n=4, P<0.01-0.05). Similarly,
in vivo studies pointed out that EPO-primed ECFC transplantation induced the recovery of ischemic
hindlimb blood flow and an increase in capillary density compared to non primed ECFC transplantation
(respectively 170±73 and 118±4; n=8, P<.,01). These in vivo effects were abolished by CD131 siRNA
silencing.
Conclusion: These results highlight the potential role of EPO primed ECFC for cell-based therapy in
critical limb ischemia and focus on the critical role of CD131 as an EPO co-receptor.
Keywords: Cell therapy, endothelial colony-forming cells, erythropoietin, CD131 receptor
Mesenchymal stem cells potentiate angiogenic activity of endothelial progenitor
cells through up-regulation of SPHK-1/S1P pathway
Stéphane POITEVIN 1, Gabrielle SARLON 1, Virginie ALBINET 2, Nathalie ANDRIEUABADIE 2, Daniel CUSSAC 2, Angelo PARINI 2, Françoise DIGNAT-GEORGE
1
and
Florence SABATIER 1
1
INSERM UMR-S 608, Faculté de Pharmacie, Université de la Méditerranée, Marseille
2
INSERM U858, Institut de Médecine Moléculaire de Rangueil, UPS, Toulouse
Endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) are good candidates
for cell-based therapy in cardiovascular diseases. Although part of the regenerative potential
of MSC involves stimulation of angiogenesis, their capacity to modulate EPC activity has not
been addressed so far. The present study was designed to evaluate whether paracrine activity
from MSC can stimulate functional properties and therapeutic potential of late-outgrowth
endothelial colony-forming cells (ECFC).
Exposure of cord blood ECFC to conditioned media from bone marrow-derived MSC
significantly increased angiogenic properties of ECFC in vitro assessed by proliferation,
migration and capillary-like structure formation. These effects were associated with upregulation of sphingosine kinase 1 (SPHK-1) expression and activity in ECFC as determined
by PCR array and radioenzymatic assay respectively. Inhibition of SPHK-1 in ECFC using
pharmacological agent (SKI) or siRNA knockdown prevented MSC induced stimulation of
ECFC in vitro. Paracrine effect of MSC on ECFC also involved upregulation of S1P receptor
1 and was abrogated in the presence of S1PR1/3 antagonist (VPC23019). Finally, pretreatment of ECFC with either MSC conditioned media or exogenously used S1P, before
transplantation in nude mice hindlimb ischemia, significantly improved recovery of blood
perfusion compared to basal ECFC.
In conclusion, paracrine factors from MSC increase vasculogenic activity of ECFC through
SPHK-1 activation and a S1P/S1PR1 dependent autocrine amplification loop. This
mechanism could contribute to the vasculotropic properties of MSC. In addition,
identification of SPHK-1/S1P as a critical pathway favoring ECFC angiogenic activity
provides attractive targets for development of optimized cell-based therapy in ischemic
diseases.
ROLE OF SCD 146 IN TROPHOBLAST INVASION
KASPI Elise(1), GUILLET Benjamin(1), ALFAIDY-BENHAROUGA Nadia(2),BRETELLE
Florence(3), BERTAUD-FOUCAULT Alexandrine(1), RAMBELOSON Laka(4), LACROIX
Odile(5), BLOT-CHABAUD Marcel(1), DIGNAT-GEORGE Françoise(1), BARDIN
Nathalie(1)
(1)Inserm UMR-S 608, faculté de Pharmacie,
(2)LAPV-U878 / iRTSV / CEA
(3)APHM, SERVICE DE GYNECOLOGIE-OBSTETRIQUE
(4)APHM, SERVICE D'IMMUNOLOGIE- Hôpital de la Conception - (5)APHM,
LABORATOIRE DE BIOLOGIE DE LA REPRODUCTION- Hôpital de la Conception
CD146, also known as Mel-CAM, MUC18 and S-Endo-1 antigen, is a member of the
immunoglobulin gene superfamily. CD146 is a membrane glycoprotein localized at the
endothelial junction. CD146 is expressed in intermediate and extra-villous trophoblasts,
having migratory and invasive properties. Recent studies suggest that CD146 is involved in
reduced invasiveness of trophoblats in pre-eclamspia and unexplained recurrent miscarriage.
Indeed, in pre-eclampsia, intermediate trophoblasts fail to express CD146. CD146 also exists
as a soluble form in plasma (sCD146), detected in vascular pathologies. Recently, sCD146
plasma levels were found significantly higher in recurrent pregnancy loss; suggesting its role
in endovascular trophoblast invasiveness.
The aim of our study was to analyse the role of sCD146 on trophoblasts invasiveness.
In vitro assays were performed with HTR-8/SVneo trophoblast cell lines and confirmed on
trophoblasts from placenta explants.
First, sCD146 setting was analysed in comparison with CD146 localisation, using
FACS and Confocal Immunofluorescence Microscopy. We showed that sCD146 binds
trophoblastic membrane, with a preferential localisation at the cell cell contact, as found for
CD146. Then, invasion property was tested in vitro with tube-like structures formation in
Matrigel, and in vivo, with villous explants from first trimester human placentae. Our results
show that sCD146 used at the concentration of 100ng/ml, significantly reduced the tube-like
structure formation, and the invasiveness of trophoblasts from placenta explants, compared to
control conditions (p=0.02 and 0.001 respectively).
Moreover, we tested the role of sCD146 on trophoblasts migration, using a Wound Healing
assay. Trophoblasts migration is significantly reduced in presence to sCD146 (p<0.001). At
least, we analysed the effect of sCD146 on trophoblastic apoptosis, in comparison with UV
radiated cells. Our study shows that sCD146 doesn’t enhance apoptosis.
In conclusion, sCD146 decreases trophoblastic invasion. The mecanism is not yet elucidated
but we can speculate that sCD146 could bind CD146 on the trophoblastic membrane and
inhibit invasion capacities. This study suggests the role of sCD146 in pathologic pregnancies,
such as pre-eclampsia, in which defective invasiveness of trophoblasts is involved.
Abstract Journée de la recherche Faculté de Pharmacie
23 mars 2011
Implication of the Fractalkine/CX3CR1 activation pathway in regulation of the circulating progenitor pool
In kidney transplant recipients
Dilyana Todorova1,2, F.Sabatier1, S. Robert1, H. Vacher Coponat3, C. Cerini1, L. Dou1, E. Doria 2, L. Lyonnet2, A. Larosa2,
Raymond Calaf, P.Charpiot1, S. Morange4, T. Legris3, M. Indries3, V. Moal3, P. Brunet3, B. Dussol3, Y.Berland3, S. Burtey3,1,
F.Dignat George1,2 and P. Paul1,2
1UMR-S U608, Université de la Méditerranée et 2Laboratoire Hématologie Conception, Direction Pr F. Dignat George, 3Service de
Néphrologie, 4CIC, CHU Conception, Direction Pr Y. Berland Introduction: Dysruption of endothelial integrity is a critical feature of cardiovascular risk associated with renal diseases.
Immunosuppressive drugs expanded access to transplant replacement therapy for endstage kidney failure. In addition to common
cardiovascular risk factors, uremia, immunosuppressive drugs and alloimmune conflict are central mechanisms promoting
endothelial injury in kidney transplant recipient (KTR). Decrease in circulating CD34+ progenitor cell numbers have been identified
as surrogate markers of vascular dysfunction and cumulative cardiovascular risk. Increasing evidence supports the concept that
the turnover and autologous replacement of endothelial cells is a major mecanism in the maintenance of vascular integrity within
the grafted kidney. Endogenous repair mecanisms that involve endothelial progenitor cells (EPC) originating from the bone marrow
may thus limit endothelial alloimmune injury, thus favouring graft survival. Our study was based on the assumption that stressinduced endothelial molecules may identify CD34 progenitor subsets reflecting EPC endothelial repair potential in the specific
context of kidney transplant. Our study focused on Fractalkine (CX3CL1), a soluble and membrane-bound CX3C chemokine
shown to be up regulated on activated endothelial cells and in rejected allografts. CX3CL1 is known to mediate immune cell
migration, adhesion and activation through engagement of its cognate polymorphic CX3CR1 receptor, predominantly expressed on
natural killer (NK) cells. The fractalkine /CX3CR1 axis was identified as a vascular gateway for immune cytotoxic effector cells
recruitment and activation, and is implicated in various pathogenic processes including renal inflammation and fibrosis, coronary
artery diseases and allograft loss.
Our working hypothesis is that upon inflammatory stimuli, uremia or immunosuppressive drugs, the induction of fractalkine in
graft cells and recipient endothelial progenitors may control CX3CR1 NK cell adhesion and graft infiltration associated to NK antiendothelial cytotoxic activity and interferon secretion in transplanted patients.
Methods: In this aim, we conducted a combined ex vivo analysis of fractalkine seric level, flow cytometry enumeration of
CX3CR1+ NK effector cells and circulating progenitor cells subsets: CD34+, CD34+CD133+ and CD34+Fract+ within peripheral blood
mononuclear cells in a cohort of 161 kidney transplanted patients, analyzed at a median of 5.8 years post graft, and randomly
included in two arms of immunosuppressive regimen (Tacrolimus/mycophenolate mofetil vs cyclosporine/azathioprine) since
transplant. KTR were further analyzed in reference to 59 gender and age-matched healthy control donors with normal renal
function. We further investigated whether Interferon-γ and TNF-α inflammatory stimuli or KTR sera were able to induce fractalkine
expression in mature glomerular endothelial cells, late EPC and adult circulating progenitor cell in vitro. We then evaluated whether
induced or transfected mb-fractalkine could target NK adhesion and cytotoxicity towards these endothelial cells.
Results: In KTR, CD34+ and CD34+133+ progenitors cell were comparable to that of sex and age matched control. Multivariate
analysis show that higher indoxyl sulfate uremic toxin levels were correlated to lower progenitor cell counts and enhanced NK cell
cytotoxic activity in KTR. CD34+ and CD34+CD133+ cell counts were highly heterogeneous within patients and their deficiency
was inversely correlated to higher occurrence of a newly defined CD34+Fract+ circulating progenitor cell subset. Moreover CD34
progenitor cells expressing fractalkine were more frequently detected in transplanted patients in reference to controls and at higher
rates in the FK/MMF treatment group. Multivariate analysis regression models showed that circulating CD34Fract+ levels, cytotoxic
NK function, CSA/Aza treatment and current smoking habits were independent parameters associated to lowered graft function. In
vitro stimulation of human renal glomerular endothelial cells and late EPCs with inflammatory cytokines induced expression of both
soluble and membrane-bound CX3CL1. Fractalkine expression could also be induced on adult peripheral blood CD34+ or CD133+
purified progenitor cells after in vitro incubation with serum of transplanted patients with high levels of CD34+Fract+.
As a mechanistic support to these findings, we provide in vitro evidence that fractalkine expression in transfected late EPC or its
induction in adult peripheral blood progenitor cells is able to target NK adhesion and cytotoxic activity.
