ÈME JOURNÉE RECHERCHE PHARMACIE 24 octobre 2019 · Heidi Kidron initially studied biochemistry,...

PROGRAMME & BROCHURE

17ÈME JOURNÉE DE LA RECHERCHE EN PHARMACIE

24 octobre 2019

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LE MOT DU VICE-DOYEN RECHERCHE

La Faculté de Pharmacie s’appuie sur 9 unités de recherche (>700 personnes) labellisées par Aix-Marseille Université, l’INSERM, le CNRS, l’Institut Paoli-Calmettes, l’IRD, l’INRA et le Ministère de la Défense, et de nombreuses plateformes technologiques et cliniques accueillant étudiants et personnels de recherche en formation. Notre recherche est naturellement tournée vers l’innovation thérapeutique et diagnostique.

Les thématiques couvrent les domaines suivants : les pathologies vasculaires, la cancérologie, les maladies infectieuses, les neurosciences, la recherche en chimie, mais aussi des recherches très interdisciplinaires se focalisant sur les nuisances environnementales et l’ingénierie galénique, une des étape-clés de la conception finale d’un médicament. L’ensemble de ces recherches repose sur une forte interface entre santé et sciences avec une dimension translationnelle et clinique en lien avec les services cliniques, biologiques et pharmaceutique de l’Assistance Publique des Hôpitaux de Marseille et du centre de lutte contre le cancer de Marseille (Institut Paoli Calmettes).

Une spécificité de la recherche au sein de la faculté est de réussir l’intégration d’approches très interdisciplinaires autour du médicament et du diagnostic, de l’identification de nouveaux mécanismes moléculaires à l’évaluation préclinique, en incluant des spécialités incontournables au développement d’un médicament comme la chimie, la pharmacie galénique et la pharmacocinétique. Pour relever le défi de la recherche translationnelle au lit du patient, la faculté de pharmacie a un rôle moteur dans d’importants programmes de recherche largement soutenus par A*MIDEX, par des financements académiques nationaux, européens et par des partenariats de valorisation avec l’industrie pharmaceutique et du diagnostic.

Cette recherche s’appuie par ailleurs sur des plateformes technologiques de dernière génération (imagerie médicale-CERIMED, protéomique-IBiSA, pharmacocinétique, imagerie intravitale, cytométrie de flux et tri cellulaire) et des plateformes cliniques permettant d’amener les résultats de l’innovation au lit du patient (Centre d’Investigation Clinique, biothérapies, biobanques).

La Journée de la Recherche a pour objectifs de sensibiliser les étudiants à la recherche et de renforcer la communication et les synergies scientifiques autour des axes forts de la faculté. Cette édition 2019 est la 17ème édition de cet événement majeur de notre Faculté.

Jean-Paul BORG Vice-Doyen Recherche Faculté de Pharmacie Aix-Marseille Université

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PROGRAMME DE LA 17ÈME ÉDITION

14h00 – 14h30 (Amphi Pisano) – Jean-Paul Borg Introduction de la Journée de la Recherche Présentation générale de la Recherche à la Faculté (axes/thématiques, bilan de la production

scientifique récente) Présentation des Ecoles Doctorales et Masters affiliés à la Faculté

14h30 – 15h00 (Amphi Pisano) La Recherche en Pharmacie à l'International Avec Lasha MSKHILADZE , Professeur Associé à la Tbilisi State Medical University (Géorgie) , Heidi KIDRON, chercheur à la Faculté de Pharmacie de l’Université d’Helsinki (Finlande) et Jadwiga HANDZLIK, professeur à la Faculté de Pharmacie de la Jagiellonian University (Pologne).

15h00 – 15h50 (Amphi Pisano) Ma Recherche en 300 secondes 1 représentant de chaque équipe de recherche de Pharmacie présente en 5 minutes et 5 diapos le travail de son équipe en recherche

C2VN CRCM (Jean-Paul BORG) ICR (Nicolas PRIMAS) IMBE (Sok-Siya BUN, Pierre-Henri VILLARD) INP (Hervé KOVACIC) MCT (Jean-Michel BOLLA) MEPHI (Jean-Marc ROLAIN) MMG (Camille DESGROUAS) VITROME (Nadine AZAS, Aurélien DUMETRE)

15h50-16h30 (Amphi Pisano) Comment je suis tombé sur la thèsePrésentation en 5 minutes et 5 diapos de 4 doctorants en Pharmacie sur leur parcours de recherche. Comment ils en sont arrivés là ? Suivi de questions et échanges avec l’audience. Avec Laetitia GANIER (CRCM), Camille DESGROUAS (MMG), Johan REVOL-TISSOT(MCT) et Raphael COATMEUR (IMBE).

16h30-18h00 (Grand Hall) Discussion devant les posters autour d’une collation

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HEIDI KIDRON, HELSINKI

Heidi Kidron initially studied biochemistry, and received a PhD in 2006 on work that focused on protein homology modeling. In 2009, she started with a post-doctoral research project at the Faculty of Pharmacy in the University of Helsinki, to work on drug transporters and in silico models of drug permeation over barriers in the human body. In 2012, she was awarded an Academy Research Fellowship and during the five-year fellowship period set up an independent research group focused on drug transporters. Currently, she is employed as a University researcher at the Division of pharmaceutical biosciences at the Faculty of Pharmacy in the University of Helsinki.

Heidi KIDRON Chercheur Faculté de Pharmacie Université d’Helsinki Finlande

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JADWIGA HANDZLIK, CRACOVIE

Professor Jadwiga Handzlik currently heads the Department of Technology and Biotechnology of Drugs at the Faculty of Pharmacy in Krakow. After a PhD in medicinal chemistry from the Jagiellonian University of Krakow in 2006, she became the same year Assistant Professor in the Department of Technology and Biotechnology of Drugs. After habilitation in 2013, she became Professor of the Jagiellonian University. Her expertise focuses on medicinal chemistry areas, especially the direct or indirect computer-aided drug design, chemical synthesis for drug discovery and drugability prediction. Her topic research interest focuses on modulation of multidrug resistance mechanisms in bacteria and cancer cells, GPCRs antagonist ligands (adrenergic-, serotonin-, adenosine-, histamine- and dopamine receptors), Antiarhythmic hERG K+ inhibitory action. His research work has been valued by 70 publications and 2 patents. She is member of Polish Medicinal Chemistry Society belonging to European Federation of Medicinal Chemistry and peer review referee in the field of medicinal chemistry, organic chemistry and natural products. She obtained several International and national awards for her scientific activities: in particular Team Award of the Minister of Health for a series of publications on the study of histamine H3 receptor in 2012 and Gold Medal for the project entitled “New compounds increasing the efficiency of antibiotics and anticancer drugs”, of the 8th International Invention and Innovation show INTARG 2015.

Jadwiga HANDZLIK Professeur Faculté de Pharmacie Jagiellonian University-Pologne

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LASHA MSKHILADZE , TBILISI

Lasha Mskhiladze , Docteur en Pharmacie , est professeur au département de Pharmacognosie et Botanique à la Tbilisi State Medical University (TMSU) . Il est également directeur de l'Institut de Pharmacochimie Iovel Kutateladze,, chef de laboratoire de recherche scientifique et compétences pratiques à la TMSU ainsi qu’expert du Bureau National Médecine-Légale de Géorgie.

Son projet de recherche se centre sur l’étude phytochimique et biologique des espèces endémiques de Géorgie, la préparation et normalisation des plantes médicinales de Géorgie et les Anticancéreuses, Antiplasmodiques et Antitrypanosomiennes activités des espèces endémiques de Géorgie.

Lasha MSKHILADZE Professeur Associé Tbilisi State Medical University Géorgie

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C2VN

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Design and validation of a 68Ga‐radiolabelled PET imaging agent for in vivo evaluation of APJ expression

This study aimed at developing a 68Ga-radiolabelled PET imaging agent for assessing tissular APJ receptor expression. APJ has recently been reported to be overexpressed in various cancer types and especially during angiogenesis induction. To date, several APJ-targeted therapeutic strategies are explored. In this work, we designed AP747 as a potent specific ligand of APJ for PET/CT imaging of its expression in oncological applications.

Materials and Methods : (68Ga)Gallium chloride solution was obtained from fractionated elutions of a Galliapharm generator (Eckert&Ziegler). AP747 was designed, produced, and radiolabeling was set up (pH 5, 5min at room temperature). Radiochemical stability in NacL 0,9% and human serum was validated up to 2h after synthesis by radiochromatography on iTLC-SG in sodium citrate 0,1M pH5 (n=3). In vitro characterization of (68Ga)Ga-AP747 specificity towards APJ was performed using a 50-fold excess of APJ ligand. Quantification of radioactivity was realized on background-corrected autoradiographs (n=3, Cyclone Perkin-Elmer).

In vivo hind-limb ischemia model was realized on 6-week-old Swiss mice by microsurgery. Matrigels (Dutscher ®) were subcutaneously implanted (with 10% FCS) to these mice as angiogenesis in vivo model. Ischemic reperfusion was followed by LASER-Doppler the same days of (68Ga)Ga-AP747 IV injections (6.185±0.6MBq) and of (68Ga)Ga-RGD2(6,53±1,4MBq), used as a reference. μPET/CT acquisitions were performed 1h after radiotracer injection on a NanoScan PET/CT (Mediso) for following these two kinds of angiogenesis models. Image analysis was performed using VivoQuant ® software (InviCRO). Regions of interest were drawn over ischemic and non-ischemic limb and on Matrigel also.

4-month-old Swiss-nude mice were subcuteanously xenographted with 106 T84 cells (n=6) andrested for 4 weeks. 5.1+/- 1.1 MBq/ 50 μL of (68Ga)Ga-AP747 were IV injected (n=6), μPET/CTacquisitions performed 1h after radiotracer injection on a NanoScan PET/CT (Mediso). 24hlater, in vivo blocking was realized by injecting 50 μg of non-radioactive APJ ligand 30 minbefore injecting (68Ga)Ga-AP747 (n=6). Image analysis was performed using VivoQuant ®software (InviCRO). Regions of interest were drawn over tumor and muscle for each animalquantified and corrected by the tissue volume (MBq/mm3).

Experiments were performed according to an Ethics Committee agreement by trained and qualified operators.

Results : (68Ga)Ga-AP747 was produced with radiochemical purity of 96.7% +/- 2.0 and stability in serum evaluated at 2h (95.0+/- 1.5%). Autoradiographs showed APJ selective binding of (68Ga)Ga-AP747 on T84 cells (1,396±0,3833) reversed by blocking (0,4975±0,2296 p=0,0003). Dynamic PET imaging showed favorable pharmacokinetic profiles with fast renal clearance and low background in liver, lungs and digestive system.

μPET/CT imaging of angiogenesis models showed a significantly higher (68Ga)Ga-AP747 accumulation in Matrigel than RGD2 on day 10 (p=0.0362), day 13 (p=0.0064) and day 21 (p=0.0016). (68Ga)Ga-AP747 accumulation in hind-limb ischemia that peak at Day 7 is better regarding level of fixation (P=0.066) and this fixation differs from those of RGD2 whose peak at Day 10

μPET/CT imaging of mice ectopic colon adenocarcinoma xenografts showed a high (68Ga)Ga-AP747 tumor-to-muscle ratio (793,3± 217,2) reversed after blocking in the same animals (217,2±31,37, *P=0,0480, n=3).

Conclusion : This is proof of concept shows (68Ga)Ga-AP747 might be a useful candidate for imaging APJ expression in angiogenesis models as in tumors. (68Ga)Ga-AP747 showed a specific and strong uptake in hind-limb ischemia, a complex model of angiogenesis and Matrigel an easier one but also in colon adenocarcinoma tumor model in vitro and in vivo, with a low background in healthy organs. Upcoming studies will focus on the interest of this tracer for theranostic purposes.

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Targeting Soluble CD146 in Glioblastoma: A New Step for Personalized Therapy.

Ahmad Joshkon1, Wael Traboulsi1, Jimmy Stalin1, Richard Bachelier1, Alexandrine Bertaud1, Aurelie

Leroyer1, Francoise Dignat-George1, Nathalie Bardin1, and Marcel Blot-Chabaud1

1 INSERM UMR-1263, INRA UMR-1260, Aix-Marseille University, C2VN, UFR Pharmacy, Marseille,

France

Background: Glioblastoma is a highly aggressive and malignant grade IV tumor that originates

from astrocytes in the CNS. Despite the multifaceted therapeutic interventions, including chemo,

radio, and immuno-therapies, it remains the most resistive and lethal tumoramong primary brain

tumors. Currently, Avastin an “anti-VEGF mab” is the most common therapy prescribed for

newly diagnosed patients. However, glioma tumor cells have developed manifold mechanisms to

counteract the anti-angiogenic effect of this drug. Previous studies from our group suggested that

CD146 (MCAM) could participate to these escape mechanisms.

Methods: U87 and U373 cells were grown in serum reduced DMEM for 48hrs. Soluble CD146

concentration in the media was measured by ELISA. Recombinant human sCD146 was used at a

final concentration of 100ng/ml in all experiments. Cell Proliferation was assessed by WST-1

reagent. Mice were ectopically injected with U87 and tumor volume was monitored by calliper.

Results: Under serum starving condition,glioma cell lines U87 and U373shed the membrane

form of MCAM (CD146) to produce soluble CD146, which directly induce their proliferation,

migration, invasion, and metastasis in-vitro, and indirectly provide an alternative way for

vascularization in response to anti-angiogenic therapies. We have also shown that the use of our

patent fully humanized anti-sCD146 (H2L3) was able to reverse these effects both in-vitro and

in-vivo in nude mice xenografted with U87.

Conclusion: For the first time, we have demonstrated that sCD146,which plays a major role in

resistant to current chemotherapies, is secreted by Glioma U87 and U373 cell lines. Targeting

sCD146 with H2L3 not only protects from the aforementioned effects, but also shows an

additive therapeutic effect with Avastin, thus constituting a key breakthrough in Glioblastoma

therapy.