Conclusions
We suggest that in inflammatory conditions relating to uremia and alloimmune conflict, increased expression of fractalkine on
CD34+ progenitor cells targets their depletion by recipient NK-cells expressing CX3CR1. This immune depletion of the circulating
progenitors could thus impair endothelial repair of vascular lesions. Better understanding of the fractalkine dependent mechanisms
controlling homeostasis of KTR progenitor pool may contribute to refine non-invasive biomarkers identifying patients at higher risk
of renal failure and cardiovascular disease. It may also bring new insights to develop therapeutic approaches interfering with the
CX3CL1/CX3CR1 pathway to limit progression of cardiovascular–renal disorders and improve EPC regenerative based therapy.
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Gq-coupled Purinergic Receptors Inhibit Insulin-like Growth FactorI/Phosphoinositide 3-Kinase Pathway-dependent Keratinocyte Migration
Emilie Faure, Françoise Garrouste, Sylvie Monferran, Fabrice Parat, Salma Taboubi, Gilbert
Pommier, Jean-Claude Hubaud*, Hervé Kovacic, and Maxime Lehmann.
INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie,
Faculté de Pharmacie, Aix-Marseille Université, 13005 Marseille, France
*DIPTA, Aix en Provence, France
Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an
essential pathway for keratinocyte migration that is required for epidermis wound healing. We
have previously reported that activation of Gα(q/11)-coupled-P2Y2 purinergic receptors by
extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of
P2Y2 receptors by extracellular UTP inhibits the IGF-I–induced p110α-PI3K activation.
Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic
effects on PI3K pathway are mediated by Gα(q/11)—and not G(i/o)—independently of
phospholipase Cβ. Purinergic signaling does not affect the formation of the IGF-I receptor/
insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted
active p110α mutant, indicating that UTP acts downstream of PI3K membrane recruitment.
UTP was also found to efficiently attenuate, within few minutes, the IGF-I–induced PI3Kcontrolled translocation of the actin-nucleating protein cortactin to the plasma membrane.
This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits
keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active
p110α mutant, in a Gα(q/11)-dependent manner both. These findings provide new insight into
the signaling cross-talk between receptor tyrosine kinase and Gα(q/11)-coupled receptors,
which mediate opposite effects on p110α-PI3K activity and keratinocyte migration.
Extracellular nucleotides stabilize β4 integrin into hemidesmosomes
Emilie Faure, Françoise Garrouste, Sylvie Monferran, Fabrice Parat, Salma Taboubi, Gilbert
Pommier, Jean-Claude Hubaud*, Hervé Kovacic, and Maxime Lehmann.
*DIPTA, Aix en Provence, France
The α6β4 integrin has opposite dual functions depending on its cellular membrane
localization and regulated by serine and tyrosine phosphorylation of its cytoplasmic tail. In
normal resting epithelial cells, β4 is mainly localized into the hemidesmosomes (HDs)
wherein it possesses a mechanistic function promoting stable adhesion of the epithelium to the
underlying basement membrane. In contrast, in cancer cells, oncogenic-RTK such as c-Met,
ErbB2 or EGF receptor induce phosphorylation of the β4 cytoplasmic tail which, in turn, go
outside HDs and amplifies RTK-signaling pathways to promote pathological angiogenesis
and tumor progression (Guo et al 2006 ; Giancotti et al 2007). In the present work, we used
normal human epithelial cells wherein EGF has been reported to phosphorylate β4 and to
disrupt HDs (Margadant et al 2008). We identified a new signaling crosstalk between a Gprotein coupled receptor (P2Y2 receptor) and EGF receptor. Using immunofluorescence (IF)
analysis by confocal microscopy, we showed that even in presence of EGF, P2Y2 receptor
activation by extracellular UTP stablized α6β4 integrin into mature HDs. Using both siRNA
and a pharmacological inhibitor (YM) we showed that activation of the Gα(q/11) protein was
required for this effect. Pharmacological studies indicated that EGF induced HD dissolution in
a Raf/MEK/Erk1/2 dependent pathway whereas UTP inhibited Raf/MEK/Erk1/2 activation in
a P2Y2 receptor and Gα(q/11) protein dependent pathway.
Our future goals are : to assess whether activation of Gα(q/11)-coupled-P2Y2 purinergic
receptor induce stabilization of β4 integrin into HDs by inhibiting EGF-induced MAPK
Erk1/2 pathway; to identify the pathway downstream of Gq protein that is involved in MAPK
Erk1/2 inhibition; to determine the impact of purinergic signaling on β4 integrin signaling
function. This new crosstalk pathway between growth factor receptors and G protein-coupled
purinergic receptor should improve our knowledge of the biochemical mechanisms regulating
β4 integrin signaling function in tumor progression.
Interaction of stathmin / microtubule: a quantitative analysis by FRET
imaging in cell
Roqiya Nouar±, Gilles Breuzard±, François Devred±, Anne-Marie Monges#, Vincent Peyrot±
±
# Laboratoire de Biophysique
Stathmin/Op18 is a key regulator of microtubule (MT) instability. However, the role
of stathmin in the destabilization of MT remains unclear especially since difficult to
apprehend in cell in a pharmacological context. In this way, Fluorescence Resonance Energy
Transfer (FRET) coupled to a confocal imaging system appears us a well-adapted method to
demonstrate beyond a simple optical co-localization, the stathmin/MT interaction. Our
observations displayed a distribution of stathmin in spot, which some of them were colocalized with MT. A quantification of stathmin/MT interaction by FRET showed a
significant energy transfer both in huge spots in lamellipodia and at the (+) end of MT.
Interestingly, a treatment with MT-stabilizing paclitaxel resulted in an increase of the
stathmin phosphorylation that could be correlated to a decrease of the energy transfer. To
conclude, results clearly demonstrated for the first time in cell an interaction of stathmin with
MT that could be modulated by pharmacological agents. Overall, stathmin would force
changes of conformation and/or structure of tubulin at the (+) end of MT unfavourable to its
growth.
Bcl-2 overexpression in A549 cells enhances unusually sensitivity to microtubuletargeting agents through an increase in mitochondrial fragmentation mediated by Bim
SAVRY A, CARRE M, ROVINI A, CHACON C, BRAGUER D and BOURGAREL-REY V.
Apoptosis or programmed cell death is defined as a mechanism of cellular suicide involved
in the regulation of tissue homeostasis. A disruption of apoptosis is implicated in the
development of cancer. Therefore, the ability of tumor cells to evade engagement of apoptosis
can play a significant role in their resistance to conventional therapeutic drugs. The members
of Bcl-2 family are central regulators of apoptosis. Among them, Bcl-2, an anti-apoptotic
member, is commonly overexpressed in various tumors and often associated with unfavorable
outcome. However, we and others have previously reported that a low Bcl-2 expression could
be associated with a paradoxical resistance to microtubule-targeting agents (MTAs). Thus,
the role of Bcl-2 in the anticancer activity of MTAs, never fully evaluated, remains unclear.
To further investigate this role, we generated various cellular models overexpressing Bcl-2.
We first showed that SHEP Bcl-2, neuroblastoma cells transfected with a Bcl-2 expression
vector, exhibit classical resistance to MTAs. In contrast, A549 Bcl-2, human lung carcinoma
cells, were paradoxically more sensitive (2-fold) to MTAs than control cells. This enhanced
sensitivity, specific of MTAs, is confirmed by a more important mitotic block. Next, we
studied the mitochondrial network structure, essential organelle for MTAs-induced apoptosis.
After paclitaxel treatment, a classical mitochondrial fragmentation is observed in different cell
lines. Interestingly in A549 line, mitochondrial fragmentation is 3 fold more important in cells
overexpressing Bcl-2 than in control cells. Thus, this enhanced mitochondrial fragmentation
was correlated to the highest cytotoxicity of paclitaxel. To understand this unusual sensitivity,
we realized DNA microarray analysis across both A549 cell lines. We showed an induction of
the mitochondrial pro-apoptotic Bim. This increase, confirmed at protein (total and
mitochondrial) levels only in A549 Bcl-2 is also associated with the increased sensitivity to
MTAs. Moreover, the microtubular localization of Bim suggests its critical role in MTAs
apoptosis. We then showed that the silencing of Bim by SiRNA prevents mitochondrial
fragmentation and reverses the MTAs sensitivity. Therefore, we highlighted Bim involvement
in enhanced sensitivity of A549 Bcl-2 cells, through a decrease of mitochondrial
fragmentation. Indeed, Bim protein level seemed to be a better determinant of MTAs
sensitivity than Bcl-2 status in pulmonary epithelial tumor. Thus, it appeared that Bim
expression may be an effective biomarker in predicting MTAs treatment’s efficacy. Its
involvement in the MTAs-induced mitochondrial fragmentation strengthens the mitochondrial
major role in the apoptotic pathway.
Soumis au meeting de l’American Association for Cancer Research Orlando 2011
Microtubule dynamics is involved in the control of angiogenesis by VEGF through EB1
localization at their + ends
Géraldine GAUTHIER, Stéphane HONORE, Pascal VERDIER-PINARD, Alessandra
PAGANO and Diane BRAGUER
INSERM 911, Centre de Recherche en Oncologie biolobique et Oncopharmacologie,
Université de la méditerranée, 27 bd Jean Moulin 13005 Marseille
Microtubules (MT) are dynamic cytoskeletal elements that control a wide range of
fundamental cellular functions, including cell division, cell migration and angiogenesis. We
have previously shown that MT-targeting agents (MTAs) produce their anti-migratory/antiangiogenic effects through an increase in endothelial interphase microtubule dynamics, a
decrease of EB1 comet length at microtubule + ends and a decrease in microtubule pause at
adhesion sites. Vascular Endothelial Growth Factor (VEGF) is a crucial regulator of neoangiogenesis in cancer, promoting endothelial cell proliferation and migration. We analyzed
the effect of VEGF on microtubule and EB1 dynamics in living Human Umbilical Vein
Endothelial Cells (HUVEC). Autocrine VEGF inhibition using VEGF trap led to an alteration
of microtubule-cell cortex targeting and a strong increase in microtubule dynamic instability
(+ 43%). In contrast, exogenously added VEGF (10 ng/ml) increased microtubule-cell cortex
targeting and suppression of microtubule dynamic instability (- 29%). Interestingly, we found
that the suppression of MT dynamics by VEGF occurred through their + end stabilisation at
focal adhesion sites. Moreover, we demonstrated that VEGF and the MTA Vinflunine
differentially altered the localisation of EB1 at microtubule + end. VEGF increased EB1
comets length by 32 % and 10 nM Vinflunine reduced EB1 comet length by 39%.
Interestingly, low antiangiogenic concentration of Vinflunine completely abolished the effect
of VEGF on EB1 comets. Finally, we demonstrated the first time by 2D electrophoresis that
EB1 existed with several post-translational modifications in endothelial cells. Impact of
VEGF and MTAs on such post-translational modifications will be presented. Altogether, our
results demonstrate that VEGF inhibitors share a common anti-angiogenic mechanism of
action with microtubule targeted drugs and revealed the potential pivotal role EB1 protein in
angiogenesis.
Présenté à Embo Conference “Microtubules: structure, regulation and functions”
Heidelberg Juin 2010
Analyse directe par spectrométrie de masse MALDI des protéines de haut poids
moléculaire : application aux isotypes α and β de tubuline
David Calligaris, Claude Villard, Lionel Terras et Daniel Lafitte
La caractérisation par analyse protéomique des isotypes/isoformes des protéines est un
objectif majeur pour mieux comprendre la régulation de nombreux processus biologiques. De
plus, ces isotypes/isoformes peuvent être impliqués dans des pathologies diverses. La
tubuline, protéine de 50 kDa, est une des principales cibles en chimiothérapie et dispose de 16
isotypes différents. Chaque isotype de tubuline se caractérise par leur extrémité C-terminale et
plus particulièrement par les 15-20 derniers acides aminés. Cette extrémité, présentant un taux
élevé d’acides aminés acides, est le site de nombreuses modifications post-traductionnelles.
Plusieurs études ont indiqué que la sélection isotypique s’opérant au niveau de certaines
cellules peut être à l’origine de processus de tumorogénèse. De plus, la surexpression de
l’isotype βIII de la tubuline peut influencer la dynamique des microtubules et induire des
phénomènes de résistance des cellules tumorales aux agents anticancéreux. La spectrométrie
de masse MALDI-TOF, et plus particulièrement une approche appelée in-source decay (ISD),
peut être une technique intéressante pour la caractérisation des isotypes de tubuline. Ce
processus, se produisant dans la source MALDI avant l'extraction des ions vers l’analyseur
TOF, est une fragmentation rapide qui peut être induite par deux voies principales : une voie
radicalaire et une voie thermique. L’utilisation de l’ISD couplé à une approche dite de T3séquencing a déjà permis de caractériser tous les isotypes d’une solution de tubuline de
cellules HeLa ainsi que certaines modifications post-traductionnelles telles que des
polyglutamylations et polyglycylations d’une solution de tubuline de cerveau d'agneau. De
plus, l'isotype βIII, biomarqueur tumoral, a pu être caractérisé. La fragmentation en source de
la tubuline par ISD se réalise préférentiellement dans le plasma en expansion lors du
phénomène de désorption laser. Elle se produit par des mécanismes de collision et produit des
ions de serie y. Cette étude est la première étude montrant la fragmentation par ISD de
l'extrémité C-terminale d'une protéine de 50 kDa. Elle ouvre une nouvelle voie pour la
caractérisation de biomarqueurs dans le domaine de la recherche sur le cancer et des
applications potentielles en imagerie par spectrométrie de masse MALDI.
Signature des peptides carbonylés par spectrométrie de masse MALDI : Dérivatisation et études
MS/MS
Lyna Sellami1, Claude Villard1, Pascale Barbier2, Vincent Peyrot2, Daniel Lafitte1
1. INSERM UMR 911, Centre de Recherche en Oncologie biologique et Oncopharmacologie;
Université Aix-Marseille; Protéomique et Innovation Technologique Timone, Faculté de
Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
2. INSERM UMR 911, Centre de Recherche en Oncologie biologique et Oncopharmacologie;
Université Aix-Marseille; Faculté de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille
Cedex 5, France
Résumé
La carbonylation des protéines est une modification post traductionnelle irréversible due aux espèces
réactives de l’oxygène, affectant principalement les chaines latérales des acides aminés arginine,
lysine, proline et thréonine. Elle est associée à plusieurs pathologies telles que la maladie
d’Alzheimer, la maladie de Parkinson, le cancer et certaines maladies inflammatoires. Plusieurs
techniques permettent le dosage des carbonyles totaux dans un échantillon mais la localisation
exacte des sites de carbonylation reste une tâche difficile. L’analyse par spectrométrie de masse
semble propice à ce genre d’étude mais elle nécessite des étapes d’enrichissement. Ceci a été réalisé
sur un peptide carbonylé modèle et un peptide que nous avons carbonylé. Ils ont été dérivatisés à la
biotine hydrazide (TAG-Mass). L’analyse comparée de ces peptides par spectrométrie de masse
MALDI TOF en mode CID a permis de déterminer des fragments rapporteurs caractéristiques du TAGMass. La tubuline est une protéine du cytosquelette sensible à l’oxydation. Nous avons prouvé sa
carbonylation par le dosage au 2,4 DNPH. Cette protéine sera un modèle pour le développement de
notre stratégie.
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Titre : Efficacité antibactérienne des dérivés aminostéroïdiens inhalés dans un modèle
d’infection pulmonaire chronique à Pseudomonas aeruginosa chez le rat
Auteurs: S Hraiech, JM Brunel, JM Rolain* , F Bregeon, H Lepidi, L Papazian, A Roch.
Corresponding author: Pr. Jean-Marc ROLAIN, URMITE CNRS UMR 6236 IRD 198, Faculté de Médecine
et de Pharmacie, Université de la Méditerranée, 27, Boulevard Jean Moulin 13385 Marseille cedex 5.
Introduction : Les dérivés aminostéroïdiens (DAS) sont des molécules de synthèse analogues de la
squalamine, substance extraite du requin. De précédents travaux ont montré leur efficacité
antibactérienne in vitro sur les cocci Gram + et les bacilles Gram – et ils pourraient être une
alternative intéressante pour la décontamination bronchique des patients insuffisants respiratoires
chroniques colonisés à P. aeruginosa. Ces molécules n’ont cependant jamais été évaluées in vivo. Le but
de ce travail était d’étudier l’efficacité anti-bactérienne des DAS inhalés après avoir mis au point un
modèle de pneumonie chronique à P. aeruginosa chez le rat.
Matériels et méthodes
Modèle d’infection chronique : Nous avons synthétisé des microbilles d’agar porteuses de P. aeruginosa
afin de réaliser un modèle d’infection chronique non létale jusqu’au 14ème jour.Vingt rats « SpragueDawley » mâles ont subi une intubation oro-trachéale après anesthésie par sévoflurane et ont reçu
150 µl d’une solution de microbilles d’agar contenant 108 unités formant colonie (UFC)/ml. A J1, J3, J7
et J14, cinq animaux étaient sacrifiés et leur poumons prélevés. L’aspect macroscopique était noté et
le poumon droit mis en culture dans un milieu TSA. La charge bactérienne était déterminée par
technique de dilutions en cascade.
Aérosols de DAS inhalés : Après infection, une série de 24 animaux (3 groupes de 8) ont été traités par
aérosols biquotidiens d’anti-infectieux (DAS de synthèse appelé NV503, squalamine ou colistine) du
lendemain de l’infection (J1) jusqu’à J6. Huit animaux infectés le même jour ne recevaient pas de
traitement (groupe témoin). A J7, les 32 animaux étaient sacrifiés et les poumons prélevés. Le poumon
droit était broyé et mis en culture pour détermination de la charge bactérienne. Le poumon gauche
était conservé pour étude anatomopathologique.
Résultats
Modèle d’infection chronique : Les 20 animaux infectés ont survécu à l’infection. Après une première
période de perte de poids et d’asthénie, les animaux retrouvaient une croissance pondérale normale.
Tous les poumons prélevés présentaient des lésions macroscopiques : œdème, hémorragies, abcès,
non retrouvés chez les animaux témoins. Après euthanasie des animaux, le poumon droit était broyé
au moyen d’un UltraTurrax™ et mis en culture. La charge bactérienne restait supérieure à 105 UFC à
J7 et supérieure à 103 UFC à J14. Il existait un portage chronique non létal de la bactérie jusqu’à J14
au moins.
Aérosols de DAS inhalés : Les aérosols de NV503, squalamine et colistine permettaient une réduction
significative de la charge bactérienne pulmonaire après 6 jours d’aérosols biquotidiens par rapport au
groupe témoin (charge bactérienne à J7 de 5. 103 UFC/poumon pour NV503, 103 UFC/poumon pour
squalamine , 103 UFC/poumon pour colistine , 105 UFC/poumon pour le groupe témoin (p < 0,01
pour les 3 groupes)). Il n’existait pas de surmortalité dans le groupe des animaux traités. Ces
résultats sont retrouvés avec les 2 types de DAS ainsi qu’avec la colistine utilisée comme molécule de
référence.
Conclusion : Le traitement par aérosols biquotidiens de dérivés aminostéroïdiens permet une
réduction significative de la charge bactérienne pulmonaire à J7 dans un modèle de pneumonie
chronique à P. aeruginosa non létale chez le rat. Les aérosols étaient bien supportés et n’ont pas
occasionné de surmortalité. Il existe une activité anti-bactérienne in vivo des DAS. Ces résultats
doivent être confrontés aux données anatomopathologiques (type de lésions observées, évolution
après traitement) ainsi qu’aux résultats de l’étude de pharmacologie et de toxicité en cours.
Title: MALDI-TOF Mass Spectrometry: an useful tool for typing Staphylococcus aureus
strains in the context of cystic fibrosis.
Authors: N. Murillo, F. Bittar, M. Reynaud-Gaubert, J.C. Dubus, N. Stremler, D. Raoult, J.M.
Rolain
Corresponding author: Pr. Jean-Marc ROLAIN, URMITE CNRS UMR 6236 IRD 198, Faculté de Médecine
et de Pharmacie, Université de la Méditerranée, 27, Boulevard Jean Moulin 13385 Marseille cedex 5.
Objectives Respiratory infections remain a major threat to cystic fibrosis (CF) patients and lungs
represent a specific ecological niche that is chronically colonized by various bacteria especially
Staphylococcus aureus that are known to be highly adapted to this microenvironment during
evolution of the disease. In this study, we used MALDI-TOF MS on whole cells as a typing method to
look for the possibility of a specific clustering of S. aureus isolates recovered from sputum samples of
CF patients as compared to non-CF clinical isolates of S. aureus.
Methods A total of 523 S. aureus isolates were analyzed including 324 isolates from CF patients
collected from 2006 to 2010 at Marseille (CF group), as well as 195 isolates from other patients in
Marseille and four reference strains (non-CF group). All strains were cultivated on COS petri dishes
at 37°C overnight before analysis by MALDI-TOF MS (AutoFlex apparatus, Brucker Daltonics®).
Each of four replicates of spectra was used to create profiles that were compared and analyzed using
Biotyper 2.0 software (Brucker Daltonics ®) to generate a dendrogram.
Results All isolates were correctly identified by MALDI-TOF MS with scores > 1.9. The dendrogram
obtained using Biotyper software clustered the strains in five different clusters with an arbitrary
distance level > 500. Statistical analysis revealed that cluster 1, 2 and 3 were significantly associated
to isolates recovered from the CF group (p < 10-3)(Figure). Cluster 4 was composed of 42 strains
both from CF and non-CF group. Conversely, cluster 5 was significantly composed of isolates
recovered from the non-CF group (p < 10-6)(Figure). Looking more precisely at the composition of
these clusters for the CF group we found that cluster 3 was significantly associated to isolates from
adults (age >18 years, p = 0.007).
Conclusion Our results suggest that MALDI-TOF MS may be useful for the differentiation of
isolates of S. aureus isolates from CF patients. This should be compared with isolates from other CF
centers to exclude the possibility of a specific epidemiology of strains in our region. The difference of
clustering of isolates observed between children and adults CF patients may be due to selective
pressures in the CF patient's lungs during chronic infection, such as host responses and repeated
antibiotic treatment that may act as a driver for microevolution.
Titre : L’hépatite Delta au CHU de Marseille.