Journée de la Recherche 2019 – Faculté de Pharmacie, Aix-Marseille Université

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Le décollement endothélial au cours du purpura thrombotique thrombocytopénique : implication de la VE-Cadhérine et des métalloprotéinases matricielles

Cauchois R, Tellier E, Faccini J, Desseaux A, Arnaud L, Poullin P, Leroyer A, Dignat-George F, Kaplanski G.

Introduction

Le purpura thrombotique thrombocytopénique auto-immun (PTT-a) est une microangiopathie thrombotique sévère liée à un déficit en ADAMTS-13. L’agression endothéliale joue également un rôle majeur dans la physiopathologie et entraîne un décollement cellulaire entraînant la détection de cellules endothéliales circulantes (CEC) plasmatiques.

Hypothèse

Nous avons étudié les mécanismes du décollement endothélial au cours du PTT-a, en faisant l’hypothèse selon nos précédents travaux, que le décollement était la conséquence d’une activation endothéliale calcium-dépendante et qu’il impliquait un relâchement des jonctions intercellulaires via des modifications de la VE-Cadhérine et une perte de l’interaction avec la matrice sous-endothéliale faisant intervenir MMP-2, la principale métalloprotéinase matricielle endothéliale.

Résultats

Chez 39 patients, nous avons confirmé que le taux des CEC est associé à la sévérité clinique des crises de PTT-a et corrélé à l’intensité du flux calcique endothélial induit par les mêmes plasmas in vitro. De plus, nous avons reproduit in vitro, le décollement cellulaire endothélial en présence des plasmas de PTT-a. En accord avec ces résultats, nous avons observé que les plasmas PTT-a induisent la phosphorylation et l’internalisation de la VE-Cadhérine in vitro de façon calcium-dépendante et augmentent la perméabilité vasculaire dans un modèle murin. De plus, les plasmas PTT-a induisent (lors d’une stimulation plus longue) une majoration de l’activité de MMP-2 dépendante d’une modulation transcriptionnelle de ses régulateurs MT1-MMP et TIMP2. Enfin, l’effet des plasmas PTT-a sur la VE-Cadhérine et sur MMP-2 est reproduit par la fraction IgG des plasmas.

Conclusion

Nos résultats suggèrent que les IgG des patients PTT-a induisent l’internalisation calcium-dépendante de la VE-Cadhérine (activation endothéliale de type 1) et une majoration de l’activité de MMP-2 d’origine transcriptionnelle (activation endothéliale de type 2), favorisant un relâchement des interactions intercellulaires et matricielles sous-endothéliales, conduisant au décollement cellulaire endothélial.


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Immuno-Magnetic Separation to extract microvesicle subsets for measurement of TF-dependent procoagulant activity.

Franco C1,2, Judicone C1, Roca E4, Abdili E2, Astoul P4, Dignat-George F2,3, Lacroix R2,3, Poncelet P1.

1 : Research and Technology Department, Biocytex, Marseille, France 2 : Aix-Marseille University, INSERM, INRA, C2VN, Marseille, France 3 : Department of Hematology and Vascular Biology, CHU La Conception, APHM, Marseille, France 4 : Department of Thoracic Oncology, Pleural Diseases and Interventional Pulmonology, Hôpital Nord, Aix-Marseille University, Marseille, France Background :

Pleural effusions arise around lungs in either inflammatory or cancerous pathological conditions and are removed for diagnostic and therapeutic purposes. They contain cells and cell-derived microvesicles (MVs) from a wide diversity of cell origins, mainly leucocytic, endothelial and tumoral. MVs distribution is very different from that found in blood samples, and in cases of carciSnomas, EpCAM+ tumor-MVs can be well represented (Roca et al Oncotarget 2016). Evaluation of the tissue factor-dependent procoagulant activity (TF-PCA) of MVs from these fluids may be informative on the tumor’s propensity to generate thrombosis in tissues and/or peripheral blood in case of metastasis. A new hybrid bio-assay involving immuno-magnetic separation (IMS) of MVs is here tested on such samples to measure TF-PCA on either total MVs or selected MV subsets and define which ones bear the activity.

Material & Methods

Our MV-TF Activity assay is a new version of FXa generation assay that uses FVII rather than FVIIa and the anti-TF Mab SBTF1 for higher specificity and sensitivity. Its measuring range [10fM to 1.5pM rec-TF] and linearity with pre-test dilution allows dosing up-to 200pM TF. Biotin-labelled MAbs are coated on 1μm streptavidin magnetic beads for easy manual (and possibly automatizable) Ag-specific MV recovery. MVs bound to IMS beads are tested into the FXa generation assay in comparison with the overall load of MVs pelleted from the same fluid volume by high-speed centrifugation (24,000g 1h) that is used as a 100% control of available TF-PCA. In parallel, distribution of MV subsets is qualified by high-sensitivity flow cytometry (4 laser CytoFLEX), standardized for 75 nm-eq. threshold with calibrated beads (Gigamix-Plus, BioCytex).

Results

Pleural fluids contain very high MV-TF PCA levels (up-to 200pM TF vs < 20fM in normal blood). Targeting the widely distributed Ags CD29 and CD59 together with bispecific beads provides PCA levels always = or > to those for total MVs obtained by centrifugation, illustrating efficient extraction and better reproducibility. High levels are also gained using anti-EpCAM IMS beads, in line with high representation of tumor-MVs. Specific extraction reveals MVs-TF activity of CD146, HLA DR and CD326 subpopulation. Flow cytometry images after lineage specific extraction can’t distinguish the coexpression case of Ags on tumor MVs or the presence of aggregate between CD146, HLA DR and CD326.

Conclusion

IMS allows efficient global or Ag-specific extraction of MVs that can be measured into a TF- PCA bio-assay. This automatizable technology targeting CD29+59 provides TF-PCA levels superior to the extraction by centrifugation, suggesting higher overall recovery. Moreover, the selectivity of IMS may help clarify the cellular origin of TF-bearing MVs in biological fluids. However, expression of multiple Ags due to MV aggregates or Ag transfer complexifies the interpretation of data from theoretically lineage specific extractions.

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CRCM

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Abstract

Precision medicine in hematology-oncology: CDA as a predictive marker for Cytarabine exposure in AML patients

M. Donnettea, G. Ventonb, L. Farnaultb, R. Costellob,, B. Lacarellea,, J Ciccolinia LH Ouafikc, and R. Fanciullinoa

a SMARTc- CRCM UMR Inserm 1068 AMU.

b Hematology Unit Assistance Publique des Hôpitaux de Marseille

cTransfert Oncology Unit, Assistance Publique des Hôpitaux de Marseille

E-mail of corresponding author: [email protected]

Objectives: Cytarabine (Ara-C) remains the backbone of the vast majority of protocols for treatment of acute myeloid leukemia (AML). We have previously demonstrated that clinical outcome with cytarabine was markedly influenced by genetic polymorphisms affecting CDA, the enzyme responsible for its detoxification to Ara-U in the liver. Interestingly, AML patients with CDA PM phenotype exhibited both high risk of severe/lethal toxicities upon cytarabine treatment, but a trend towards longer progression-free and overall survival as well. We can hypothesize that PM patients had probably higher circulating cytarabine plasma levels and lower circulating plasma levels of inactive Ara-U. To confirm this, we have monitoring drug and main metabolite level. The monitoring cytarabine concentrations in plasma required highly sensitive bioanalytical methods especially during induction phase with low dose cytarabine. We have developed a new LC-MS/MS method that meets these requirements.

Methods: Ara-C and Ara-U concentrations were determined in plasma samples using a new LC-MS/MS method. Blood samples were withdrawn from 7 patients treated for AML with 200mg/m2 Ara-C at as part of a study approved by the institutional review board of the Conception Hospital (Marseille, France) registered as # 2017-A00070-53. Patients were phenotyped for CDA status prior to starting the infusion following a spectrophotometric method previously described and categorized as Poor Metabolizer (PM) or Extensive Metabolizer following CDA activity. Patients were sampled at the end of the administration, then 5 min, 10 min, 1H, 2H and 6H after the end of the infusion.

Results: 4 patients were CDA deficient (i.e., CDA ≤ 2 U/mg, aka PM ), and 3 patients were CDA no-deficient (i.e., CDA > 2 U/mg, aka EM). For Ara-C, AUCs were 3312 ± 326 ng/ml.min and 1502 ± 497 ng/ml.min, for PM and EM patients, respectively. The difference was statistically different (p>0.024, t test). For Ara-U, AUCs were 7.3.105 ± 2.1.105 ng/ml.min and 4.8.105 ± 0.8.105 ng/ml.min, for PM and EM patients, respectively. The difference was not statistically different (p>0.05, t test). Metabolization ratio between AUC of Ara-C and AUC of its metabolite Ara-U was calculated. The mean metabolization ratio was 255 ± 103 and 460 ± 218 for PM and EM patients, respectively. The 1.8-fold difference was not statistically different (p>0.05, t test).

Conclusions: This method was successfully applied to determine the pharmacokinetic profile of Ara-C and Ara-U in 7 patients and evidenced marked differences in both drug levels and metabolic ratio depending on patient’s CDA status. CDA status could be further used as a covariate to tailor durg dosage so as to ensure an optimal efficacy/toxicity balance in patients with AML. Evaluation of Ara-C pharmacokinetics as part of a prospective clinical trial is currently ongoing

Relevant references:


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CDA as a predictive marker for life-threatening toxicities in patients with AML treated with cytarabine. Fanciullino et al. 2018

Cytidine deaminase residual activity in serum is a predictive marker of early severe toxicities in adults after gemcitabine-based chemotherapies, Ciccolini et al. 2010

Simultaneous determination of cytosine arabinoside and its metabolite uracil arabinoside in human plasma by LC-MS/MS: Application to pharmacokinetics-pharmacogenetics pilot study in AML patients, Donnette et al. 2019

KEYWORDS: Cytarabine, Cytidine deaminase (CDA), pharmacokinetic, LC-MS/MS, Acute Myeloid Leukemia


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TARGETING OF THE PTK7 TYROSINE KINASE RECEPTOR IN BREAST CANCER Authors: Laetitia Ganier (1), Stéphane Betzi (1), Carine Derviaux (1), Christophe Muller (1), Philippe

Roche (1), Xavier Morelli (1), Jean-Paul Borg (1)

(1) Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, CNRS, Institut Paoli-Calmettes, Marseille, F-13009, France, Aix-Marseille University, Marseille, France

Breast cancer is a major public health issue and impact 1 for 9 women during their life. Breast cancer is a heterogeneous disease and four main subtypes can be described associated to different clinical behaviours. Among those, basal breast cancer is a very aggressive subtype with poor prognosis outcome due to lack of targeted therapies (1). Deregulation of cell polarity is a hallmark of cancer in solid tumors that participates to tumor development. The alteration of tissue organization and its homeostasis results in perturbation of signalling pathways. In breast cancer, cancer cells frequently reactivate developmental pathways including the Wnt/Planar cell polarity (PCP) signalling pathway which plays an important role during tumorigenesis. PTK7 is a membrane receptor belonging to the tyrosine kinase receptor superfamily and is involved in Wnt/PCP signalling regulation (2,3). However, PTK7 is described as a pseudokinase due to a lack of kinase activity. PTK7 overexpression was found associated to poor prognosis and has been also involved in resistance to chemotherapy in basal breast cancer (4) rendering PTK7 a promising new therapeutic target. This project aims to offer an alternative to therapeutic monoclonal antibodies against PTK7 which have already moved into clinical development (5) and is based on the discovery of chemical compounds specific to the PTK7 kinase domain. Because of the lack of enzymatic activity, we decided to eliminate PTK7 expression in cancer cells with compounds using the Proteolysis-Targeting Chimera (PROTAC) technology which induces the degradation of the target. In order to identify chemical compounds that interact specifically with PTK7, we have developed different screening strategies. Several chemical libraries have been screened in vitro using Thermal Shift Assay as a first approach. In parallel, we also developed a second strategy in cellulo based on the NanoBRET technology. Selected ‘hit’ compounds will be validated and optimized by medicinal chemistry. In this first objective, we will also evaluate chemical derivatives specific for E3 ubiquitin ligases. The second objective will be to further assess the efficacy of the identified compounds in vitro and in vivo using basal breast cancer cell models.

Reference(s) 1. Bertucci F, Finetti P, Birnbaum D. Basal breast cancer: a complex and deadly molecular subtype. Curr Mol

Med. janv 2012;12(1):96‑110.

2. Martinez S, Scerbo P, Giordano M, Daulat AM, Lhoumeau A-C, Thomé V, et al. The PTK7 and ROR2 ProteinReceptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway. J Biol Chem. 18 déc2015;290(51):30562‑72.

3. Lhoumeau A-C, Puppo F, Prébet T, Kodjabachian L, Borg J-P. PTK7: a cell polarity receptor with multiplefacets. Cell Cycle Georget Tex. 15 avr 2011;10(8):1233‑6.

4. Ataseven B, Angerer R, Kates R, Gunesch A, Knyazev P, Högel B, et al. PTK7 expression in triple-negativebreast cancer. Anticancer Res. sept 2013;33(9):3759‑63.

5. Damelin M, Bankovich A, Bernstein J, Lucas J, Chen L, Williams S, et al. A PTK7-targeted antibody-drugconjugate reduces tumor-initiating cells and induces sustained tumor regressions. Sci Transl Med. 11 janv2017;9(372).


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Repositioning beta-blockers as an anticancer agents for the treatment of medulloblastoma: new hope for children with high-risk brain tumors?