Auteurs : Morgane Plumelle1*, Anne Motte1, Mireille Henry1,2, Marie-Hélène Romera1,
Christian Tourrès1, Audrey Ferretti1, Catherine Tamalet1,2, P. Colson1,2
Affiliations : 1 Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de
Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, 264, rue Saint-Pierre 13385 Marseille
cedex 5 ; 2 URMITE CNRS UMR 6236 IRD 198, Faculté de Médecine et de Pharmacie, Université de la
Méditerranée, 27, Boulevard Jean Moulin 13385 Marseille cedex 5
Rationnel et objectifs : Le virus de l’hépatite Delta (VHD), ou agent Delta, constitue une
préoccupation clinique supplémentaire pour les patients chroniquement infectés par le virus de
l’hépatite B (VHB). En effet, on considère que le VHD est responsable de formes sévères d’hépatites
virales et qu’une réponse virologique prolongée ne peut être obtenue que dans 25% des cas et
seulement avec l’interféron- alpha. En France, les études sur l’hépatite delta sont rares, alors que la
prévalence de l’infection chronique par le VHB y est estimée à 0.65% et que la couverture vaccinale
anti VHB y est faible (30-40%). Nous présentons la prévalence et les caractéristiques virologiques et
épidémiologiques de l’hépatite delta chez les patients porteurs de l’antigène de surface du VHB (HBs)
(chroniquement infectés par le VHB) diagnostiqués au CHU de Marseille.
Patients et Méthodes : La prévalence des anticorps anti-VHD et de l’ARN du VHD ont été
étudiées à partir des échantillons de sang périphérique collectés de 1998 à 2010. Les anticorps totaux
et les IgM anti-VHD ont été détectés par des techniques immuno-enzymatiques commercialisées.
L’ARN du VHD a été détecté par une technique de PCR en temps réel mise au point au laboratoire,
et par PCR conventionnelle puis séquençage. Le génotype du VHD a été déterminé par analyse
phylogénétique. Le diagnostic d’infection chronique par le VHD a reposé sur la mise en évidence
d’IgM anti-VHD ou sur la détection de l’ARN du VHD.
Résultats : Des résultats sérologiques ont été disponibles pour 881 sérums collectés à partir de 772
patients. Les anticorps anti-VHD totaux et les IgM anti-VHD étaient positifs chez 61 (7.9%) et 23
(3.0%) patients respectivement. La proportion des patients VIH positifs était significativement plus
élevée chez les séropositifs VHD par rapport aux séronégatifs (48% contre 19% ; p<10^-6). En outre,
la proportion des hommes était significativement plus élevée chez les patients séropositifs VHD que
chez les séronégatifs (82% contre 63% ; p=0.003), et les patients séropositifs VHD étaient
significativement plus nombreux dans la tranche d’âge des 30-60 ans que les patients séronégatifs
(64% contre 21% ; p<10^-3). Une primo-infection par le virus delta a été documentée en 2008.
L’ADN du VHB a été mesuré parallèlement aux marqueurs virologiques du VHD. Aucune différence
significative portant sur la charge virale du VHB n’a été observée entre les patients séropositifs VHD
et les patients séronégatifs VHD. Au total, l’ARN du VHD a été détecté par PCR temps réel et/ou
séquençage dans les sérums de 24 patients. Il a été séquencé pour 19 patients : 18 VHD étaient de
génotype I, 1 de génotype V.
Conclusion : Le virus Delta infecte 3% des patients chroniquement infectés par le VHB suivis dans
les hôpitaux de Marseille, principalement ceux co-infectés par le VIH. Ces résultats incitent à étudier
de manière plus précise l’impact clinique du VHD sur la progression de la maladie hépatique.
Titre : Infection de souris et de cellules intestinales humaines par un virus de plantes, le
Pepper Mild Mottle Virus
Auteurs : Fanny Balique1, Khatoun Al Moussawi1, Audrey Ferretti2, Claude Nappez1, JeanLouis Mège1, Hervé Lecoq3, Didier Raoult1, Philippe Colson1,2
Affiliations : 1 URMITE CNRS UMR 6236 IRD 198, Facultés de Médecine et de Pharmacie, Université de la
Méditerranée, 27, Boulevard Jean Moulin 13385 Marseille cedex 5 ; 2 Pôle des Maladies Infectieuses et
Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire
Timone, 264, rue Saint-Pierre 13385 Marseille cedex 5 ; 3 Institut National de la Recherche Agronomique
(INRA), Unité de Recherche (UR) 407, Unité de Pathologie Végétale, Domaine St-Maurice, BP 94, 84143,
Montfavet cedex,
Rationnel et Objectifs: Le Pepper Mild Mottle Virus (PMMoV) est un virus de plantes à ARN simple
brin de polarité positive, appartenant au genre Tobamovirus. Récemment, il a été détecté dans des
produits alimentaires à base de piments. Par une approche de métagénomique, ce virus a été identifié
comme étant le virus à ARN le plus prévalent dans des selles humaines non diarrhéiques. De plus, sa
présence dans les selles de patients a pu être associée à une réponse immunitaire et à des
symptômes cliniques (fièvres, douleurs abdominales et prurit). Par ailleurs, l’infectiosité du virus
detecté dans les selles et les produits alimentaires a été démontrée. Ces résultats soulèvent donc
des questions sur l’éventuel rôle pathogène du PMMoV pour la santé humaine. Le présent travail
avait pour objectif d’étudier l'infection du PMMoV dans un model expérimental de souris et dans des
cellules intestinales humaines.
Matériels et Méthodes: Des cultures contenant 4.105 cellules Caco2 (cellules d'adénocarcinome
de l’épithélium colorectal humain) ont été incubées pendant 12 jours avec 1e10 particules de PMMoV
infectieux. Dans le modèle expérimental, les souris ont reçu par voie orale 1e10 particules de virus
infectieux avec ou sans traitement préalable avec 0,2 mg de capsaïcine (molécule irritante du piment)
par souris pendant 5 jours avant l'ingestion du PMMoV. Quatre groupes de souris ont été définis
incluant un groupe contrôle (n=9), un groupe traité à la capsaïcine (n=6), un groupe traité avec du
PMMoV (n=10) et un groupe traité avec de la capsaïcine et du PMMoV (n=5). Le PMMoV a été
recherché quotidiennement dans les selles de souris en utilisant une technique de RT-PCR en temps
réel. Sept et 14 jours après l'ingestion de virus, les souris ont été sacrifiées afin de rechercher le virus
dans les organes (le foie, la rate, le colon et l’intestin grêle). Afin d’évaluer la survenue de diarrhées,
le rapport poids de selles hydratées/poids de selles déshydratées a été mesuré à partir des selles
provenant du colon des souris. L'infectiosité virale a été testée en réalisant des inoculations sur des
plantes hôtes du PMMoV, et des anticorps anti-PMMoV ont été recherchés dans les sérums des
souris par test ELISA.
Résultats: Du PMMoV a été détecté jusqu'à 12 jours après l’infection dans le milieu de culture des
cellules Caco2, et dans les cellules lavées. Dans le milieu de culture, nous avons observé une
diminution de 3.5 Log (copies/ml) du titre viral entre les jours 0 et 6 (11.1 Log à 7.6 Log), puis une
diminution de 1.4 Log a été observée entre les jours 6 et 12. Dans les cellules Caco 2, nous avons
observé une diminution de 2.2 Log entre les jours 0 et 6 (8.4 Log à 6.2 Log), puis le titre viral n'a pas
diminué entre les jours 6 et 12. Concernant le modèle expérimental de souris, du PMMoV a été
détecté dans les selles entre les jours 1 et 7 après l'ingestion de virus. Le titre viral a diminué de 5
Log (de 1e10 à 1e5copies/ml). L’infectiosité du virus dans les selles de souris a pu être mise en
évidence jusqu'à 2 jours après l’infection. Le PMMoV n'a pas été détecté dans les organes de souris
testés dans ce travail (foie, rate, colon, intestin grêle). Des anticorps IgG anti-PMMoV n'ont pas été
détectés dans les sérums des souris. D’autre part, nous n’avons pas observé la présence de diarrhée
chez les souris testés.
Conclusion: Ces résultats confirment que le PMMoV reste infectieux après son transit dans
l’appareil digestif. Le PMMoV serait capable d’infecter des cellules de vertébrés.
Title: Squalamine tablets for rapid disinfection of home nebulizer from cystic fibrosis
patients.
Authors : Lamia Djouhri-Bouktab, Kamel Alhanout, Véronique Andrieu, Nathalie Stremler,
Jean-Christophe Dubus, Didier Raoult, Jean-Marc Rolain and Jean-Michel Brunel*
* Corresponding author : Jean-Michel Brunel, Unité de Recherche des Maladies Infectieuses et tropicales
Emergentes (URMITE) UMR 6236, CNRS, Faculté de Médecine et de Pharmacie, Université de la
Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France. E-mail: [email protected]
Background: Bacterial contamination of nebulizers represents a major problem for cystic fibrosis
patients leading to reduced nebulizer performance and increasing the risk of patient reinfection by
the contaminant bacteria.
Objective: We investigated herein the potent use of broad spectrum antimicrobial squalamine in a
nebulizer disinfection model in vitro.
Methods: Nebulizers were infected by bacterial and fungal suspension and disinfected by immersion
in squalamine solution. Glutaraldehyde and korsolex peracetic acid were used as inhibition control.
Result: We found that squalamine at a 0.5g/L were able to reduce 5 log10 of S. aureus, P. aeruginosa
and 4 log10 C. albicans viable cells in 20 min while these compounds were able to reduce 4 log10 of A.
niger cells in 6 hours with a concentration of 2 g/L. Finally, a formulation of squalamine water soluble
disinfecting tablets at 2.5 % was developed and successfully applied for nebulizer disinfection.
Conclusion: Our result suggest that this family of compounds may be used by cystic fibrosis patients
for home nebulizer disinfection and that water soluble tablets may be developed for this purpose.
Keywords: nebulizer, cystic fibrosis (CF), squalamine, disinfection
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DESIGN OF A NOVEL SERIES OF BENZOXAZOLONE USING TDAE
STRATEGY
a,b,c
Aïda Rebaïa Nadji-Boukrouche,
c
c
b
Omar Khoumeri, Thierry Terme, Messaoud Liacha,
Patrice Vanelle
c
a) Département de Génie des Procédés, Université de Guelma, BP 401, 24000 Guelma, Algérie.
b) Laboratoire de Synthèse et de Biocatalyse Organique (LSBO), Faculté des Sciences, Université Badji MokhtarAnnaba, BP 12 El-Hadjar, 23000 Annaba, Algérie.
c) Laboratoire Chimie Provence - UMR 6264 CNRS- Universités d’Aix-Marseille I, II et III
Equipe Pharmaco-Chimie Radicalaire, Faculté de Pharmacie – 27 Bd Jean Moulin,
13885 Marseille Cedex 5, France
Courriel :[email protected]
Benzoxazol-2(3H)-one heterocycles have attracted considerable attention as a result of their
1
medicinal properties. Several potentially useful drugs and pharmacological tools based on this
2
pharmacophore have been developed in recent years.
3
Tetrakis(dimethylamino)ethylene (TDAE) is an organic reducing agent which reacts with
haloalkyl derivatives to generate an anion under mild conditions via two sequential transfers of one
4
electron. Since 2003, we have introduced a new program directed toward the development of original
5
synthetic methods using TDAE methodology in medicinal chemistry. In continuation of this program,
we synthesized the 5-(bromomethyl)-3-methyl-6-nitrobenzo[d]oxazol-2(3H)-one and investigate its
reactivity in the presence of various electrophiles via the TDAE strategy, leading to a novel series of 5substituted benzoxazolones.
Br
O2N
Br
O2N
R
Me
N
OHC
O +
R
O
O
HO
O2N
R = 2-NO2, 2-Br, 3-Br
4-NO2, 4-Br, 4-CN
O
44-52%
Me
N
O +
O
Me
N
TDAE
O
O
R1
OEt
O
TDAE
Me
N
R1
EtO
HO
O2N
O
R1 = H, CO2Et
O
52-72%
References
1. (a) J. S. Jadhav, V. A. Chatpalliwar, S. C. Khadse, R. R. Patil, Indian J. Heterocycl. Chem. 2008, 17, 343. (b) Z.
Moussavi, D. Lesieur, C. Lespagnol, J. Sauzieres, P. Olivier, Eur. J. Med. Chem. 1989, 24, 55. (c) V. Kalcheva, Z.
Mincheva, P. Andreeva, Arzneim. Forsch. 1990, 40, 1030.
2. (a) P. Carato, J. P. Bonte, D. Lesieur, D. Depreux, M. Millan, A. Newman-Tancredi, M. C. Rettori, D. H,
Caignard, Drug Des. Discov. 2000, 173. (b) O. Diouf, P. Carato, I. Lesieur, M. C. Rettori, D. H, Caignard, Eur. J.
Med. Chem. 1999, 34, 69.
3. M. Mahesh, J.A. Murphy, F. LeStrat, H. P., Wessel, Beilstein J. Org. Chem. 2009, 5, 1.
4. N. Takechi, S. Ait-Mohand, M. Medebielle, W. R. Dolbier, Tetrahedron Lett. 2002, 43, 4317.
5. (a) G. Giuglio-Tonolo, T. Terme, M. Médebielle, P. Vanelle, Tetrahedron Lett. 2003, 44, 6433. (b) O. Khoumeri,
M. Montana, T. Terme, P. Vanelle, Tetrahedron 2008, 64, 11237. (c) O. Khoumeri, M. Montana, M. D. Crozet, T.
Terme, P. Vanelle, Tetrahedron Lett. 2009, 50, 6372.
ORIGINAL SNAr REACTION VIA TDAE STRATEGY:
A NEW WAY TO THE 2-TRICHLOROMETHYLQUINAZOLINE
ANTIPLASMODIAL PHARMACOMODULATION
M. Since, P. Verhaeghe, O. Khoumeri, T. Terme, P. Vanelle
Laboratoire Chimie Provence - UMR 6264 CNRS- Universités d’Aix-Marseille I, II et III
Recently, our team has developed a program focusing on new quinazoline derivatives bearing
1-3
antiplasmodial properties.
Therefore, some 4-anilino-2-tricholoromethylquinazolines display
interesting in vitro antiplasmodial activity on the W2 chloroquino-resistant P. falciparum strain,
combined with a safe toxicological profile on the HepG2 human cell line. Replacing the amino group of
such derivatives could result in promising biological results. However, contrary to amines, we found
4
very little literature which mention SNAr reactions involving carbanions with 4-chloroquinazoline.
Tetrakis(dimethyl-amino)ethylene (TDAE) is a reducing agent which reacts with halogenated
derivatives to generate a carbanion, which is able to react with various electrophiles via nucleophilic
5
addition reactions with aldehydes, α-ketoesters, ketomalonates and α-keto lactam derivatives. Our
team also extended the reactivity of carbanion obtained with TDAE to the SN2 mechanism with α6
haloesters and α-haloamides.
We report herein an original SNAr reaction via a TDAE strategy on 4-chloro-2trichloromethylquinazoline, leading to new 4-nitrobenzyl-2-trichloromethylquinazoline derivatives with
potential antiplasmodial activity.
O2N
Cl
NO2
R
Cl
N
+
R= H
4-CH3
4,5-(OCH2O)
4,5-(OMe)2
R
TDAE, DMF, N2
N
CCl3
N
-20°C 1h, 50°C 1h
N
CCl3
62-80 %
References
1) Y. Kabri, N. Azas, A. Dumètre, S. Hutter, M. Laget, P. Verhaeghe, A. Gellis, P. Vanelle. Eur. J. Med. Chem.
2010, 45, 616.
2) P. Verhaeghe, N. Azas, S. Hutter, C. Castera-Ducros, M. Laget, A. Dumètre, M. Gasquet, J.-P. Reboul, S.
Rault, P. Rathelot, P. Vanelle. Bioorg. Med. Chem. 2009, 17, 4313.
3) P. Verhaeghe, N. Azas, M. Gasquet, S. Hutter, C. Ducros, M. Laget, S. Rault, P. Rathelot, P. Vanelle. Bioorg.
Med. Chem. Lett. 2008, 18, 396.
4) a) S. P. Govek, A. K. Shiau, S. A. Noble, D. J. Thomas. PCT Int. Appl. 2008, WO 2008006052. b) T. Higashino,
E. Hayashi, Chem. Pharm. Bull. 1970, 18, 1457.
5) a) G. Giuglio-Tonolo, T. Terme, M. Médebielle, P. Vanelle, Tetrahedron Lett. 2003, 44, 6433. b) Giuglio-Tonolo,
G.; Terme, T.; Médebielle, M.; Vanelle, P. Tetrahedron Lett. 2004, 45, 5121.
6) M. Since, T. Terme, P. Vanelle. Tetrahedron 2009, 65, 6128.
PALLADIUM(0)-CATALYZED SUZUKI-MIYAURA CROSS-COUPLING
OF ELECTRON-DEFICIENT NITRO(FLUOROARYL)IMIDAZOLE AS
POTENTIAL TRICHOMONACIDAL AGENTS
Laura ZINK, Maxime D. CROZET, Vincent REMUSAT, Patrice VANELLE
Laboratoire Chimie Provence - UMR 6264, CNRS - Universités d’Aix-Marseille I, II, III - Equipe Pharmaco-Chimie
Radicalaire, Faculté de Pharmacie - 27, Bd Jean Moulin, 13385 Marseille Cedex 5; France,
E-mail: [email protected], Fax: (33) 4 91 79 46 77
The 5-nitroimidazole scaffold is well-known for displaying major anti-infectious activities. Several 5nitroimidazole-containing active principles are commonly used in medecine such as metronidazole,
secnidazole and ornidazole. These chemotherapeutic agents inhibit the growth of both anaerobic
bacteria and some anaerobic protozoa. Nowadays, the most clinically used drug-compound for the
treatment of both infections caused by protozoa such as Trichomonas vaginalis, Entamœba histolytica,
Giardia intestinalis and infections induced by anaerobic bacteria is metronidazole. However, the 5nitroimidazoles have been found to possess a high mutagenic activity in prokaryotic micro-organisms. A
nitroimidazole possessing good pharmacological activities with no mutagenicity would be of great
interest, not only from a safety point of view, but would also provide a basis for further investigations on
the mechanism involved in their mutagenicity. Moreover, emergence of metronidazole-resistant
Trichomonas vaginalis results in decreasing the success of current therapies. These refractory cases are
usually treated with higher doses of metronidazole, which lead to an increase in the occurrence of side
effects. So, alternative curative therapies are needed.
Arylimidazoles possess high in vitro antimicrobial action; several compounds were fungicidal towards
pathogenic organisms but introduction of a nitro group into the imidazole ring largely destroyed this
1
activity and simultaneously bestowed on some of the compounds very high antitrichomonal properties.
2,3
During the course of our studies on the synthesis and antiprotozoal activity of some nitroimidazoles,
we observed a high reactivity versus SNAr and Suzuki-Miyaura cross-coupling in nitro(fluoronitroaryl)imidazole series. Aryl fluorides, however, have long been considered inert to Pd(0)-catalyzed coupling
4
reactions. A search of the primary literature yielded only few relevant studies. In 1999, Widdowson and
6
co-workers demonstrated that η -Cr(CO3)-fluorobenzene underwent Stille and Suzuki couplings in the
5
presence of Pd2(dba)3 and PMe3 in good yields. In this communication, we would like to report our
studies on the Pd(0)-catalyzed Suzuki coupling of electron-deficient imidazole bearing an aryl fluoride
group.
Ar
F
Nu
O2N
O2N
O2N
N
O2N
CH3
N
CH3
NuDMF
N
O2N
CH3
N
CH3
ArB(OH)2
Pd(PPh3)4 (3.4mol%)
Cs2CO3
MW 150W
DMF
N
O2N
CH3
N
CH3
The structure of the synthesized imidazoles, the methodologies employed and the initial biological
evaluation on Trichomonas vaginalis will be reported and discussed.
References
1) Ellis, G. P.; Epstein, C.; Fitzmaurice, L.; Golberg, L.; Lord, G. H. J. Pharm. Pharmac. 1967, 19, 102.
2) Crozet, M. D.; Botta, C.; Gasquet, M.; Curti, C.; Rémusat, V.; Hutter, S.; Chapelle, O.; Azas, N.; De Méo, M.;
Vanelle, P. Eur. J. Med. Chem. 2009, 44, 653.
3) Crozet, M. D.; Zink, L.; Remusat, V.; Curti, C.; Vanelle, P. Synthesis 2009, 3150.
4) Kim, Y. M.; Yu, S. J. Am. Chem. Soc. 2003, 125, 1696.
5) Widdowson, D. A.; Wilhelm, R. Chem. Commun. 1999, 2211.
EFFICIENT SYNTHESIS OF SPIROCYCLIC COMPOUNDS USING
MANGANESE(III) ACETATE METHODOLOGY
Ahlem Bouhlel, Christophe Curti, Patrice Vanelle
Laboratoire Chimie Provence - UMR 6264 CNRS- Universités d’Aix-Marseille I, II et III
Among the extensive therapeutical applications of spirocyclic compounds, properties of
spiro[5.5]undecane and 1-oxaspiro[4.5]decane derivatives in analgesic area have been widely
1
investigated. Oxidative radicalar cyclizations mediated by manganese(III) acetate between βdicarbonyl compounds and alkenes afford original access to a large variety of molecular structures,
2
3
including spirocycles. Syntheses of spiro[4.5]decane and 1-oxaspiro[4.5]dec-2-ene were already
reported, and we propose herein an efficient synthesis of two other spirocyclic structures using the
manganese(III) acetate methodology.
H3 C
O
O
H3C
O
O
O
O
H3C
O
CH3
O
Mn(OAc)3, 2.1 eq.
O
AcOH, N2
O
O
O
CH3
1
2
O
O
O
CH3
O
CH3
3
Using different experimental conditions, we obtained 1 (yielding from 17% to 51%), 2 (yielding
from 32% to 79%) and 3 (yielding from 0% to 30%). Therefore, products 2 and 3 allow us to obtain
several spirocyclic functionalized compounds of therapeutic interest.
References
1. a) Zemolka, S.; Nolte, B.; Frormann, S.; Hinze, C.; Linz, K.; Schroeder, W.; Englberger, W.; Schick, H.;
Sonnenschein, H. World Patent 2009, 118173, Chem. Abstr. 2009, 151, 1204343. b) Merla, B.; Oberboersch, S.;
Sundermann, B.; Englberger, W.; Hennies, H.-H.; Koegel, B.-Y.; Graubaum, H. Ger. Offen. 2007, 102006019597,
Chem. Abstr. 2007, 147, 1239392. c) Vonvoigtlander, P.F.; Lewis, R.A. J. Pharmacol. Exp. Ther. 1988, 246, 259.