M. Rossi1, J. Talbot2, M.P. Montero1, P. Piris1, D. Buric1, O. Ayrault2, M. Carré1 and N. André1,3

1Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm UMR1068, CNRS UMR7258, Aix-Marseille Université UM105, Institut Paoli Calmettes – Marseille, France 2Institut Curie, Inserm U1021, Université Paris-Saclay UMR 3347 – Orsay, France 3AP-HM, Service d’hématologie et d'oncologie pédiatrique de la Timone Enfants – Marseille, France

E-mails: [email protected] & [email protected]

Medulloblastoma (MB) is the most frequent brain malignancy of the childhood. Their current multimodal treatment – surgery, radiotherapy and chemotherapy – allows 60% of children to survive up to 10 years. However, this comes at the expense of serious and often long-lasting side effects. Moreover, alternative treatment options are still urgently needed for MB with a very poor prognosis, i.e. for TP53-mutated SHH and c-myc-amplified group 3 patients. Drug repositioning, which consist oftesting already approved drugs for new medical indications, represents an increasingly popularstrategy to fast-track anti-cancer therapy with very little toxicity in the clinic.Here, we investigated the preclinical efficacy of beta-blockers and of their original combination withradiotherapy for the treatment of MB. We first showed that propranolol, carvedilol and nebivolol –lipophilic β-blockers that cross the blood-brain barrier – decreased both MB cell survival andmigration in 2D and 3D spheroid models. These beta-blockers are able to disrupt MB bioenergetics,i.e. both glycolysis and mitochondrial respiration, in agreement with our previous results in infantileneuroblastoma.We then showed that low doses of propranolol, carvedilol or nebivolol potentiate clinically-relevantradiation protocols (1.8 Gy/day, 5 days a week) in 2D cultures and 3D micromasses of human MB celllines. All these results were confirmed in MB cells from group 3 patient-derived xegrografts (PDX)cells. Lastly, to evaluate the most promising combinations in more in vivo-like situations, we havedeveloped a tumor-grafted organotypic cultures of cerebellar tissue: stably DsRed-expressing MBspheroids are grafted in mice healthy cerebellum slices. Progression of the MB cells exposed toradiotherapy + β-blockers is being analyzed over time in these innovative co-culture models, using alive cell analysis system (JULIStage) and a sensitive microplate reader with an advanced matrixscanning feature (PHERAstar).Our results provide a rationale for further study of β-blockers in combination with conventionalradiotherapy in medulloblastoma, with the ultimate goal of improving the quality of life of childrenwith high-risk brain tumors.


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Conditional generation of free radicals by selective activation of alkoxyamines: towards more effective and less toxic targeting of brain tumors.

D. Buric1, P. Piris1, T. Yamasaki2, MP. Montero1, M. Rossi, N. André, S. Combes, P. Brémond1 and M.Carré1

1Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm UMR1068, CNRS UMR7258, Aix-Marseille Université UM105, Institut Paoli Calmettes – Faculté de Pharmacie, Marseille, France 2Institute of Radical Chemistry, CNRS UMR7273, Aix-Marseille Université – Faculté des Sciences, Marseille, France E-mails: [email protected] & [email protected]

Glioblastoma multiform (GBM) is the most common intracranial tumor observed in adults. Despite a significant progress in clinical management over the last decade, these tumors have one of the poorest prognoses for survival due to a poor response to therapeutic strategies. GBMs are thus aggressive tumors that present unique challenges for identification of new drugs. Our previous results revealed the theranostic properties of alkoxyamines (R1–ONR2R3). These molecules can undergo homolysis to generate: 1) a highly toxic alkyl radical (R1•), which trigger the cell death process in GBM cells and 2) a non-toxic nitroxide (R2R3NO•), which can be used to enhance the MRI signal. In the present project, we propose to improve the efficacy of alkoxyamines while controlling their homolysis through the activation of matrix metalloproteases (MMP). Therefore, we have synthesized a library of 85 novel alkoxyamines as putative prodrugs for GBM. End-point and real-time cytotoxicity assays on 2D cultures and 3D spheroids of GBM cells led to a selection of the most efficient molecule, i.e. K1. The latter was more active in GBM cell lines and GBM stem cells than in cells from other cancer types (neuroblastoma, lung and breast carcinomas). We showed that K1-induced apoptosis resulted from the early generation of reactive oxygen species and a significant mitochondrial network fragmentation. K1 was then bioconjugated to a peptide selectively recognized by MMP. This bioconjugate successfully inhibited survival, proliferation, migration and invasion of the GBM cells. To further characterize its activity, we developed an innovative organotypic model based on the graft of stably-fluorescent GBM spheroids in ex vivo mice normal brain slices. Response to treatment was daily monitored (over 14 days) by live cell microscopy and high resolution fluorescence well scanning, and showed that K1-bioconjugate was very promising in inhibiting GBM progression in this co-culture system. No severe side-effects to the healthy tissue was observed at experiment completion. A great part of these results has also been extended to medulloblastoma, which is the most common malignant brain tumor in children. Results from a physiologically-based pharmacokinetic (PBPK) modeling supported that K1 is a good candidate for further preclinical tests in brain tumors. Based on structure-activity relationships, new halogenated derivatives are being synthesized to further increase the activity of this alkoxyamine.


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Development of a web application for detecting Y-site drugs compatibilities: an in silico decision-making tool for managing multi-drugs infusion

G. Sicard* 1, V. Cuplov1, R. Fanciullino1, 2

1SMARTc, Faculté de Pharmacie, 2Pharmacie, Hôpital de la Conception, Marseille, France

Background: In patients receiving multi-drugs infusion, knowledge, management and anticipation of physical and chemical incompatibilities are essential. These interactions are found responsible for the majority of errors and/or accidents related to parenteral drug delivery when several drugs are to be administered simultaneously. Our goal was to develop a simple and free web application to help prescribers, nurses and pharmacists in detecting these incompatibilities.

Materiel and Methods: First, we conducted a literature review in order to develop an in silico tool to detect these incompatibilities. A total of 112 drugs, 41 cytotoxics, 7 therapeutic monoclonal antibodies, 3 solvents and 1 parenteral nutrition mix were screened. Combinations of drugs were next classified into 4 categories: “compatible”, “incompatible”, “variable compatibility” (i.e. when conflicting data were reported in literature) and “no data available”. The web application was finally written in Python and deployed on the web hosting service PythonAnywhere.

Results: From all the selected drugs, we listed the main interactions for each pair of drugs. A total of 13 366 pairs of interactions were analyzed: 2 362 were compatible (17.7%), 679 were incompatible (5.1%), 95 presented a variable compatibility (0.7%) and 10 230 for which compatibility was not described yet (76.5%). For each incompatible couple, the underlying mechanism (e.g. turbidity, precipitate form, color change, drug loss…) was described and integrated in the web application. Two modules were created: one for drug-drug Y-site compatibility and another one specific to drug-chemotherapy Y-site compatibility.

Conclusion: In this study we have built a free, simple and intuitive tool for detecting physicochemical interactions, which is a key element of pharmaceutical analysis. Of note, this type of analysis is currently not included in prescription assistance softwares. In addition to the analysis of patient's biological data or PK/PD-based drug-drug interactions, the development and use of this tool allows to detect physical interactions related to the co-administration of intravenous drugs during routine pharmaceutical analysis. This web application is accessible on any computer or smartphone and is an asset for physicians and clinical pharmacists that could be used as a decision-making tool when managing multi-drugs infusions.


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Population pharmacokinetic model of irinotecan and its four main metabolites in patients treated with FOLFIRI or FOLFIRINOX regimen

Laure Deyme1*, Litaty Céphanoée Mbatchi2,3, Dominique Barbolosi1, Nicole Tubiana4,Joseph Ciccolini1, Alexandre Evrard2,3 and Florence Gattacceca1

* 2nd year PhD student in Aix-Marseille University1 SMARTc Unit, CRCM, Inserm UMR1068, CNRS UMR7258, Aix-Marseille University,

Marseille, France. 2 Laboratoire de Biochimie et Biologie Moléculaire, CHU Nîmes-Carémeau, Nîmes, France. 3 IRCM, Inserm, Université de Montpellier, ICM, Montpellier, France. 4 Centre Hospitalier Universitaire de Limoges, Limoges Cedex, France.

Background: Irinotecan (CPT-11) is an anticancer drug included in first-line treatments for patients with metastatic colorectal cancer and pancreatic cancer in combination with leucoverin, 5-fluorouracil (FOLFIRI) and oxaliplatin (FOLFIRINOX). CPT-11 is a prodrug which is converted into the cytotoxic form, SN38 (7-Ethyl-10-hydroxy-camptothecin), by carboxylesterases in the liver and into two inactive forms, APC (7-ethyl-10-[4-N-(5-aminopentanoicacid)-1-piperidino] carbonyloxycamptothecin) and NPC (7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycampto-thecin) by CYP3A4/5 [1]. SN38 is detoxified by UGT1A1 into SN38G (glucuronidated SN38), an inactive metabolite, before biliary excretion. The aim of this study is to determine the population pharmacokinetic (PK) parameters of CPT-11 and its four main metabolites in metastatic colorectal cancer patients treated with FOLFIRI and FOLFIRINOX regimens and quantify and explain their inter-individual variability.

Methods: A multicenter study of 109 metastatic colorectal cancer patients treated with FOLFIRI and FOLFIRINOX regimen was conducted. 506 plasma samples were collected at five different times during the first cycle of treatment. Plasma concentrations of CPT-11 and its metabolites were measured by high-performance liquid chromatography-fluorescence method [2]. Population PK analysis was performed using Monolix2019R1 software. Model selection and qualification were performed by both statistical and graphical methods. Once the compartmental and random effects models were selected, covariates were tested to explain the inter-individual variability in PK parameters.

Results: A three-compartment model was selected to describe CPT-11 PK with three first-order rate constants for CPT-11 elimination, transformation into SN38 and into APC and a Michaelis-Menten kinetics for transformation into NPC. Two-compartment model was chosen for the PK of SN38 metabolite with a first order rate constant for the conversion of SN38 into SN38G. A one-compartment model with first-order elimination best described SN38G, APC and NPC PK. Residual error was best described by a proportional error model for CPT-11, SN38 and APC and a combined error model for SN38G and NPC. Covariate analysis suggested that the SN38G central volume of distribution is affected by the patient’s performance status. Additional covariates were evaluated but not retained: regimen (FOLFIRI/FOLFIRINOX), sex, body surface area, weight, height and comedication with therapeutic antibody.

Conclusion: In FOLFIRI and FOLFIRINOX regimen, CPT-11, SN38, SN38G, APC and NPC were correctly described by our eight-compartmental model. The next step is to establish the relationship between irinotecan PK and its toxicity, taking into account associated drugs (5-fluorouracil and oxaliplatin). This forthcoming PKPD model will allow to perform in silico simulations to define the optimal administration protocol (i.e. dosing, scheduling, sequencing) for a maximal benefit to risk ratio.


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Evaluation of the suitability of prior information on a new target population

Anna Chan Kwong◊*,1,2,3, Amaury O’Jeanson◊,2, David Fabre3, Romaric Larcher4,Florence Gattacceca1 and Sonia Khier2

◊ ACK and AOJ contributed equally to this work1 Aix-Marseille University, INSERM, CNRS, CRCM SMARTc2 Montpellier University, IMAG (Alexander Grothendieck Institute)3 SANOFI R&D Montpellier4 CHU Lapeyronie Montpellier

Background: Before implementing a pharmacokinetic model in a therapeutic drug monitoring (TDM) program, its predictive performances have to be tested on the target population. The pharmacokinetic model can be a model built on the target population (i), a model from the literature (ii) or a model from the literature adapted to the target population (iii). The three approaches were tested on TDM concentrations of meropenem. Methods: TDM samples of meropenem were collected in critically ill patients hospitalized in the medical intensive care unit of Montpellier hospital (CHU Lapeyronie). Data were split into an “estimation dataset” (four fifth of the patients) and a “prediction dataset” (one fifth of the patients).

(i) A model was built on the “estimation dataset” (model (i)).(ii) Besides, literature was screened for appropriate NONMEM-built population

pharmacokinetic (popPK) models of meropenem. In order to choose themost suitable published model (model (ii)), we used the methodologypreviously proposed by Knøsgaard et al. (1): the models applied on the“estimation dataset” were ranked based on Akaike Criterion (AIC) ofBayesian estimation, Visual Predictive Checks (VPC) and NormalizedPrediction Distribution Error (NPDE).

(iii) Then, the selected literature model was adapted to the “estimation data”with the $PRIOR NWPRI subroutine (model (iii)).

To replicate real-life conditions, only the first observation of each patient in the “prediction dataset” was used to estimate individual PK parameters with Bayesian method. The next observed values were compared to the Bayesian-based predictions from the three models to evaluate their respective accuracies.

Results: The “estimation dataset” and the “prediction dataset” consisted in 85 concentrations from 46 patients and 30 concentrations from 12 patients respectively. Six NONMEM-built population pharmacokinetic (popPK) models of meropenem were selected in the literature (2–7). AIC of Bayesian estimation on the “estimation dataset” ranked from 594 to 1008. After checks of VPC and NPDE, we chose as model (ii) the model from Dhaese et al. (5) (AIC=594). Estimates of model (ii) were adapted to the “estimation dataset” using the $PRIOR NWPRI subroutine: the adapted model was model (iii). Model (iii) displayed the best predictive performances compared to models (i) and (ii), globally showing the smallest difference between Bayesian-basedpredictions and observations of the “prediction dataset”.

Conclusions The adaptation of a model from the literature to the target population using the $PRIOR subroutine (iii) was the most suitable approach for TDM of meropenem, compared to the direct application of a model from the literature (ii) or the use of a new model entirely built on the target population (i).


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References: 1. Knøsgaard KR, Foster DJR, Kreilgaard M, Sverrisdóttir E, Upton RN, van den Anker JN.

Pharmacokinetic models of morphine and its metabolites in neonates:: Systematiccomparisons of models from the literature, and development of a new meta-model. Eur JPharm Sci. 2016 Sep 20;92:117–30.

2. Li C, Kuti JL, Nightingale CH, Nicolau DP. Population pharmacokinetic analysis anddosing regimen optimization of meropenem in adult patients. J Clin Pharmacol. 2006Oct;46(10):1171–8.

3. Roberts JA, Kirkpatrick CMJ, Roberts MS, Robertson TA, Dalley AJ, Lipman J.Meropenem dosing in critically ill patients with sepsis and without renal dysfunction:intermittent bolus versus continuous administration? Monte Carlo dosing simulations andsubcutaneous tissue distribution. J Antimicrob Chemother. 2009 Jul 1;64(1):142–50.

4. Ulldemolins M, Soy D, Llaurado-Serra M, Vaquer S, Castro P, Rodríguez AH, et al.Meropenem population pharmacokinetics in critically ill patients with septic shock andcontinuous renal replacement therapy: influence of residual diuresis on doserequirements. Antimicrob Agents Chemother. 2015 Sep;59(9):5520–8.

5. Dhaese SAM, Farkas A, Colin P, Lipman J, Stove V, Verstraete AG, et al. Populationpharmacokinetics and evaluation of the predictive performance of pharmacokineticmodels in critically ill patients receiving continuous infusion meropenem: a comparison ofeight pharmacokinetic models. J Antimicrob Chemother. 2019 Feb 1;74(2):432–41.

6. Jaruratanasirikul S, Thengyai S, Wongpoowarak W, Wattanavijitkul T,Tangkitwanitjaroen K, Sukarnjanaset W, et al. Population pharmacokinetics and MonteCarlo dosing simulations of meropenem during the early phase of severe sepsis andseptic shock in critically ill patients in intensive care units. Antimicrob Agents Chemother.2015;59(6):2995–3001.

7. Mattioli F, Fucile C, Del Bono V, Marini V, Parisini A, Molin A, et al. Populationpharmacokinetics and probability of target attainment of meropenem in critically illpatients. Eur J Clin Pharmacol. 2016 Jul;72(7):839–48.


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Development of protein-protein interactions inhibitors: application at the Bromodomains

Kendall Carrasco a, Laurent Hoffer a, Yuliia V a. Voitovich a, Brigitt Raux a, Christophe Muller a, Carine Derviaux a, Stéphane Betzi a, Yves Collette a, Sébastien Combes a, Philippe Roche a, et

Xavier Morelli a

a Centre de Recherche en Cancérologie de Marseille, U1068 INSERM, UMR7258 CNRS, Institut Paoli Calmettes, UM105 Université Aix-Marseille , Laboratoire de Chimie Biologie et Structurale Intégrative

E-mail: [email protected]

Bromodomains (BRD) are conserved protein-protein interaction modules which can recognize and bind to acetylated lysine residues and particularly in the histones tails [1]. In the Human, there are 61 bromodomains which are divided in 8 families [1]. In my thesis project and this poster, I focused on the family II, the Bromo and Extra-Terminal domain or BET family. This family is composed of 4 proteins called BRD4, BRD3, BRD2 and BRDT, each protein is composed of two BRDs called BD1 and BD2. BETs proteins are very interesting therapeutic targets. In fact, there are involved in the regulation of gene transcriptions and implicated in many diseases such cancers or inflammations [2]. BET proteins are widely studied all over the world and it already exists many inhibitors of BET proteins, some of them are engaged in clinical trials. In my thesis project, I have searched to develop a BET inhibitor with a particular profile, called selective BD1. This type of inhibitors inhibits only the BD1 of BETs bromodomains. For this purpose, we use an identification method, called the Homogeneous Times-Resolved Fluorescence (HTRF), which permits us to screen a data base of 1600 molecules. Molecules which are identified in this primary screen are validated by two experiments, the Thermal Shift Assay (TSA) and the Isothermal Titration Calorimetry (ITC). The binding mode of the compound in the BRD is evaluated by crystallography. The crystallographic structure of complex BRD/compound permits me to drive the compound optimization. These optimizations are essentials to increase the affinity and the selectivity with the goal to obtain a BD1 selective biological probes.

[1] Filippakopoulos, P.; Knapp, S. The bromodomain interaction module. FEBS Lett. 2012,586, 2692−2704[2] Belkina, A. C.; Denis, G. V. BET domain co-regulators in obesity, inflammation andcancer. Nat. Rev. Cancer 2012, 12, 465−477.


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Population pharmacokinetic model of sorafenib and application to a case report Ba-Hai LE1,2, Nadège Néant1, Benoit Blanchet 3, François Goldwasser3, Joseph Ciccolini1, Florence Gattacceca1

1: Aix Marseille Univ, INSERM, CNRS, CRCM SMARTc, F-13005 Marseille, France; 2: Hanoi University of Pharmacy, Ha Noi, Viet Nam; 3: CERIA, Paris, France

Introduction: High inter- and intra-individual variabilities (61-65% and 44-47% respectively) of pharmacokinetics (PK) of sorafenib were observed during phase I studies, as well as in some population pharmacokinetic (pop-PK) studies, which could explain the unstable response and the unintended toxicities that occur in some patients under a recommended dosage of sorafenib [1-4]. Therefore, we aimed to develop a sorafenib pop-PK model based on a large population treated by sorafenib during a long period. This pop-PK model was then used to predict sorafenib individual plasma PK based on sparse therapeutic drug monitoring using Bayesian approach, then to explore the relationship between sorafenib plasma exposure and toxicity outcome in patients from La Timone hospital.

Method: Sparse PK data available from 267 patients treated with sorafenib between 2008 and 2018 have been included in this multicentric study (10 French hospitals). The PK data were analyzed using nonlinear mixed-effect modeling (NONMEM software version 7.3). Model evaluation was performed using standard goodness-of-fit plots, and simulation-based tools such as visual predictive check (VPC).

The final pop-PK model was applied to provide explanations for the onset of severe sorafenib-related toxicities in one patient, then to evaluate the rationale of the associated empirical dose reductions in this patient. The individual parameters were estimated using Bayesian method in NONMEM and used to simulate the exposure to sorafenib over the course of the treatment in R studio.

Results: 1310 plasma concentrations collected at steady-state were available to build the model. A 1-compartment structural model with first-order absorption and linear elimination described the data satisfactorily. Bioavailability (F1) was found to vary as a function of the dose and to decline over time on sorafenib treatment. Typical values (RSE %) of the final model parameters were as follows: clearance (CL) 1.41 L/h (9.4%), distribution volume (V) 50.3 L (21.1%), absorption rate constant (ka) 0.635 h-1

(35.9%). The high inter-patient variability was confirmed in this study with 44.4 % for CL (46.5%), V(93.7%) and ka (100% fixed). The lack of information regarding the absorption phase and covariatessuch as food intake may account for the extremely large variability of ka.

Based on the final pop-PK model, the individual parameters of the patient were estimated and used to describe the PK of sorafenib over the course of the treatment. The steady-state AUC from 0 to 12h (AUC0-12) post dose was calculated: values of 109.4, 82.3 and 78.9 mg*h/L were obtained with the dosages 400mg twice daily, 400mg daily and 200mg daily respectively. These values are particularly high when compared to the mean AUC0-12 of 57.7±28.6 mg* h/L obtained in patients experiencing grade 3–4 adverse events in a previous study [4] and are consequently consistent with the observed severe toxicities. These results document the decision to continue reducing the sorafenib dose by expanding the time between doses with the 200mg dosage.

Conclusion and perspectives: The high unexplained inter-individual variability in our population could be partly explained by covariates that will need to be collected in further prospective studies. However, the established population model allows reliable prediction of pharmacokinetics based on Bayes estimation. The population model could be used in the context of therapeutic drug monitoring to support dose adjustments in patients.

References:

1. Hornecker, M., et al., Saturable absorption of sorafenib in patients with solid tumors: apopulation model. Invest New Drugs, 2012. 30(5): p. 1991-2000.

2. Jain, L., et al., Population pharmacokinetic analysis of sorafenib in patients with solid tumours.Br J Clin Pharmacol, 2011. 72(2): p. 294-305.

3. Awada, A., et al., Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 dayson/7 days off in patients with advanced, refractory solid tumours. Br J Cancer, 2005. 92(10): p.1855-61.

4. Boudou-Rouquette, P., et al., Variability of sorafenib toxicity and exposure over time: apharmacokinetic/pharmacodynamic analysis. Oncologist, 2012. 17(9): p. 1204-12.


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METHODE ALTERNATIVE POUR LE PHENOTYPAGE DE LA DPD (DIHYDRO PYRIMIDINE DÉHYDROGÉNASE) CHEZ LES PATIENTS TRAITES PAR PYRIDIMES

Clémence MARIN1, Anis KRACHE1, Chloé PALMARO1, Mathilde LUCAS1,Valentin HILAIRE1, Renée UGDONNE1, Bénédicte DE VICTOR1, Sylvie QUARANTA1, Caroline SOLAS1, Bruno LACARELLE1, Joseph CICCOLINI11 : Laboratoire de Pharmacocinétique et Toxcilogie, CHU Timone - APHM,

Bâtiment F 264 rue saint pierre, 13005 Marseille

Introduction : La détection des patients déficitaires en DPD est devenue une préoccupation majeure en oncologie. Cette préoccupation a pris de l ́ampleur lorsque les autorités de santé ont recommandé la sécurisation de l ́administration des pyrimidines (5-FU et capécitabine) par un test prospectif en décembre 2018. Le suivi des concentrations physiologiques en uracile (U) et/ou du ratio dihydro-uracile/uracile (UH2/U) est une alternative fréquente à la mesure de l ́activité enzymatique de la DPD. Avec l ́augmentation du nombre de patient à phénotyper, le développement d ́une méthode compatible avec le screening en routine d ́un grand nombre de patient est devenu un enjeu critique.

Matériels & méthode : Nous avons donc développé et validé une méthode simple et robuste par UPLC-UV, avec un court temps d ́analyse (12 minutes par run), et compatible avec les pré-requis d ́un screening à grande échelle.Les niveaux de détection de cette méthode sont de 5 à 500 ng/mL (265 nm) pour l ́U, et de 40 à 500 ng/mL (210 nm) pour l ́UH2, sans interférences chromatographiques, y compris à 210 nm.

Résultats & discussion : Lors d ́un screening de routine cette méthode est capable de différencier les patients non déficitaires (U < 16 ng/mL et/ou ratio UH2/U élevé) des patients déficitaires qui présentent un risque élevé de toxicité sévère (U > 16 ng/mL et/ou ratio UH2/U bas). De plus, les cas de déficit totaux pouvant conduire à des décès toxiques peuvent également être détectés (U > 150 ng/mL et/ou ratio UH2/U proche de 0). Cette nouvelle méthode, qui utilise une extraction solide-liquide simple, est basée sur l ́utilisation d ́une UPLC standard. Celle-ci est facilement implantable dans n ́importe quel laboratoire désireux de démarrer une activité de phénotypage de la DPD en routine.

Conclusion : La détection précoce des déficits en DPD est au cœur de l ́exercice de l ́oncologie médicale lors de l ́administration de fluoro-pyrimidine. Ici nous avonsprésenté une méthode UPLC-UV rapide, simple et abordable qui permet de quantifier l ́Uet le UH2 pour déterminer le phénotypage DPD. Lorsqu ́elle est utilisée en routine cettetechnique permet de traiter un grand nombre de prélèvement provenant de patientatteint de cancer de manière à sécuriser leur traitement.


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Impact of trastuzumab coating when prototyping immunoliposomes in breast cancer models: the more the merrier?

Rodallec Anne1*, Franco Corentin2, Stéphane Robert3, Sicard Guillaume1, Giacometti Sarah1, Brochier Camille4, Lacarelle Bruno1, Ciccolini Joseph1, P. Poncelet2, Fanciullino Raphaelle1

1: SMARTc Unit, CRO2 U-911, Aix Marseille Univ, 27 boulevard Jean Moulin, 13005 Marseille,

France 2: Biocytex, Marseille, France 3 : VRCM, AMUTICYT platform, Aix Marseille Univ, 27 boulevard Jean Moulin, 13005 Marseille,

France. 4: Institut Roche, 30 Cours de l'Ile Seguin - F-92650 Boulogne Billancourt Cedex, France.

Objectives: To improve drug delivery in oncology, developing targeted nanoparticles loaded with cytotoxics is a rising strategy in oncology. Antibodies are the most commonly used targeting agents. However, determination of their optimal density on nanoparticles surface remains an open question, mainly due to the difficulty in measuring accurately the actual level of coating. We have developed a stealth immunoliposome encapsulating docetaxel and harboring anti-Her2 trastuzumab. To better prototyping the nanoparticles, we have developed a method for the absolute quantitation of trastuzumab, to compare the performance of the immunoliposomes in terms of efficacy in breast cancer depending on the number of antibodies. Methods: Using flow cytometry optimized for submicron particle analysis and beads covered with known amounts of human IgG-kappa, mimicking various amounts of trastuzumab, we developed a quick and quantitative assay to measure the amount of trastuzumab coated per docetaxel immunoliposome. Three batches of immunoliposomes exhibiting different densities of coated trastuzumab were synthesized using the standard thin-film method and a maleimide linker. Quality control was operated regarding size, docetaxel encapsulation and trastuzumab coating levels, including stability studies. In vitro and in vivo efficacy studies were performed on MDA-MB-453 used as a canonical model of Her-2+ human breast cancer cells. Results: The three batches of Immunoliposomes exhibited 330 ± 30 (batch1), 480 ± 110 (batch2) and 690 ± 80 (batch3) trastuzumab IgG molecules per liposome, respectively (p<0.01, One-Way Anova). In 2D apoptosis induction experiments, no difference was observed between free drugs (i.e., trastuzumab + docetaxel) and immunoliposomes, regardless of trastuzumab density. When shifting to 3D spheroids, higher antiproliferative efficacy was observed with batch2 as compared with other immunoliposomes or free drugs (+76%) or reference T-DM1 (+85%). Next, batch2 was further tested in mice bearing MDA-MB-453 xenografts. Immunoliposomes achieved a tumor reduction of 89 % when compared to free drugs and 66% as compared with T-DM1 (p<0.05, One-Way Anova). Conclusion: Our new fully calibrated assay based on flow cytometry of submicron particles can accurately quantify the number of coated antibodies on single nanoparticles. This single particle analysis quantitative approach was used for quality control of different immunoliposomes batches targeting Her2+ breast cancer cells. In vitro studies demonstrated that maximal density of targeting agent on nanoparticles was not a prerequisite for maximal therapeutic effect. In vitro spheroids and in vivo studies in mice demonstrated higher efficacy of our immunoliposomes when compared to free trastuzumab docetaxel used in combo and antibody-drug conjugate T-DM1. Beyond the current project, this new quantitation method could be valuable when prototyping nanoparticles or conjugated drugs using monoclonal antibodies as targeting agent.