2. Snider, B.B.; Buckman, B.O. Tetrahedron 1989, 45, 6969.
3. Fujino, R.; Nishino, H. Synthesis 2005, 731.
PHARMACOCHIMIE ANTI-INFECTIEUSE EN SERIE QUINAZOLINE
P. Verhaeghe,a N. Azas,b C. Castera-Ducros,a A. Dumètre,b Y. Kabri,a S. Hutter,b P. Garrigue,a
M. Laget,b A. Gellis,a A. Cohen,b L. Mbatchi,a M. Maillard-Boyer,a F. Sifredi,a C. HopkinsSibley,c M. Phillips,d D. Dorin-Semblat,e C. Doerig,e S. Rault,f P. Rathelota and P. Vanellea
a
Laboratoire Chimie Provence, UMR CNRS 6264 Université d’Aix-Marseille I, II et III, Laboratoire de Pharmacochimie
Radicalaire LPCR, Faculté de Pharmacie, 27 Bd J. Moulin, 13385 Marseille Cedex 05, France.
b
Relation Hôte-Parasites, Pharmacologie et Thérapeutique, UMR MD3, Université de la Méditerranée, Marseille, France.
c
Department of Genome Sciences, University of Washington, Seattle, USA.
d
Southwestern Medical Center, University of Texas, Dallas, USA.
e
INSERM U609, Ecole Polytechnique Fédérale de Lausanne, Suisse.
f
Centre d’études et de Recherche sur le Médicament de Normandie, UPRES EA4248, Université de Caen, France.
Dans le but d’identifier de nouvelles molécules antiplasmodiales possédant un mécanisme
d’action novateur, une série de 300 quinazolines a été synthétisée et criblée in vitro sur la souche
multi-résistante W2 de Plasmodium falciparum. En parallèle, afin d’apprécier la sélectivité (IS) de
leur action antiplasmodiale (IC50), l’évaluation de la cytotoxicité (CC50) de ces mêmes molécules a été
réalisée in vitro, notamment sur la lignée hépatocytaire humaine HepG2, permettant ainsi de définir
les index de sélectivité correspondants. 6 « hits » ont ainsi pu être mis en évidence, présentant des
CI50 comprises entre 0,4 et 2,5 µM, des CC50 comprises entre 16 et >100 µM et des index de
sélectivité variant de 40 à >125, par comparaison à des principes actifs antimalariques comme la
chloroquine (CI50=0,7 µM; CC50=30 µM ; IS=43) et la doxycycline (CI50=6,5 µM; CC50=20 µM ; IS=3).
Le mécanisme d’action de ces molécules a ensuite été recherché, en commençant par l’étude
des principaux mécanismes d’action connus pour les molécules antimalariques commercialisées
(inhibition de la cristallisation de l’hème, inhibition de la PfDHFR, polarisation de la membrane
mitochondriale, genèse d’espèces radicalaires). Parmi les 6 « hits » identifiés, 2 molécules ont
présenté des propriétés inhibitrices de la cristallisation de l’hème (mécanisme d’action des
aminoquinoléines comme la chloroquine).
Le(s) mécanisme(s) par le(s)quel(s) les 4 autres molécules exercent leur action
antiplasmodiale est (sont) en cours de détermination. Nous nous focalisons d’une part sur certaines
kinases plasmodiales (PfPK5, PfPK6, PfPK7, Pfmap2, PfCK2α, Pfnek1) et d’autre part sur la
dihydroorotate
deshydrogénase
plasmodiale
(PfDHOD),
cibles
parasitaires
originales
et
prometteuses, dans le cadre de collaborations et de contrats transfert de matériel avec des équipes
suisse et américaine référentes.
Références bibliographiques :
- Verhaeghe, P.; Azas, N.; Gasquet, M.; Hutter, S.; Ducros, C.; Laget, M.; Rault, S.; Rathelot, P.; Vanelle, P. Bioorg. Med. Chem. Lett. 2008, 18, 396.
- Verhaeghe, P.; Azas, N.; Hutter, S.; Castera-Ducros, C.; Laget, M.; Dumètre, A.; Gasquet, M.; Reboul, J-P.; Rault, S.; Rathelot, P.; Vanelle, P. Bioorg.
Med. Chem. 2009, 17, 4313.
- Kabri, Y.; Azas, N.; Dumètre, A.; Hutter, S.; Laget, M.; Verhaeghe, P.; Gellis, A.; Vanelle, P. Eur. J. Med. Chem. 2010, 45, 616.
- Kabri, Y.; Verhaeghe, P.; Gellis, A.; Vanelle, P. Molecules 2010, 15, 2949.
- Maillard-Boyer, M.; Castera-Ducros, C.; Verhaeghe, P.; Sifredi, F.; Rathelot, P.; Vanelle, P. Molecules 2010, 15, 2719.
- Castera-Ducros, C.; Azas, N.; Verhaeghe, P.; Hutter, S.; Garrigue, P.; Dumètre, A.; Mbatchi, L.; Laget, M.; Remusat, V.; Sifredi, F.; Rault, S.;
Rathelot, P.; Vanelle, P. Soumis à Eur. J. Med. Chem. janvier 2011.
- Verhaeghe, P.; Azas, N.; Castera-Ducros, C.; Hutter, S.; Dumètre, A.; Laget, M.; Gellis, A.; Yzombard, J.; Prieri, M.; Fersing, C.; Rault, S.;
Rathelot, P.; Vanelle, P. en préparation pour Bioorg. Med. Chem. Lett.
- Since, M.; Khoumeri, O.; Verhaeghe, P.; Terme, T.; Vanelle, P. en préparation pour Tetrahedron Lett.
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Interest of Cepharanthine (Stephania rotunda) in the fight against malaria: mechanism of action,
pharmacokinetic, pharmacodynamic.
Camille DESGROUAS, Nicolas TAUDON, Evelyne OLLIVIER, Daniel PARZY
IRBA antenne Marseille
The global burden of malaria affects about 250 million people every year. In the global anti-malaria
strategy, chemotherapy takes an important place in the control efforts. An effective antimalarial
therapy enables not only to reduce infection injuries but also reduces risk of resistance.
Unfortunately, the development and the propagation of the parasite resistant to drugs, jointly with
the lack of new drugs and new therapeutic targets these last few years generated an urgent need of
new therapeutic solutions. In front of this major need, the Ethnobotany approach, based on the
traditional use of plants as remedies, may offer a new hope by characterization of original potential
drugs in this high biodiversity.
In this context, collaboration between Cambodian (Phnom-Phen) and French (Marseille) faculties
allows an inquiry on Cambodian plants used in traditional medicine. Thirty plants were selected
amongst them Stephania rotunda from which a few molecules were extracted and purified and more
particularly a bisbenzylisoquinoline alkaloid named Cepharantin. First in vitro studies performed with
this molecule show an interesting and probably original antiplasmodial activity (CI50 = 0.6 µM) and a
synergy with chloroquine.
The aim of this study is to characterize more precisely the antiplasmodial activity of Cepharanthin. A
microarray approach will be performed to try to identify the Plasmodium biomechanisms affected by
it. This technology needs previously to study the impact of incubation of the drug with the parasitic
growth. In a second step, bioanalytical methods for the quantification of cepharantin in biological
matrices will be developed and validated. These methods allow performing pharmacokinetic and
phamacodynamic studies, on infected and uninfected rodent, to characterize the corresponding
parameters. In conclusion of these studies we should be able to confirm if Cepharantin have an
original antiplasmodial mechanism of action and if it could be an appropriate lead for
pharmacomodulation. This study will then consist in generating structural analogues to improve the
parameters of activity and/or pharmacokinetic.
Use of generic High Dose Buprenorphine (HDB): about a qualitative survey.
Y. El Haïk, E. Frauger, N. Tanti-Hardouin, J. Micallef, X. Thirion
Introduction:
Appeared in France in 1995, generic drugs are in full development. Health authorities encourage the
use of generic drugs in order to reduce costs. Indeed, in 2008 the national market penetration rate
was 82%, but however, the rate of HDB (since 2006, buprenorphine has been marketed in generic
forms) is only 31.8% in 2008 on the French market.
What factors may explain the low use of buprenorphine generic in the treatment of opiate
substitution? What are heath care providers' and patients' attitudes towards this substitution?
This study aimed to assess professionals’ and patients’ feelings towards effectiveness, tolerability,
acceptance and clinical impact of generic buprenorphine.
Methods:
To be able to analyze this perception towards buprenorphine, a qualitative method based on the
realization of semi-directive conversations was held.
14 health professionals (General Practitioners, Specialists, Pharmacists, others health professionals)
and 10 patients were interviewed.
Results:
The healthcare professionals are unanimous on the fact that generic drug of HDB presents
undeniable economic benefits and galenic advantages (as new dosages). They claim to offer this
generic at first prescription or with stabilized patients, otherwise they know that the change of
molecule is more difficult with patients who diverted the product, or when they are accustomed to
the brand product.
Some patients prefer the brand product because of its galenics (as size or taste), or of the perception
of greater efficiency. For most of them, this generic is perceived as a lower-quality drug.
Conclusion:
This study reveals that there is a certain distrust compared to the generic of HDB.
If professionals seem to encourage the prescription of buprenorphine generic for some patients, it is
necessary to properly support the prescription of this drug in order to strengthen its image.
Indeed patients would feel safer if healthcare professionals had a more active role in informing them
about these drugs.
Microtubule dynamics is involved in the control of angiogenesis by VEGF through EB1 localization
at their + ends
Géraldine GAUTHIER, Stéphane HONORE, Pascal VERDIER-PINARD, Alessandra PAGANO et Diane
BRAGUER
Laboratoire UMR911 Pr BRAGUER
Microtubules (MT) are dynamic cytoskeletal elements that control a wide range of fundamental
cellular functions, including cell division, cell migration and angiogenesis. We have previously shown
that MT-targeting agents (MTAs) produce their anti-migratory/anti-angiogenic effects through an
increase in endothelial interphase microtubule dynamics, a decrease of EB1 comet length at
microtubule + ends and a decrease in microtubule pause at adhesion sites. Vascular Endothelial
Growth Factor (VEGF) is a crucial regulator of neo-angiogenesis in cancer, promoting endothelial cell
proliferation and migration. We analyzed the effect of VEGF on microtubule and EB1 dynamics in
living Human Umbilical Vein Endothelial Cells (HUVEC). Autocrine VEGF inhibition using VEGF trap led
to an alteration of microtubule-cell cortex targeting and a strong increase in microtubule dynamic
instability (+ 43%). In contrast, exogenously added VEGF (10 ng/ml) increased microtubule-cell cortex
targeting and suppression of microtubule dynamic instability (- 29%). Interestingly, we found that the
suppression of MT dynamics by VEGF occurred through their + end stabilisation at focal adhesion
sites. Moreover, we demonstrated that VEGF and the MTA Vinflunine differentially altered the
localisation of EB1 at microtubule + end. VEGF increased EB1 comets length by 32 % and 10 nM
Vinflunine reduced EB1 comet length by 39%. Interestingly, low antiangiogenic concentration of
Vinflunine completely abolished the effect of VEGF on EB1 comets. Finally, we demonstrated the first
time by 2D electrophoresis that EB1 existed with several post-translational modifications in
endothelial cells. Impact of VEGF and MTAs on such post-translational modifications will be
presented. Altogether, our results demonstrate that VEGF inhibitors share a common anti-angiogenic
mechanism of action with microtubule targeted drugs and revealed the potential pivotal role EB1
protein in angiogenesis.