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ICR

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Pharmacomodulation study using ring variation strategy and simplification in trichloromethylated-heterocycles series against Plasmodium falciparum

Dyhia Amrane,a Nicolas Primas,a Sébastien Hutter,b Nadine Azas,b Pierre Verhaeghec and Patrice Vanellea

a Aix-Marseille Univ, CNRS, ICR UMR 7273, PCR, Faculté de Pharmacie, 13385 Marseille. b Aix-Marseille Univ, IHU Méditerranée Infection, UMR VITROME, 13005 Marseille.

c Université Paul Sabatier, CNRS UPR 8241, LCC, 31077 Toulouse. E-mail: [email protected]

Malaria is still the leading cause of death among parasitic infections worldwide.1 The emergence and the expansion of Plasmodium strains resistant to the Artemisinin-based Combination Therapies (ACTs) are now threatening the efficacy of malaria treatment notably in the Greater Mekong Subregion. Therefore, new molecules displaying original mode of action are urgently required. Aiming at developing new antiplasmodials, our laboratory previously described the synthesis and the biological activities of a library of 2-trichloromethylquinazoline derivatives which highlighted a Hit molecule 1 (IC50 = 0.4 µM, CC50 = 16 µM).2 Moreover, a scaffold hopping strategy showed that the replacement of the quinazoline moiety by a quinoxaline one improved the cytotoxicity profile.3 Thus, we synthetize a new series of 2-trichloromethylquinoxaline analogues. The in vitro biological evaluations against the multi-resistant K1 P. falciparum strain highlighted two new hit molecules substituted by an electron-withdrawing group inpara-position. According to these results, we prepared new 2-trichloromethylquinoxaline analoguesbearing other electron-withdrawing group such as -CF3, -SF5, or -OCF3.

In parallel, to complete the global SAR study using the scaffold hopping approach, we replaced the quinoxaline ring by a phthalazine one. The importance for the antiplasmodial activity of the phenyl ring contained in the bicyclic quinazoline and quinoxaline scaffolds was also studied by structural simplification leading to the related pyrimidine and pyrazine analogues. The synthesis details of the new analogs of Hit 1 in various series and the biological results will be described in the poster.

This work was supported by the ANR NINTARMAL project, grant ANR-17-CE11-0017.

References

1. WHO, World Malaria report, 2018, http://www.who.int/malaria/publications/world-malaria-report-2018/report/en/

2. Cominetti, M. D. D.; Hughes, D. L.; Matthews, S. E. Org. Biomol. Chem. 2016, 14, 10161-10164. 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, 4313-4322.

3. Desroches. J.; Kieffer, C.; Primas, N.; Hutter S.; Gellis, A.; El-Kashef, H.; Rathelot, P.; Verhaeghe, P.; Azas, N.;Vanelle, P. Eur J. Med. Chem. 2017, 125, 68-86.

N

N

CCl3

X

R

X = S , O, NHQuinoxaline series

N

N

CCl3

X

Pyrazineseries

NN

CCl3

X

N

N

HN

CCl3

ClCl

Hit 1

N

N

X

CCl3Pyrimidine

seriesQuinazoline Phthalazine series

RR

R

Ring simplification

Scaffoldhopping

Figure 1. Ring variation strategy adopted in order to explore SARs


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mailto:[email protected]

Efficient selenocyanation of electron-rich heterocycles

and antileishmanial activities Anne Roly Obah Kosso,a Julie Broggi,a Anita Cohen,b Nadine Azas,b

Sébastien Redon,a Patrice Vanelle.a a) Institut de Chimie Radicalaire (ICR), équipe PCR, UMR 7273, Faculté de Pharmacie,

Aix-Marseille Université, Marseille, France. b) IHU Méditerranée Infection, UMR VITROME, Faculté de Pharmacie,

Aix-Marseille Univ., Marseille, France.

Recently, organic selenocyanates (RSeCN) have attracted the interest of medicinal chemists, due to their remarkable biological activities especially as leishmanicidal1a and anticancer agents.1b In continuation of our research program centered on the medicinal chemistry of electron-rich heterocycles, we investigated the reactivity of triselenodicyanide for the selenocyanation of various imidazoheterocycles and chromones. Selenocyanate function can be introduced using KSeCN, as electrophilic source with N-iodosuccinimide,2a or as radical source with an oxydant.2b These methodologies present several disadvantages: using very smelly and air sensitive KSeCN or tedious column chromatography. In order to develop a more attractive industrial process, it is of high importance to offer a straightforward, odorless, inexpensive and scalable method. To do so, the triselenodicyanide represents the ideal electrophilic source, especially since its novel generation from malononitrile and odorless selenium dioxide.3 We present here an efficient methodology for the regioselective C-3 selenocyanation of imidazoheterocycles,4a a one pot C-3 selenocyanation of chromen-4-ones derivatives4b and an ipso-selenocyanation of arylboronic acids.4c

SeO2 +

N

NRHet

18 exemples80-99%

N

NRHet

SeCN

Het = pyridine, pyrimidine,thiazole, benzothiazole

CN

CN, DMSO

25°CO

SeCNO

R

16 exemples56-81%(2 steps)

OH

O

R N

25°C

B(OH)2

SeCN

60°C Ar

Ar

22 exemples29-98%

These selenocyanations are characterized by the use of odorless and inexpensive starting materials and an ease of purification by filtration. In this study, we also explored interesting reactivities of the SeCN function toward decyanations. Preliminary in vitro activities against Leishmania donovani promastigotes and their cytotoxicities will be presented. ___________________________________________________________________________ [1] a) Baquedano, Y.; Alcolea, V.; Toro, M. A.; Gutierrez, K., J.; Nguewa, P.; Font, M.; Moreno, E.; Espuelas, S.; Jimenez-Ruiz, A.; Palop, J. A.; Plano, D.; Sanmartín, C. Antimicrob. Agents Chemother. 2016, 60, 3802. b) Plano, D.; Karelia, D. N.;Pandey, M. K.; Spallholz, J. E.; Amin S.; Sharma, A. K. J. Med. Chem., 2016, 59, 1946.[2] a) Muniraj, N.; Dhineshkumar, J.; Prabhu K. R. ChemistrySelect 2016, 5, 1033. b) Mitra, S.; Ghosh, M., Mishra, S.;Hajra, A. J. Org. Chem. 2015, 80, 8275.[3] Kachanov, A. V.; Slabko, O. Y.; Baranova, O. V.; Shilova, E. V.; Kaminskii. V. A. Tetrahedron Lett. 2004, 45, 4461.[4] a) Redon, S.; Obah Kosso, A. R.; Broggi, J.; Vanelle P. Tetrahedron Lett. 2017, 58, 2771. b) Obah Kosso, A. R.; Broggi,J.; Redon, S.; Vanelle P. Synlett 2018, 29, 1215. c) Redon, S.; Obah Kosso, A. R.; Broggi, J.; Vanelle, P. Synthesis 2019, 51,3758.


28

https://en.wikipedia.org/wiki/Leishmania

Anti-infectious pharmacochemistry of nitroimidazooxazole

derivatives Mathias, F. (1), Kabri, Y. (1), Crozet, M. D. (1), Cohen, A.(2), Okdah, L.(3),Rolain, J.-M.(3), Azas,

N.(2), Rathelot, P.(1), Vanelle, P. (1),*

(1) Aix-Marseille Université, Institut de Chimie Radicalaire ICR, UMR CNRS 7273, Laboratoire de Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin – CS 30064, 13385 Marseille Cedex05, France

(2) Aix-Marseille Université, IHU Méditerranée Infection, UMR VITROME - Tropical Eukaryotic Pathogens,19-21 Boulevard Jean Moulin, 13005 Marseille, France.

(3) Aix-Marseille Université, IHU Méditerranée Infection, Unité de Recherche sur les Maladies Infectieuseset Tropicales Emergentes, URMITE UMR 63, CNRS 7278, IRD 198, Inserm 1095, 19-21 Boulevard JeanMoulin, 13005 Marseille, France

Currently, pharmacomodulation work in nitroimidazooxazole series focused on 6-

nitroimidazooxazole compounds1 since the discovery of their antitubercular and antileishmanial

properties.2-3 We report herein the synthesis of nitroimidazooxazole compounds for novel structure

activity relationship study: 5-nitroimidazooxazole, 7-nitroimidazo[5,1-b]oxazole and 5-substituted 6-

nitroimidazooxazole derivatives.

We synthesized 7-nitroimidazo[5,1-b]oxazole and 6-substituted 5-nitroimidazooxazole series using

an intramolecular cyclization and Suzuki-Miyaura cross-coupling reactions under microwave

irradiation.4-5 For the 5-substituted 6-nitroimidazooxazole serie, we worked in the optimization of a CH

activation reaction.

The methodology used and the biological results of these compounds on C. difficile and L. donovani

will be presented.

1. D. Kumar, B. Negi, D. Rawat. Future Med. Chem. 2015, 7, 1981.2. K. Nagarajan, R.G. Shankar, S. Rajappa, S. J. Shenoy, R. Costa-Pereira. J. Med. Chem. 1989, 24, 631.3. A. M. Thompson, P. D. O’Connor, A. Blaser, V. Yardley, L. Maes, S. Gupta, D. Launay, D. Martin, S. G. Franzblau,B. Wan, Y. Wang, Z. Ma, W. A. Denny. J. Med. Chem. 2016, 59, 2530.4. F. Mathias, Y. Kabri, M. D. Crozet, P. Vanelle. Synthesis 2017, 49, 2775.5. F. Mathias, Y. Kabri, M. D. Crozet, P. Vanelle. Synth. Commun. 2018, 48, 1213.


29

Novel 2,4-disubstituted 5-nitroimidazoles : synthesis and biological

evaluation against Clostridium difficile

Spitz, C.(1), Mathias, F.(1), DOAN, T. H. D.(2), Péchiné, S.(2), Innocent, J.(1), Pellissier, S.(1), Di Giorgio, C.(3),

Crozet, M. D.(1), Janoir, C.(2), Vanelle, P.*(1)

(1) Aix-Marseille Université, Institut de Chimie Radicalaire ICR, UMR CNRS 7273, Laboratoire de Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin – CS 30064, 13385 Marseille Cedex 05,France

(2) Univ Paris-Sud, Université Paris-Saclay, EA4043 Faculté de Pharmacie, Chatenay-Malabry, France(3) Aix-Marseille Université, CNRS, IRD, Avignon Université, IMBE UMR 7263, Laboratoire de Mutagénèse

Environnementale, 13385 Marseille, France

For decades, metronidazole (the most used 5-nitroimidazole marketed) was one of the first-line

treatments for non-severe Clostridium difficile infections (CDI). However, 38% therapeutic failures and

6.8% resistance to metronidazole were described, leading to recent updating of American guidelines

to recommend metronidazole only as an alternative to vancomycin or fidaxomicin for an initial non-

severe CDI episode.1 Research for new potent 5-nitroimidazoles against Clostridium difficile is urgently

needed.

Previously, it was shown that an ethylenic function in 2-position of the 5-nitroimidazole scaffold

allowed to offer better activity than metronidazole against anaerobic bacteria with higher

mutagenicity.2 On the other hand, pharmacomodulation studies have suggested that the

functionalization of the 4-position could modulate the mutagenicity of 5-nitroimidazole compounds.3

So, we report herein the synthesis of new 2,4-disubstituted 5-nitroimidazole compounds using a

Vicarious Nucleophilic Substitution of hydrogen (VNS) reaction to introduce a phenylmethylsulfone

group in 4-position and a Unimolecular Radical Nucleophilic Substitution (SRN1) reaction to introduce

an ethylenic function in 2-position of the 5-nitroimidazole scaffold:

In vitro studies demonstrated that three compounds have a higher activity than metronidazole on

four C. difficile strains (O12, O27, O85 and O87). The methodology used and the biological results of

these compounds on C. difficile will be presented.

1 L.C. McDonald, D.N. Gerding, S. Johnson, J.S. Bakken, K.C. Carroll, S.E. Coffin, E.R. Dubberke, K.W. Garey, C.V. Gould, C. Kelly, V. Loo, J. Shaklee Sammons, T.J. Sandora, M.H. Wilcox, Clin. Infect. Dis. 2018, 66, 987–994. 2 P. Vanelle, M. Crozet, J. Maldonado, M. Barreau, Eur. J. Med. Chem. 1991, 26, 167–178. 3 M.D. Crozet, C. Botta, M. Gasquet, C. Curti, V. Rémusat, S. Hutter, O. Chapelle, N. Azas, M. De Méo, P. Vanelle, Eur. J. Med. Chem. 2009, 44, 653–659.


30

DO YOU KNOW THAT CARBENES CAN PROMOTE

REDUCTION REACTIONS?

Guillaume TINTORI, Pierre NABOKOFF, Yuxi ZHAO, Sébastien REDON, Patrice VANELLE and Julie BROGGI*

Aix Marseille Univ, CNRS, Institut de Chimie Radicalaire ICR Faculté de Pharmacie, 13005 Marseille, France.

e-mail address: [email protected]

The equilibrium between a carbene and the corresponding dimeric form,

known as the Wanzlick equilibrium, is a fascinating process. Depending on the steric

or electronic hindrance of the heterocycle, the equilibrium shifts preferentially toward

one form or the other.1 Starting from the imidazolium salt precursor or the carboxylate

adduct, both the carbene and the dimer have been used as source of N-heterocyclic

carbene ligands.2 We recently demonstrated that both forms can also be used as

source of Organic Electrons Donors (OEDs) to promote reduction reactions.3

OEDs are powerful organic reducing agents capable of spontaneous and

chemoselective single- or double-electron transfers to organic substrates under mild

conditions.4 They thus allow C-, N-, O-, and S-heteroatom bond dissociations and the

generation of fundamental radical or anionic intermediates. Aminopyridinylidenes

and imidazolylidenes have both been used to promote reduction reactions albeit

adopting different preferential form in the Wanzlick equilibrium.3,5 The different

mechanisms leading to the formation of the organic electron donors will presented.

References

[1] R. W. Alder, M. E. Blake, L. Chaker, J. N. Harvey, F. Paolini, J. Schütz, Angew.

Chem. Int. Ed., 2004, 43, 5896-5911.

[2] F. E. Hahn, M. C. Jahnke Angew. Chem. Int. Ed. 2008, 47, 3122-3172.

[3] G. Tintori, P. Nabokoff, R. Buhaibeh, D. Bergé-Lefranc, S. Redon, J. Broggi, P.

Vanelle, Angew. Chem. Int. Ed., 2018, 57, 3148-3153.

[4] a) J. Broggi, T. Terme, P. Vanelle, Angew. Chem. Int. Ed. 2014, 53, 384-413. b) J.

A. Murphy, J. Org. Chem. 2014, 79, 3731-3746.

[5] a) J. A. Murphy, J. Garnier, S. R. Park, F. Schoenebeck, S.-Z. Zhou, A. T. Turner,

Org. Lett. 2008, 10, 1227-1230. b) P. I. Jolly, S. Zhou, D. W. Thomson, J. Garnier, J.

A. Parkinson, T. Tuttle, J. A. Murphy, Chem. Sci. 2012, 3, 1675-1679.


31

IMBE

32

Porous materials for the detection of low concentrated biomolecules

C. Belliardoa,b, V. Hornebecq b, F. Chaspoula, D. Bergé-Lefranca

aUMR CNRS 7263 IMBE Unité Chimie Physique, Prévention des Risques et Nuisances Technologiques

Aix-Marseille Université, Faculté de Pharmacie, Marseille bMADIREL, Campus Saint Jérôme, 141 Traverse Charles Susini, 13013 Marseille

The detection of low-concentrated biomolecules is a key issue in many areas of life sciences such as drug intoxication, medical diagnostic assistance, prevention of pathology’s appearance and environmental integrity monitoring. Currently, in case of biological matrices, the main analytical methods used are either immunological or chromatographic. These two technics are powerful from both qualitative and quantitative point of view. However, some points limit their routine use for preventive purposes. Chromatographic methods require high qualified personnel, equipment and their maintenance are expensive and pre-treatment phases are time-consuming. Immunological methods are rapid, sensitive but have some limitations such as problem of interferences with other molecules or the impossibility of detecting certain biomolecules or drugs. The introduction of a solid/liquid interface to promote a controlled adsorption phenomenon can lead to the development of a complementary method for the detection of molecules at low concentration. Adsorption has the advantage of simultaneously allowing an overconcentration and a specific (or selective) interaction with a targeted molecule for routine uses with an acceptable costs and accuracy. In this presentation, two examples will be developed to illustrate the use of porous materials to detect, after adsorption, low concentrated biomolecules. In the first example, the surface of a porous silica material was functionalized with specific functions able to realize “key/lock” type interaction with the target. Using an innovative synthesis procedure, hybrid mesoporous silicas with uniformly and densely covered polypeptides (glutathione) functions on its surface were prepared starting from a diblock copolymer composed of a polylactide hydrophobic block and a polypeptides hydrophilic block. The reactivity and accessibility of the confined glutathione functions towards benzoquinone that is a main metabolite of benzene were studied from a thermodynamic point of view. Adsorption parameters, such as the affinity between benzoquinone and porous materials functionalized with polypeptides and maximal adsorption capacities, were obtained. The crosschecking with the results of calorimetric experiments led to the energetic aspect of the interaction. These thermodynamic parameters were found similar to those determined in solution for the same interaction. In the second example, gold nanoparticles were immobilized in the pores of the silica material (Figure 1). The resulting porous nanocomposite was used as a substrate to detect oxazepam, a benzodiazepine and a benzodiazepine metabolite. This detection is based on the SERS effect that corresponds to a Raman signal enhancement observed when Raman-active molecules are adsorbed on “nanostructured” metal surfaces. The Raman response of oxazepam was coupled with its adsorption properties on such substrates allowing to understand the synergistic interplay between materials parameters/adsorption process and the drug detection threshold.

Figure 1: Transmission Electronic Microscopy picture of Au-based porous nanocomposite


33

INP

34

Institut de NeuroPhysiopathologie (INP), UMR CNRS 7051

L'Institut de NeuroPhysiopathologie (INP UMR7051) est dirigé par Michel KHRESTCHATISKY. Il est localisé sur le site Timone de la Faculté de Médecine de Marseille et a pour tutelles Aix-Marseille Université (AMU) et le CNRS. L’INP rassemble 11 équipes représentant 150 personnes. Les principaux objectifs de l’Institut sont d’étudier à un niveau fondamental, à travers des modèles animaux et cellulaires (y compris la reprogrammation / modélisation de cellules iPS), à différents niveaux d’intégration (du moléculaire aux modèles animaux) et dans divers contextes pathologiques, les interactions entre les cellules neuronales, gliales, endothéliales et immunitaires, et leurs rôles dans la plasticité des réseaux de neurones, dans les horloges biologiques et dans la fonction cognitive. Les équipes de l’Institut étudient, au cours du développement et du vieillissement différentes maladies, dont la maladie d’Alzheimer, la sclérose en plaques, les processus neuro-inflammatoires et neurodégénératifs, le glioblastome, la tumorigenèse, l’angiogenèse et des altérations de la barrière hémato-encéphalique (BHE).

Les équipes de l'INP promeuvent le développement d'approches diagnostiques et thérapeutiques, basées notamment sur l'identification de bio-marqueurs et de cibles thérapeutiques, sur le développement de nouvelles molécules thérapeutiques et sur des stratégies de thérapie cellulaire impliquant des cellules souches olfactives et des cellules iPS. Les équipes ambitionnent la valorisation et les partenariats avec l'industrie pharmaceutique, notamment à travers la création d'un « Laboratoire Commun de Recherche (LabCom) » avec la société de biotechnologie VECT-HORUS. Ce partenariat vise à développer de nouvelles stratégies de vectorisation dans le système nerveux central, ainsi que des agents thérapeutiques ou d'imagerie novateurs, qui bénéficieront de ces stratégies pour améliorer leur délivrance et leur efficacité cérébrales.

Les équipes de l'INP et de VECT-HORUS partagent un large éventail d'approches et de technologies complémentaires, notamment: la biologie moléculaire; la chimie (nanoparticules, contribution de VECT-HORUS à la synthèse / purification de peptides, associée à la chimie organique, développement d'agents d'imagerie vectorisés pour l’imagerie PET, etc.); la biologie cellulaire (lignées de cellules neurales, lignées tumorales, cultures de cellules neurales primaires, neurones, astrocytes, microglies, cellules endothéliales, cellules souches adultes et cellules iPS, modélisation de la BHE in vitro); l’imagerie (fluorescence, confocale, TIRF, super-résolution, MET, IRM, TEP); l’étude intégrée de modèles animaux de diverses maladies (maladie d'Alzheimer, sclérose en plaques, glioblastome, autisme, etc.), y compris d'animaux transgéniques ou invalidés pour des gènes d'intérêt, avec l'étude du comportement, l'apprentissage (développement de prototypes automatisés pour l'étude de comportement) et l’évaluation des agents thérapeutiques précliniques.

Sur le plan de la formation, l’INP est largement impliquée dans le Master Santé, le Neuroscience Master International, le PhD Program in Integrative & Clinical Neuroscience et la NeuroSchool récemment créé.

Les projets de l’INP sont développés dans le cadre de collaborations nationales et internationales et sont financés par des contrats publics nationaux ou européens (ANR, CE, BPI, etc.), par des fondations et des partenaires de l’industrie.

L’équipe « Cytosquelette et physiopathologie » (H. Kovacic/ V. Peyrot) et « Gliomagénèse » (D ; Figarella-Branger) et la plateforme PINT sont dynamisées par des personnels de la Faculté de Pharmacie.


35

Plateforme NeuroTimone (PFNT)

Plateforme Technologique Aix-Marseille Université

Institut de NeuroPhysiopathologie (INP)

Les neurosciences sont des disciplines hautement technologiques, qui nécessitent des équipements coûteux et complexes. La mise en place de plateformes mutualisées permet à l’ensemble de la communauté des chercheurs du pôle NeuroTimone d’accéder à des techniques de pointe pour développer des projets ambitieux, mondialement compétitifs dans leur domaine de recherche. Ces projets se concrétisent par un volume de publications scientifiques de haut niveau, ainsi qu’une valorisation par le biais de brevets et la création d’entreprises innovantes.

La Plateforme NeuroTimone PFNT s’inscrit dans cette dynamique forte et propose des outils de pointe en neurobiologie organisés autour de trois composantes principales :

Le « Neuro Cellular Imaging Support » NCIS est une des deux composantes de la Plateforme “Photonic Imaging NeuroTimone” PImaNT qui propose un large éventail de techniques d’imagerie photonique permettant d’aborder l’étude du cerveau et du système nerveux dans toute sa complexité. Les technologies proposées par le NCIS s’articulent autour de la microscopie super-résolution, l’imagerie sur cellules vivantes à haut débit et la microscopie confocale rapide à haute résolution. La deuxième composante de PImaNT le « In vivo neuronal photonic imaging » INPHIM hébergé par l’Institut des Neurosciences Timone (INT), réunit un plateau de microscopie biphotonique in vivo et in vitro, de microscopie à feuille de lumière, et d’imagerie optique mésoscopique et optogénétique.

La « Plateforme Interactome NeuroTimone » PINT est un plateau centré sur la détection et l'étude des interactions protéines-protéines par des approches orthogonales et complémentaires de biophysique : la résonnance plasmonique de surface (SPR), la microcalorimétrie, l’ultracentrifugation analytique (UCA) et la spectrométrie de masse.

Le « Stem Cell center NeuroTimone » SCeNT est une infrastructure de culture cellulaire (notamment cellules humaines, niveau L2) pour la préparation de cellules souches pluripotentes induites (iPSCs), à partir de cellules somatiques de patients, et les modèles cellulaires de pathologies qui en dérivent.

Ces 3 composantes sont complétées par un plateau de PCR quantitative, PTQPCR.

Conformément à la charte des « Plateformes technologiques d’Aix-Marseille » labellisées, l’offre en prestations technologiques de la PFNT est accessible aux laboratoires publics et privés.

La PFNT bénéficie, pour son développement, du soutien de l’Etat via la Délégation Régionale à la Recherche et la Technologie, de la Région Provence Alpes Côte d’Azur, du Département des Bouches du Rhône et de la Ville de Marseille. Ces soutiens inscrits au CPER Contrant de Plan Etat Région sont complétés par un soutien en cours d’instruction de l’Europe dans le cadre du programme FEDER.

Le laboratoire de Biophysique de la Faculté de Pharmacie de Marseille est partie prenante de la plateforme PINT


36

nanoDSF : a new tool for studying microtubules assembly

Romain La Rocca1,2, Philipp O. Tsvetkov1, François Devred1,2

1 Aix-Marseille Université, Plateforme Interactome Neurotimone (PINT), Faculté de Médecine, 13385 Marseille 2 Aix-Marseille Université, CNRS UMR 7051, Institut de Neurophysiopathologie, Faculté de Médecine, 13385 Marseille

In vitro formation of microtubules has been extensively studied since the discovery of the self-assembly properties of the αβ-heterodimer of tubulin. Among the methods to monitor microtubule formation, turbidimetry, which consists in measuring the Absorbance at 350nm, is one of the key steps of the traditional workflow to characterize potential microtubule targeting agents. Indeed, not only it can be used to measure several intrinsic polymerization parameters such as the critical concentration or the amount of tubulin polymerized, but also the impact of several small molecules (ions, nucleotide, pH, …) or microtubule associated proteins (MAPs) and microtubule targeting agents (MTAs).

Here we present a new application of nanoDSF instrument that will significantly facilitate microtubule polymerization studies. Equipped with up to 48 capillaries, the Nanotemper Prometheus originally designed to study thermostabtility of purified proteins, turns out to be also a powerful tool to monitor microtubules polymerization using 30 times less tubulin. Through examples of tubulin polymerization in the absence or in the presence of MTAs or MAPs, we will show how we can easily determine the same parameters than with classical turbidimetry, and additional ones (including melting temperature of tubulin, and of microtubules) that bring new insights into this long studied process.


37

New insights on zinc-induced aggregation of proteins involved in neurodegenerative diseases

Romain La Roccaa,b, Andrei Yu. Romana,b, Deborah Byrnec, Natalia N. Ninkinad, HervéKovacica,b, Vincent Peyrota, Philipp O. Tsvetkova,b, François Devreda,b

a Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Fac Médecine, Marseille, France;bAix-Marseille Univ, Plateforme Interactome Neurotimone (PINT), Fac de Médecine, Marseillec Institut de Microbiologie de la Méditerranée, CNRS, FR3479, Aix-Marseille Univ, Marseille,d School of Biosciences, Cardiff University, Sir Martin Evans B., Museum Avenue, Cardiff, UK.