MANAGEMENT WITH LITHIUM CARBONATE OF CLOZAPINE-INDUCED NEUTROPENIA: A CASE
STUDY
Golé C, Verine A, Bongrand MC
PUI Conception
Introduction:
Clozapine, an atypical antipsychotic drug, is the most effective medication for treatment-resistant
schizophrenia. It is also usefull for patients with severe side effects with others antipsychotics drugs.
Its use is however limited by the high risk of neutropenia and agranulocytosis and requires regular
and strict white blood count (WBC).
Lithium carbonate, used in first line treatment in bipolar disorder, has an opposite side effect:
leukocytosis.
Several studies show encouraging results about the use of the combination of clozapine and lithium
carbonate to prevent the risk of agranulocytosis. However, could premature discontinuation of
lithium increase this risk?
Materials and methods:
Our presence in a psychiatry unit has resulted in the retrospective study of a case of agranulocytosis
with clozapine following the discontinuation of lithium.
Results:
Mrs. R., 35, suffered from bipolar disorder. After the inefficiency of various neuroleptic drugs
(loxapine, olanzapine, amisulpride) and mood stabilizers including lithium, clozapine treatment was
initiated. The dose was increased until reaching an efficient dose of 600mg/day in 7 weeks.
Lithium was gradually stopped two months after initiation of clozapine. At the end of these two
months, the granulocyte rate of the patient was normal.
Less than 15 days later, WBC was 1.6G/L and neutrophils count was 0.33G/L. Treatment with
filgrastim was initiated immediately and a prophylactic antibiotherapy continued until neutrophil
recovery. The patient has not developed infection. Lithium was finally reintroduced as monotherapy
and the patient returned at home 15 days later.
Discussion/Conclusion:
Lithium may be used for treatment of some diseases such as neutropenia of Felty syndrome. Several
studies on the coadministation of lithium and clozapine have demonstrated a significant reduction of
the risk of iatrogenic neutropenia. This case could be an additional argument to the value of this
combination in this indication and brings a new element: the increased risk of agranulocytosis if
discontinuation of lithium is too premature. Yet we can not be certain about the causal relationship
between the two.
The coadministration of lithium and clozapine seems particularly interesting when reintroducing
clozapine after neutropenia. However, associations that increase the risk of agranulocytosis as
carbamazepine, valproate, or risperidone must be avoided and the increased risk of neuroleptic
malignant syndrome must be taken into account.
Further studies are needed to confirm the interest and safety of this association and the potential
risk of stopping lithium associated with clozapine.
Therapeutic Drug Monitoring of Raltegravir in experienced HIV-infected patients.
Chloé GOUGUET, Sylvie BREGIGEON , Christel PISSIER, Sylvie QUARANTA, Bruno LACARELLE,
Caroline SOLAS
Introduction: Raltegravir (RAL), the first drug of a new class of antiretrovirals (ARV), the integrase
inhibitors, is available since 3 years. RAL is mainly metabolized in the liver by the UGT1A1 and is
neither substrate nor inducer/inhibitor of CYP3A4. However, a wide interindividual pharmacokinetic
variability was reported during clinical trials. At the standard dose of 400 mg bid, the RAL mean trough
concentration (Ctrough) reported is 63 ng/ml (range: 29-118). We evaluated RAL Ctrough in a context
of real life and the interindividual variability according to the drug coadministrated
Materials and Methods: Retrospective study on 54 patients (24 women and 30 men) with a median
age of 46 years receiving RAL as part of their ARV regimen. RAL Ctrough was determined using a
liquid chromatography – tandem mass spectrometry (LC-MS/MS) method (limit of quantification of 5
ng/ml).
Results: Data were collected during 14 months. RAL dose was 400mg bid (n=53), 800mg bid (n=1).
During the follow-up, 2 patients were switched to 800mg bid following the introduction of rifampicine.
Patients have received RAL in association with an optimized background therapy (OBT) as follow:
2NRTIs (A, reference group, n=35), DRV/r±NRTIs (B, n=8), ETR±NRTIs (C, n=5), ATV/r±NRTIs (D,
n=4) and TPV/r±NRTIs (E, n=2). Overall 100 RAL Ctrough were determined at 12 ± 3 hours after the
last drug intake. The median (IQR, CV %) of RAL Ctrough for all groups was 113 ng/mL (45-320; 182).
Median Ctrough values were (A): 152 ng/ml (66-353; 122), (B): 45 ng/ml (24-107; 177), (C): 155 ng/ml
(121-402; 121), (D): 60 ng/ml (37-69; 90) and (E): 56 ng/ml (41-59; 68), respectively. RAL Ctrough
was always close or above the mean Ctrough reported of 63 ng/ml. A significantly higher RAL Ctrough
was observed when RAL was only associated with 2 NRTIs compared with group 2 (p=0.001), group 4
(p=0.04), group 5 (p=0.02). We did not find a higher RAL Ctrough with the coadministration of ATV/r,
probably du to the small number of patients. No decrease in the RAL Ctrough was observed when
ETR was associated.
Discussion/Conclusion: Our results confirmed a large interindividual variability in the RAL Ctrough and
confirmed no significant drug-drug interactions. Therefore, RAL may be safely coadministrated with
other antiretroviral agents. RAL variability increase with food and coadministration of drugs like the
proton pump inhibitor suggesting an important variability at the absorption level. Therefore, the
therapeutic drug monitoring of RAL may be of interest if pharmacodynamic/pharmacokinetic
relationships are demonstrated
Efficacy of carboxypeptidase G2 in the treatment of methotrexate severe intoxication
1*
1
1
2
A. James , B. Deluca-Bosc , S. Honoré , B. Lacarelle .
1
2
pharmacie Timone 1er étage, laboratoire de pharmacocinétique toxicocinétique, Hôpital de la
Timone, 264 rue St Pierre, 13385 Marseille cedex 05
Introduction: High-dose methotrexate (HDMTX) is used for treatment of some cancers (non
Hodgkin’s lymphoma, children’s acute lymphoblastic leukemia, osteosarcoma). This therapy can
induce a severe nephrotoxicity. Precautionary measures are associated with HDMTX: alkalinization,
hydration, preventive treatment of toxicity by leucovorin and pharmacokinetic methotrexate follow-up.
Despite these measures, in cases of severe acute intoxication, an antidote is available in France with
nominative Autorization for Temporary Use (ATU): carboxypeptidase G2 (CPDG2), a bacterial enzyme
that cleaves methotrexate into non-cytotoxic metabolite 4~deoxy-4-amino-N10-methylpteroic acid
(DAMPA).CPDG2 is a very expensive drug (€ 7.000 per vial 1000 IU) used at a dose of 50 UI / kg in
one intravenously infusion. CPDG2 administration must be done in maximum 96 hours after the date
of administration of high-dose methotrexate.
Materials & Methods: The purpose of this study was to examine the efficacy of this antidote through
various cases. The study was conducted over one year (April 2009 - April 2010) at the Timone
Hospital. During this period, 3 patients were treated with CPDG2.
Blood levels of MTX measured using florescent polarization immunoassay (FPIA) and high-pressure
chromatography (HPLC) before and after CPDG2 administration were chosen as markers of treatment
efficacy. Before CPDG2 administration, MTX concentrations are measured by FPIA. The inactive
metabolite of CPDG2 (DAMPA) cross-reacts in MTX immunoassays, so after carboxypeptidase
infusion, MTX concentrations have to be measured by HPLC.
Results: After HDMTX infusion and before the use of CPG2, patients had concentrations of MTX 33.3
µmol / L (24 hours after HDMTX), 12.9 µmol / L (48 hours after) and 3.97 µmol / L (48 hours after). 12
hours after injection of CPDG2, concentrations of MTX were respectively 0.88 µmol / L (97.4 %
reduction), 0.32 µmol / L (97.5 % reduction) and an undetectable level for the last patient, 24 hours
after antidote andministration. The average reduction rate of MTX was 98.3 % in these three patients
twelve hours after the administration of this antidote.
Discussion, Conclusion: CPDG2 allows a rapid and effective rescue of methotrexate acute
intoxication.
The ATU drug status gives at CPDG2 an additional security, avoiding misuse of this product, because
the form must be validated by the Agence Française de Sécurité Santitaire et des Produits de Santé
(AFSSaPS).Given the circumstances of use of CPDG2 and the place of supply (England), an
emergency stock for a patient was authorized by the AFSSaPS in our University Hospital.
Posaconazole tolerance and efficacy in cystic fibrosis lung disease children with fungal
refractory infection
E.Kiouris, N.Stremler le Bel, P.Pisano, B.Pourroy
CHU of La Timone, Marseille, France
a. Background
Cystic fibrosis lung disease is frequently associated to invasive, chronic and refractory fungal
infections in children. Thus, an effective antifungal treatment is required and, if it’s possible,
taken orally for improving patient quality of life. Posaconazole is an extended spectrum
triazole used for treatment and prophylaxis of refractory invasive fungal infection in adult
patients. Furthermore, it presents favourable pharmacocinetic properties, limited number of
adverse events and documented preventive and therapeutic clinical efficacy in adults
b. Purpose
Posaconazole characteristics allow us to consider children’s cases with cystic fibrosis who
presents antifungal classical treatment failure. The aim of our study is to evaluate
posaconazole use in these dedicated pediatric patients.
c. Material and Method
We studied 4 paediatric patients (6-15 years old) with cystic fibrosis and invasive fungal
infection.
d. Results
In all patients, aspergillus fumigatus strain was isolated in bronchial secretion samples. In one
patient, an initially resistance to flucytosine, itraconazole, caspofungin and amphotericin
conduced to use posaconazole in first intention. In other patients, posaconazole was used in
second or third intention after oral treatment failure or adverse effects apparition with other
antifungal drugs. Three patients received adult posology (400 mg x 2 / day) and the fourth
received 300 mg x 2 per day after plasma concentration measurement and posology
adjustement. Posaconazole improved general state in all patients and all bronchial samples
were negatives after treatment completion.
e. Conclusion
In our study, posaconazole tolerance and efficacy profile justify its paediatric use in invasive
refractory fungal infection associated with cystic fibrosis. Nevertheless, posology was
adjusted to posaconazole plasma levels and pharmacokinetic properties in children have to be
investigated to better treat these patients.
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MOROCCAN PLANTS ESSENTIAL OILS AS POTENTIAL
CHEMOSENSITIZERS RESTORING THE ANTIBIOTIC ACTIVITY IN
RESISTANT GRAM-NEGATIVE BACTERIA
Mariam Fadli, Jacqueline Chevalier, Asmaa Saad, Nour-Eddine Mezrioui, Lahcen Hassani,
Jean-Marie Pagès
UMR-MD1: Transporteurs Membranaires, Chimioresistance et Drug Design, Université de la Méditerranée,
Facultés de Pharmacie et de Médicine, 27 bd Jean Moulin, 13385 Marseille cedex 05, France.
Laboratory of Biology and Biotechnology of Microorganisms Faculty of Science, University Cadi Ayyad,
Marrakech, Morocco.
Program Averroes- Erasmus Mundus
E-mail: [email protected]
Bacterial drug resistance is a worrying problem of public health. Antibiotic efflux is the major
non-specific resistance mechanism used by bacteria and efflux pumps are involved in the
low level susceptibility of several important Gram-negative pathogens. The use of
molecules that can block these pumps is an attractive strategy, but many researches have
shown only a partial efficacy of these molecules due to a lot of limits that still remain
(stability, selectivity, bioavailability, toxicity...).
Our objective is to search natural sources of molecules able to inhibit efflux pump systems
of resistant Gram-negative bacteria (Escherichia coli, Enterobacter aerogenes, Klebsiella
pneumoniae, Salmonella enterica Typhimurium and Pseudomonas aeruginosa). The results
indicate that the studied essential oils exhibit an interesting activity against the tested
bacteria. This activity is significantly enhanced in the presence of efflux pump inhibitor such
as phenylalanine arginyl β-naphthylamide (PAβN). The role of lipopolysaccharide (LPS)
structure in the permeation of essential oils was also reported in Salmonella LPS deep
rough mutants. In addition, essential oils of Thymus maroccanus and Thymus broussonetii,
used at a low concentration (a fraction of the Mimimum Inhibitory Concentration), are able
to significantly increase chloramphenicol susceptibility of several resistant isolates. These
results demonstrate that these essential oils can block efflux pumps, and may be good
candidates to develop new drugs for chemosensitizing multidrug resistant (MDR) strains to
clinically used antibiotics.
Keywords: Antibiotics; Antibiotic resistance; Chemosensitizers; Efflux pumps inhibitors;
Efflux systems; Essential oils; Gram-negative bacteria; Multidrug resistance;
QUINAZOLINONE-BASED CHEMOSENSITISERS TO COMBAT
BACTERIAL MULTIDRUG RESISTANCE
Joanna Ombouma, Sandrine Alibert, Jacqueline Chevalier, Abdallah Mahamoud, Aurélie
Lieutaud, Jean-Michel Bolla, Jean-Marie Pagès
UMR-MD1: Transporteurs Membranaires, Chimioresistance et Drug Design, Université de la
Méditerranée, Facultés de Pharmacie et de Médicine, 27 bd Jean Moulin, 13385 Marseille cedex 05,
France.
E-mail: [email protected]
In Gram-negative bacteria, antibiotic resistance mechanisms are a worldwide
health problem. The continuing spread of multi-drug resistant (MDR) bacteria drastically
reduces the efficacy of antibiotic families and consequently increases the frequency of
therapeutic failure. Because of the clear involvement of the resistance–nodulation–cell
division (RND) transporters in the increased frequency of MDR clinical bacteria, efflux
pumps are now considered as an attractive target for the development of a
combinational therapy using antibiotic/efflux pump inhibitor (EPI) as adjuvant of usual
antibiotic.1
Using a screening assay that measures the susceptibility of antibiotic resistant
Enterobacter aerogenes strains, quinazolinone derivatives exhibiting some structural
similarities to the quinolone family have been shown to restore the activity of diverse
antibiotics that are expelled by bacterial efflux pumps.2
These compounds are not substrate of efflux pumps. Among the identified
molecules, some have a synergistic activity with antibiotic in a non-competitive manner.
Moreover, their chemosensitising activity depends on the nature of the antibiotic used.
So further pharmacomodulations are considered to improve the effect against bacterial
efflux pumps according to the synthetic pathway described in scheme 1.
Scheme 1:
O
R1
OH
NH2
R3COCl
dry pyridine
60°C / 3h
O
O
R1
N
benzoxazinone
1
O
1) R2NH2 / CHCl3
reflux / 8h
R3
2) ZnCl2 / ethylene glycol
155°C / 8h
N
R1
N
R2
R3
quinazolin-4(3H)-one
J.-M. Pagès, S. Alibert-Franco, A. Mahamoud, J.-M. Bolla, A. Davin-Regli, J. Chevalier, E. Garnotel, Curr. Top.
Med. Chem. 10 (2010) 1848-1857.
2
J. Chevalier, A. Mahamoud, M. Baitiche, E. Adam, M. Viveiros, A. Smarandache, A. militaru, M. L. Pascu, L.
Amaral, J.-M. Pagès, Int. J. Antimicrob. Agents 36 (2010), 164-168.
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Etude ethnopharmacologique de plantes antipaludiques utilisées en médecine traditionnelle
cambodgienne
Bory S1, Bun SS1, Baghdikian B1, Chea A1, Azas N2, Mahiou-Leddet V1, Elias R1, Ollivier E1
1 Laboratoire de Pharmacognosie-Ethnopharmacologie, UMR-MD3, Faculté de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille cedex 5,
France
2 Laboratoire de Parasitologie, UMR-MD3, Faculté de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France
Le développement extrêmement rapide du phénomène de résistance aux antipaludiques, même aux plus récents,
impose une recherche permanente de nouvelles molécules. L’objectif de nos travaux est d’identifier de nouvelles
substances antiparasitaires issues de la biodiversité végétale cambodgienne en appliquant une démarche
ethnopharmacologique.
Quatre enquêtes ethnobotaniques ont été conduites dans neuf provinces au Cambodge afin de répertorier les
plantes utilisées en médecine traditionnelle pour leurs propriétés fébrifuge et antipaludique. Vingt-huit plantes
réputées actives contre les fièvres et le paludisme ont été sélectionnées [1].
Parmi elles, Stephania rotunda a fait l’objet des travaux phytochimiques et pharmacologiques les plus importants
[2,3]. Ces travaux ont permis d’isoler seize alcaloïdes [4]. Au niveau pharmacologique, les extraits du tubercule de S.
rotunda exercent in vitro une activité très élevée sur la souche chloroquino-résistante de Plasmodium falciparum
W2. Ces résultats encourageants ont motivé le fractionnement bioguidé des extraits de cette plante. Ainsi, les 4
alcaloïdes majoritaires de S. rotunda (tetrahydropalmatine, xylopinine, déhydroroemérine et cépharanthine) ont fait
l’objet d’une étude de l’activité antiplasmodiale in vitro et chez des souris infestées par P. berghei [2]. La
cépharanthine et la déhydroroemérine présentent la meilleure activité antiplasmodiale sur la souche chloroquinorésistante W2 avec des CI50 égales à 0.61 et 0.36 µM respectivement. Des travaux analytiques ont ensuite permis
de déterminer les teneurs en cépharanthine, tétrahydropalmatine et xylopinine dans des tubercules de S. rotunda
récoltés dans différentes provinces cambodgiennes [5].
Références
[1] Hout S et al. (2006) Screening of selected indigenous plants of Cambodia for antiplasmodial activity. J Ethnopharmacol, 107, 12-18.
[2] Chea A et al. (2007) Antimalarial activity of alkaloids isolated from Stephania rotunda. J Ethnopharmacol, 112, 132-137.
[3] Chea A et al. (2010) Antiplasmodial activity of three bisbenzylisoquinoline alkaloids from the tuber of Stephania rotunda. Nat Prod Res,
sous presse.
[4] Chea A (2006) Doctorat de l’Université de la Méditerranée Aix-Marseille II.
[5] Bory S et al. (2010) Simultaneous HPLC determination of three bioactive alkaloids in the Asian medicinal plant Stephania rotunda. Nat Prod
Commun, sous presse.
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Role of the PTK7 receptor in normal and malignant haematopoiesis
A.C. Lhoumeau, M.-L. Arcangeli, T. Prébet, C. Arnoulet, S. Marchetto , Orsoni J.-C., F. Bardin,
M. Aurrand-Lions, and J.-P. Borg
Centre de Recherche en Cancérologie de Marseille, Inserm UMR891. Institut Paoli-Calmettes.
Université de la Méditerranée.
27 Bd Lei Roure, 13009 Marseille, France.
The pseudo tyrosine kinase receptor 7 (PTK7) is a new planar cell polarity receptor with poorly
described functions. PTK7 is an orphean tyrosine kinase receptor with a kinase-dead domain. Deletion
of the mouse ptk7 gene leads to early lethality due to a severe neural tube defect (craniorachischisis).
Initially described as an epithelial receptor, PTK7 is also expressed in human haematopoietic cells, and
particularly in myeloid progenitors. Screening leukemia cells from various origins, we found strong
expression of PTK7 at the surface of myeloid blasts. Expression of PTK7 is correlated with a poor
prognosis as patients with PTK7-positive acute myeloid leukemia (AML) are more resistant to
anthracycline-based frontline therapy with a significantly reduced relapse free survival in a multivariate
analysis model (Prebet , Lhoumeau et al, Blood,2010).
Similarly to its human homologue, murine PTK7 is expressed at the surface of hematopoietic
stem cells, myeloid and lymphoid T progenitors. Expression is lost in mature hematopoietic cells. We
show that the frequency of Lin-Sca1hic-Kithi (LSK) progenitors is decreased in the fetal liver of ptk7deficient embryos, suggesting a role of PTK7 in early hematopoiesis. We are currently testing the
reconstitution capacity of ptk7-deficient stem cells by transplantating fetal hematopoietic stem cells of
these animals in lethally irradiated mice. These data highlight a poorly described role of a planar cell
polarity protein in normal and malignant hematopoiesis. This project will contribute to a better
understanding of the functions of PTK7 in these processes. Furthermore, despite lack of catalytic
activity, PTK7 may represent a new therapeutic option in acute leukemia treatment.
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8e Journée de la Recherche – Mercredi 23 Mars 2011 – Faculté de Pharmacie de Marseille
From rare disease to anti-aging targets
Bénédicte CANTECOR1, Philippe PICCERELLE1, Marie-Pierre SAVELLI1, Vincent
BONNIOL2.
1
Laboratoire de Pharmacie Galénique
2
UMR INSERM U910
A rare disease affects a restricted number of people. In Europe a rare disease
is defined as affecting 1 over 2000 person. Progeria also known as HutchinsonGilford Syndrom is one of the 7000 rare diseases identified. This pathology
affects 1 over 8 million births. Progeria children suffer from an accelerated
aging, 10 times faster compared to a healthy person and they present a life
expectancy that exceeds rarely 13 years. Symptoms for Progeria are skin aging,
hair loss and diseases related to aging such as joint stiffness or cardiovascular
disorders. Those children mostly die of a myocardial infarction or stroke.
Progeria pathophysiological mechanism is related to the persistence of a
prenylated nuclear protein (lamin A), a phenomenon also observed during
physiological aging in healthy subjects. Therefore Progeria represents an
excellent model for studying aging.
A therapeutic treatment based on the combination of two drugs was found. This
patented association decreases accumulation and/or persistence into the cells of
the “toxic” prenylated nuclear proteins that are responsible for the cellular
aging. Since October 2008, 15 Progeria children received this drug combination
in the Progeria European Therapeutic trial.
This remarkable scientific discovery is the starting point of a research on the
effects of this active combination on the physiological skin aging.
First, we have studied the transdermal diffusion of the drugs to determine
their bioavailability and then we have developed a cosmetic formula able to carry
the active synergy through skin route.
A clinical test has been carried out on volunteer panel to show the anti-aging
effects of the cosmetic formula on some cutaneous targets. The results had
proved the anti-aging efficiency of the product.
7 mars 2011
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Untitled - Faculté de Pharmacie - Aix

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