There is a whole class of aggregation-prone proteins associated with different proteinopathies, i.e. neurodegenerative disorders (NDDs) with protein aggregation-based pathology, such as Alzheimer disease (AD) or ALS. The original trigger of aggregation in these diseases is not known. Still there are different endogenous factors such as mutations and post-translational modifications that could favor the transition of proteins into pathological misfolded conformations and favor their aggregation. But one factor, could accelerate aggregation of different proteins implicated in different NDDs. This is zinc. Thus, if not the causative agent, zinc is considered as an important factor that is implicated in the development of pathological protein aggregation in some NDs.

Here we investigated the effect of zinc on aggregation of two proteins implicated in AD and ALS, Tau and TDP43. Using set of biophysical methods, for the first time we have demonstrated the direct binding of zinc to TDP-43 RNA binding domain. Furthermore, we characterized zinc binding to Tau and TDP43 from thermodynamic point of view and investigated aggregation that accompany this binding. We have found that contrary to TDP-43 which has only one zinc binding site, Tau possesses one high affinity and three low affinity sites. Interestingly, while zinc result in irreversible aggregation of TDP-43 in Thioflavin T positive amyloid-like structures, aggregation of Tau in the presence of zinc is reversible and Thioflavin T negative. Moreover, zinc-induced Tau aggregation is strongly depends on temperature. Further investigation, allowed us to hypothesize that binding of the first zinc ion to Tau, could play an important structuring and functional role, while low affinity sites are mainly responsible to Tau aggregation. Altogether our finding allowed to reveal new insights that are important for understanding of molecular mechanisms of zinc-induced protein aggregation and development of effective inhibitors of this process.


38

Differential Scanning Fluorimetry: New approach in diagnostic of neurodegenerative diseases.

Tsvetkov PO1, Tabouret E1, Malezinski S1, Figarella-Branger D2, Chinot O1, Cécile Pereira2, Eyraud R2”, Peel T3, Devred F1.

1 Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Fac Médecine, Marseille, France;

2 Aix-Marseille Univ, Laboratoire Informatique des Systèmes (LIS), Marseille, France

3 EuraNova, Marseille, France;

For more than 10 years, several pilot studies have hypothesized that Differential Scanning Calorimetry (DSC) could be used as a potential diagnostic tool for a number of pathologies by providing disease-specific calorimetric denaturation profiles of blood plasma (1). Recently we have shown that this approach can be also used in Glioblastoma, the most frequent and aggressive primary brain tumor in adults (2). Comparing the DSC denaturation profiles of plasma samples from patients with glioblastoma with the ones from healthy individuals revealed the existence of a glioblastoma signature beyond the blood brain barrier, pointing to a potential easy-to-use non-invasive monitoring tool for glioblastoma patients.

Unfortunately, there are several obstacles inherent to the DSC itself that make this approach non-transferable to the clinics. To circumvent these limitations, we proposed to use differential scanning fluorimetry (DSF) rather than DSC to obtain denaturation profiles of plasma samples. Using nanoDSF Prometheus NT instrument, we were able to obtain denaturation profiles of human plasma similar to the ones registered by DSC. The ability of this instrument to run up to 48 disposable capillaries with only 10 µL of undiluted plasma samples makes it not only more suitable for the large-scale experiments necessary for statistically robust results but will also facilitate its transfer to clinic. In order to develop a real diagnostic tool based on nanoDSF, it is necessary to perform the complex analysis of large amount of multiparametric data extracted from these profiles. In collaboration with academic and private scientists specialized in Artificial Intelligence solutions we are developing a classification method of the plasma denaturation profiles based on deep learning algorithms to make this profile analysis automatic and applicable to a wider range of pathologies.

References

[1] Garbett NC, Mekmaysy CS, DeLeeuw L, Chaires JB. Clinical application of plasma thermograms. Utility,practical approaches and considerations. Methods. 2015; 76:41–50. doi: 10.1016/j.ymeth.2014.10.030

[2] Tsvetkov PO, Tabouret E, Roman AY, Romain S, Bequet C, Ishimbaeva O, Honoré S, Figarella-Branger D,Chinot O, Devred F. Differential scanning calorimetry of plasma in glioblastoma: toward a new prognostic /monitoring tool. Oncotarget. 2018; 9(10):9391-9399. doi: 10.18632/oncotarget.24317


39

MCT

40

TEMP017

DESIGN AND SYNTHESIS OF CHEMICAL TOOLS TO EARLY

DETECT ANTIBIOTIC RESISTANCE IN CLINIC

Johan Revol-Tissot, Sandrine Alibert, Julia Vergalli, Gérard Boyer, Jean-Marie Pagès, Jean-Michel Bolla

Aix Marseille Univ, INSERM, SSA, MCT, FAC PHARM, Marseille, France

Antimicrobial resistance (AMR) constitutes a major problem of Public Health. The 2014 WHO’s global report

[1] outlined worrying levels of AMR worldwide involving therapeutic failure of the large classes of antibiotics

as quinolones, beta-lactams, macrolides, and cyclins especially in gram-negative bacterial diseases.

Resistance is a natural response of bacteria allowing them to counteract pharmacological effects of antibiotic

agents. A lot of mechanisms as membrane permeability variations, enzymatic degradations and intracellular

target modifications contribute to the Multi-Drug Resistance (MDR) phenotypes. Especially, efflux

overexpression is an early stage trigger in the MDR setting up [2].

Resistance-Cell-Nodulation (RND) efflux pump family constitute a tripartite protein system which play a major

role in gram-negative bacteria MDR [3] because they are able to extrude structurally different substances outside

the cell space; hence antibiotics struggle to reach effective intracellular concentrations.

We are working on new natural molecules derivatives with fluorescent properties useful to detect their capacity

to accumulate in bacteria (according to a fluorescence method developed in our laboratory [4]). These

compounds are susceptible to efflux pump activity while having no cytotoxic effects on bacteria growth to

minimize their resistance impact. A medicinal chemistry approach allows us the synthesis of high molecular

variability products and the study of their Structure-Activity-Relationships to identify pharmacophores and

physicochemical properties responsible for their efflux pump interactions and their substrate quality. Our aim is

to design a real-time diagnosis trial in order to identify and prevent early bacterial resistance in clinic.

References

1) Antimicrobial Resistance: Global Report on Surveillance 2014. WHO. Available from:

http://www.who.int/drugresistance/documents/surveillancereport/en/

2) Masi M, Réfregiers M, Pos KM, Pagès JM. Mechanisms of envelope permeability and antibiotic influx and efflux in

Gram-negative bacteria. Nat Microbiol. 2017 Feb 22;2:17001.

3) S. Alibert, J. N’gompaza Diarra, J. Hernandez, A.Stutzmann, M. Fouad, G. Boyer & J-M. Pagès (2016): Multidrug efflux

pumps and their role in antibiotic and antiseptic resistance: a pharmacodynamic perspective, Expert Opinion on Drug

Metabolism & Toxicology

4) Vergalli J, Dumont E, Pajović J, Cinquin B, Maigre L, Masi M, Réfrégiers M, Pagés JM. Spectrofluorimetric

quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial

membranes. Nat Protoc. 2018 Jun;13(6):1348-1361.

41

MULTIDRUG RESISTANCE MARKS THIS DECADE!

ANTIBIOTIC ADJUVANTS TO RESCUE PSEUDOMONAS AERUGINOSA FROM RESISTANCE

Multidrug resistant (MDR) bacteria are one of the major current threats to public health. The

incidence of nosocomial infections caused by MDR has increased dramatically in the community and

is associated with a terrifying rate of morbidity, mortality and antibiotic use.

The molecular mechanisms by which bacteria, particularly Gram-negatives, became resistant to

antibiotics are diverse and complex. Nowadays, Bacteria have developed resistance to all different

classes of antibiotics discovered.

Without an innovative strategy to combat multi-drug resistant (MDR) pathogens, many fields of

medicine will be severely affected, thus, new solutions are required to enhance the antibiotic

efficiency and/or reduce the mechanism of resistance.

In this context, we aim to develop an attractive strategy consisting in the synthesis of new

polyaminisoprenyl molecules. These chemo-sensitizers are readily prepared from farnesol in a two

steps synthesis with moderate to excellent yields and they are able to restore the activity of

doxycycline against pseudomonas aeruginosa bacterial strains.

We demonstrate that among 7 analogues tested one of them presented an excellent capacity to

enhance doxycycline activity. Typically, this compound named NV716 was assayed against Gram

negative strains and proved to act in a synergetic manner with doxycycline (2mg/L) at very low

concentrations in the range of 2.5-10 µM. Thus we showed that NV716 is able to decrease the MIC of

doxycycline on various reference strains of pseudomonas aeruginosa to the sensibility level (2mg/L).

The mechanism of action of this compound is now under current investigation.


42

https://www.sciencedirect.com/science/article/pii/S0966842X16300725

AN AEROSOLIZED ANTIMICROBIAL COMBINATION FOR

TREATMENT OF Pseudomonas aeruginosa INFECTIONS

Hana DOUAFER, Véronique ANDRIEU and Jean Michel BRUNEL

During the last decades, multiple approaches have been developed to combat bacterial

resistance. One of them involves combination therapies of existing antibiotics with potentiating

adjuvants, re-empowering the activity of the antibiotic against resistant strains by improving

the permeability or suppressing antibiotic efflux.

Currently, many drug delivery systems are used and aim for an optimal drug administration by

minimizing drug degradation, decreasing its side effects and increasing its bioavailability. In

this context, pulmonary route has become an attractive pathway for infections treatment.

The aim of our study is to develop and evaluate a new potent pharmaceutical form intended for

pulmonary administration to defeat cystic fibrosis, based on a combination of an antibiotic

(Doxycycline) with an adjuvant: a polyaminoisoprenyl derivative NV716 allowing restoration

of doxycycline efficacy against P.aeruginosa strains naturally resistant to doxycycline. The

proof of concept of such a combination has been previously verified in vitro on various P.

aeruginosa strains1.

Here we report the characterization of three different aerosols: doxycycline alone, NV716 alone

and doxycycline/NV716 combination, using the Next Generation Impactor (NGI) (EP, USP).

Aerosols evaluation was carried out according to different concentrations, durations of

nebulization and nebulizers: Pari LC Plus, Pari Sprint (breath-enhanced jet nebulizers) and the

Pari Eflow (vibrating mesh nebulizer). The droplet size distribution and aerosol efficiency were

expressed in terms of Mass Median Aerodynamic Diameter (MMAD) and Fine Particle

Fraction (FPF).

Results showed MMADs (3,4-4,4 μm) in accordance with the standards recommended for

therapeutic aerosols (< 5 μm) (EP, USP) suitable for deep lung deposition, with a high FPF (>

50 %) required to maintain drug level above its Minimal Inhibitory Concentrations (MIC) at

the infection site.

References

1) Borselli, D. et al. Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P.aeruginosa to Doxycycline and Chloramphenicol In Vitro. PLOS ONE 11, e0154490 (2016).


43

Microwave Oppenauer Oxidation: Revival of an Old Reaction Sophie Négrel, Jean-Michel Brunel

Oxidation of alcohols into the corresponding carbonyl products is one of the most fundamental and important reaction in organic synthesis. Different methods were described in literature as Jones oxidation with chromium trioxide and sulfuric acid or Swern oxidation using oxalyl chloride, dimethyl sulfoxide and triethylamine. In 1937, Rupert Viktor Oppenauer found that in presence of aluminum tert-butoxide, acetone acts as a hydrogen acceptor for the oxidation of primary and secondary alcohols leading to the corresponding aldehydes or ketones. 1 (Scheme 1) A great advantage of that reaction over many oxygen-transferring oxidation processes is that functional groups such as carbon-carbon double and triple bonds, aldehydes, amino groups, halogens or sulfur atom-containing groups are not affected.

O

R1 R2

O

R1 R2

OH Al(OR3)3 OH

12h, RefluxToluene

Scheme 1: Example of Oppenauer reaction

In the last decades, Microwave Assisted Organic Synthesis (MAOS) have attracted the attention of chemists who have begun to apply this unconventional technique of material heating as a routine in their practice. Heating in microwave is based upon the ability of some liquids and solids to absorb and to transform electromagnetic energy into heat. Microwave assisted reactions in organic chemistry is found to be faster and more effective than conventional methods and is able to reduce reaction time from days and hours to minutes. Microwave heating process operates under high pressure conditions in order to make a reaction faster and to limit side reactions.

In our hands, Oppenauer oxidation performed under microwave conditions was used to synthesized ketosterol on a selective way. In classic heating conditions this oxidation was performed in 12 to 24h at reflux with conversion around 60%, whereas this reaction was achieved in few minutes with a complete conversion by using microwave irradiation. We studied numerous experimental parameters as kinetic evolution, temperature and solvents as well as the nature of aluminum derivatives and reductive ketone involved. Thus, Oppenauer oxidation under microwave conditions was optimized on methyl chenodeoxycholate as substrate model and therefore was extended to numerous alcohols in moderate to excellent chemical yields.

As exemplified, this strategy was successfully applied to the synthesis of a key intermediate involved in the synthesis of Claramine C1, a polyaminosterol compound demonstrating antibacterial activity against a large panel of both Gram-positive and Gram-negative bacteria.

1 Oppenauer, R. V., Recl. Trav. Chim. Pays-Bas, 1937, 56, 137-144


44

MEPHI

45

Les antiplaquettaires inhibent l’effet antibactérien des plaquettes sur Staphylococcus aureus.

Nadji Hannachi1, Laurence Camoin-Jau1,2. 1Aix Marseille Univ, IRD, APHM, MEPHI, IHU Méditerranée infection, Marseille, France 2Laboratoire d'Hématologie, La Timone Hospital, APHM, boulevard Jean- Moulin, 13005 Marseille, France

Contexte : Les plaquettes possèdent une activité anti-infectieuse. Cette action repose notamment sur les tPMP sécrétées par des granules alpha lors de l’activation plaquettaire. Cependant, l’effet des traitements antiplaquettaires sur la capacité antibactérienne des plaquettes est mal connue. Le but de cette étude est d’étudier l'effet antibactérien des plaquettes sur trois espèces bactériennes et d'évaluer l'effet des antiplaquettaires tels que l'aspirine, ainsi que les inhibiteurs de P2Y12 sur cet effet.

Matériel et méthodes : Des prélèvements de sang veineux ont été réalisés sur citrate de sodium chez des sujets sains et des patients traités par antiplaquettaire à long terme. Trois espèces bactériennes ont été étudiées Staphylococcus aureus, Enterococcus faecalis et Streptococcus sanguinis. Pour chaque espèce, 4 souches différentes ont été testées. Les bactéries (1.5 ; 3 et 10. 108 CFU/ mL) ont été incubées avec des plaquettes lavées (4.108/ mL) à 37°C pendant quatre heures. Après des dilutions en série, le mélange a été ensemencé sur gélose au sang pour dénombrer les colonies après 18 heures d'incubation. En parallèle, des plaquettes de sujet sains, traitées in vitro par de l'aspirine ont été testées dans les mêmes conditions. En contrôle, les souches seules ont été ensemencées dans les mêmes conditions de concentration, de température et de durée.

Résultats : L'effet plaquettaire était différent selon les espèces bactériennes. Les plaquettes ont diminué de manière significative la croissance des souches de S. aureus. Il faut cependant noter des différences de sensibilité en fonction des 4 souches testées (p< 0.05 pour les 4 souches, n =11). La croissance d’E. faecalis n’a pas été modifiée en présence de plaquettes quel que soit la souche testée (p > 0.05 pour les 4 souches, n=11). En revanche, les plaquettes ont induit une augmentation de la croissance des 4 souches de S. sanguinis testées par rapport au contrôle (p< 0.05 pour les 4 souches, n =11). En présence de plaquettes provenant de patients traités par aspirine ou d’inhibiteurs du récepteur P2Y12, la croissance de S. aureus est comparable au contrôle. Les traitements antiplaquettaires induisent donc la perte de l’activité bactéricide des plaquettes sur les 4 souches de S. aureus testées. Un résultat comparable a été obtenu à partir des plaquettes provenant de sujets sains et ayant été traitées par de l’aspirine in vitro. En revanche, les traitements antiplaquettaires ne modifient pas l’effet des plaquettes sur les souches de E. faecalis et S. sanguinis.

Conclusion : Notre étude a démontré que les plaquettes avaient un effet antibactérien différent en fonction des espèces bactériennes. En outre, les antiplaquettaires diminuent l’effet antibactérien des plaquettes sur S. aureus.


46

Title: Metagenomic analysis, an alternative approach to decipher the antibiotics

resistance genes contained in fecal samples from patients with acute leukemia.

Rym Lalaoui, Ana Djukovic, Miharimamy Rajaonison, Sofiane Bakour, Jaime Sanz, Miguel

Salavert, Jose Luis López-Hontangas, Miguel A. Sanz, Seydina M. Diene, Carles Ubeda, Jean-

Marc Rolain

Background: Colonization of intestine of immunocompromised patients with multidrug-

resistant bacteria complicates the management of bacterial infections. The aim of this study was

to describe the antibiotics resistance genes (ARGs) content in fecal sample from Spanish

leukemic patients and to understand the evolution of resistome during period of hospitalization

using metagenomic analysis.

Materials/methods: 802 fecal samples were collected between 2013 and 2015 from 133 acute

leukemia patients. The DNA from a subset of 217 fecal samples from 56 patients was extracted

using a QIAamp® Fast DNA Stool Mini kit and then sequenced on Hiseq-Illumina. Read

sequences were assembled with SPAdes assembler using the ‘meta’ algorithm. For each sample,

contigs with > 2’500 bp were selected. ARGs were screened by blasting the generated contigs

to the ARG-ANNOT database using specific parameters (≥ 90% ofidentity and coverage).

Results: As primarily results, overall, we identified 203 distinct antibiotic resistance genes

including TetQ which was the most commonly found resistance gene followed by MefA, TetW,

Aph3-III and ErmB. Fourteen different families of antibiotic resistance genes were identified,

the 5 most common of which were families conferring resistance to tetracyclines (Tet), β-

lactamines (bla), Macrolides-lincosamides-streptogramines (MLS), aminosides (AGly) and

sulfamides (Sul). The analysis of the remaining metagenomes is in progress.

Conclusions: Despite the fact that our study is not complete, it already clearly shows the

usefulness of the metagenomic analysis approach in the field of clinical setting, providing the

clinician additional information that can facilitate the choice of appropriate treatment and avoid

complications that can affect patients, particularly those at high risk.


47

Titre: Étude de la sensibilité des virus géants d’amibes à des molécules médicamenteuses de multiples classes thérapeutiques

Liste des auteurs : Céline BOSCHI1,3 , Rania FRANCIS1, Sarah AHERFI1,2, Jean-Marc ROLAIN1,2,3, Bernard LA SCOLA1,2, Philippe COLSON1,2,3

Affiliations: 1Institut Hospitalo-Universitaire (IHU) - Méditerranée Infection, 19-21 boulevard Jean Moulin, 13005 Marseille, France; 2Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), Assistance Publique - Hôpitaux de Marseille (AP-HM); Microbes, Evolution, Phylogeny and Infection (MEΦI); 3Aix-Marseille Université, UFR Pharmacie, 27 boulevard Jean Moulin, 13005 Marseille, France

Mots clés : Virus géants ; amibes ; molécules médicamenteuses

Les virus géants d’amibes sont les plus grands virus connus à ce jour. Ils ont une complexité

similaire à celle des micro-organismes cellulaires. Le premier virus géant découvert en 2003 a

été Mimivirus. Depuis, plus de 100 virus géants classés dans neuf groupes ont été isolés par

culture sur les amibes Acanthamoeba castellanii et Vermamoeba vermiformis, parmi lesquels

Marseillevirus et Faustovirus. Les virus géants sont communs dans l'eau et le sol. Un nombre

croissant de données montrent leur présence chez l'Homme, même si leur pathogénicité reste

controversée. L’objectif au cours de ce master a été d’étudier la sensibilité de virus géants

d’amibes à de multiples molécules médicamenteuses.

Un criblage systématique à haut débit a été réalisé pour une librairie de 1280 molécules

médicamenteuses (Prestwick chemical library) testées à une concentration de 10 μM en

utilisant une technique de criblage à haut contenu. Des tests de cytotoxicité ont d’abord été

réalisés sur A. castellanii et V. vermiformis suivis d'inhibition de la lyse amibienne par

Mimivirus, Marseillevirus et Faustovirus.

Huit (0.6%) des 1280 molécules testées ont montré un effet cytotoxique sur A. castellanii et

15 (1.2%) sur V. vermiformis. Les tests d’activité antivirale ont montré une inhibition de la

lyse amibienne sur les co-cultures A. castellanii/Mimivirus et A. castellanii/Marseillevirus

par une molécule, la sertraline (un antidépresseur inhibiteur sélectif de la recapture de la

sérotonine). Des PCR quantitatives ont confirmé une réduction de la réplication virale. La

sertraline a également inhibé la lyse amibienne par Legionella pneumophila, indiquant que

l'effet observé n'est pas spécifique des virus géants. Aucune molécule n’a eu d’effet sur la co-

culture V. vermiformis/Faustovirus.

Ces résultats montrent alors le potentiel d’approches systématiques massives à haut débit pour

l’étude et la caractérisation de l’effet antimicrobien de certains molécules en utilisant une

nouvelle approche pour la culture virale.


48

Titre: Détection d'une réactivation du virus de l'hépatite B et séquençage quasi-complet du génome viral par séquençage à haut débit chez un greffé du rein Liste des auteurs : Maelia Swali ETOUNDI1,3 , Valérie MOAL4, Sarah AHERFI1,2, Anne MOTTE1, Philippe COLSON1,2,3

Affiliations: 1Institut Hospitalo-Universitaire (IHU) - Méditerranée Infection, 19-21 boulevard Jean Moulin, 13005 Marseille, France; 2Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), Assistance Publique - Hôpitaux de Marseille (AP-HM); Microbes, Evolution, Phylogeny and Infection (MEΦI); 3Aix-Marseille Université, UFR Pharmacie, 27 boulevard Jean Moulin, 13005 Marseille, France; 4Assistance Publique - Hôpitaux de Marseille (AP-HM), Centre Hospitalo-Universitaire Conception, Service de transplantation rénale, 147 boulevard Baille, 13005 Marseille Mots clés : Hépatite B; réactivation; transplantation rénale; génomique Introduction : Le virus de l’hépatite B (VHB) est un virus hépatotrope à ADN de la famille Hepadnaviridae. Sa prévalence est importante dans le monde entier. En France, 7% des adultes ont une cicatrice sérologique d’infection ancienne et 0.6% ont une infection chronique. L'ADN persiste sous forme d’ADN superenroulé circulaire clos (ADNccc) dans le noyau des hépatocytes, ce qui permet une réactivation virale en cas d’immunodépression sévère. Les transplantés rénaux constituent ainsi un groupe à haut risque de réactivation VHB du fait de la forte prévalence des profils sérologiques indiquant une hépatite B ancienne guérie (jusqu’à 20%), et de la diminution progressive des anticorps anti-HBs et de l’immunodépression entrainées par le traitement immunosuppresseur. Le séquençage de nouvelle génération, qui permet de détecter les mutants du VHB avec une plus grande sensibilité et d'obtenir le génome complet plutôt que seulement des gènes, émerge progressivement en diagnostic clinique de routine. Nous décrivons ici l’obtention d’un génome quasi-complet du VHB en utilisant cette technologie directement à partir du plasma d'un transplanté rénal présentant une réactivation du VHB. Cas clinique : La réactivation VHB a été diagnostiquée chez un patient de 73 ans, originaire de Madagascar, vivant en France depuis 1975. Il avait reçu une transplantation rénale en août 2016 et a été traité par la suite par azathioprine, prednisone, ciclosporine A et thymoglobuline. Sa sérologie VHB pré-greffe montrait un AgHBs négatif, des anticorps anti-HBs négatifs, et des anticorps anti-HBc positifs (techniques Architect Abbott, Abbott Diagnostics, Chicago, IL, USA), indiquant une infection ancienne guérie. Mors du suivi systématique post-greffe, sa sérologie VHB montrait en février 2019 un AgHBs positif, des anticorps anti-HBs négatifs, et des anticorps anti-HBc positifs. La charge virale VHB était de 42,480,000 UI/mL (technique Veris DxN Beckman-Coulter Diagnostics, Brea, CA, USA). Le patient était cliniquement asymptomatique, et il n’y avait pas de cytolyse hépatique. Le séquençage du génome quasi-complet a été réalisé directement à partir de l'extrait ADN obtenu du plasma par séquençage de nouvelle génération par la technologie Illumina sur automate MiSeq avec le protocole paired-end (Nextera XT protocol, Illumina Inc, San Diego, CA, USA). Le génome obtenu a une taille de 3182 nucléotides et la couverture moyenne (±écart-type) est de 753±269 reads/position nucléotidique (min.-max.: 155-1200). La diversité nucléotidique observée est en moyenne de 0,4±2,1% (0,0-45,8). Le génotype viral est D2. Des substitutions d'acides aminés dans l’AgHBs (P120Q/L (10% des quasi-espèces); P142L (40%); R122K (59%), et G145R (100%)) décrites comme altérant son antigénicité et associées à un échappement immunitaire, ainsi qu’une substitution dans la transcriptase inverse (I169L) associée à la résistance aux médicaments antiviraux ont été observées. Le patient a été traité par Entécavir 0,15 mg/j, adaptés à la fonction rénale, en association à une diminution de l’azathioprine de 75 à 50 mg/j puis à son arrêt en avril 2019, avec une augmentation de la ciclosporine de 50 mg 2x/j à 75 mg 2x/j. Discussion et perspectives : Ce cas montre l'importance d'une surveillance systématique des réactivations VHB chez les transplantés du rein présentant un profil sérologique indiquant une hépatite B ancienne guérie, et l'intérêt du séquençage de nouvelle génération en diagnostic de routine afin de caractériser de manière plus exhaustive les virus impliqués dans ces réactivations.


49

Massive analysis of 64’628 bacterial genomes to decipher a water reservoir and origin of

mobile colistin resistance variants: is there another role for these enzymes?

Mariem Ben Khedher, Sophie Alexandra Baron, Toilhata Riziki, Raymond Ruimy , Seydina

M. Diene, Jean-Marc Rolain

Abstract

Since 2015, new worrying colistin resistance mechanism, mediated by mcr-1 gene has been

reported worldwide along with eight newly described variants (mcr-2 to mcr-9) but their

source(s) and reservoir(s) remain largely unexplored. Here, we conducted a massive

bioinformatic analysis of bacterial genomes to investigate the reservoir and origin of mcr

variants. First, we identified 13’658 homologous sequences to MCR-1 in a total of 494 bacterial

genera. Secondly, our analysis of 64’628 bacterial genomes from 60 bacterial genera that

include 1’047 species, allows identifying a total of 1’386 significant hits with the nine MCR

protein variants from 14 different genera (aa identity ≥ 90% and coverage ≥ 98%). A high

number of MCR-1 was observed in Escherichia coli (n=862). Interestingly, while almost all

variants were identified in bacteria isolated from different sources (i.e. human, animal, and

environment), the last variant, MCR-9, was exclusively detected in bacteria from human.

Moreover, with BlastP thresholds (i.e. 50% < aa identity > 90% and coverage > 90%), an

additional subset of 5’265 MCR hits in 25 bacterial species were identified; those can being

putative MCR variants that have not yet been discovered. Although these variants could be

identified in bacteria from human and animal sources, we found plenty MCR variants in

unsuspected bacteria from environmental origin, especially from water sources. The ubiquitous

presence of mcr variants in bacteria from water likely suggests another role in the biosphere of

these enzymes as an unknown defense system against natural antimicrobial peptides and/or

bacteriophage predation.


50

Title: Inactivation of mgrB gene regulator and resistance to colistin is becoming endemic

in carbapenem-resistant Klebsiella pneumoniae in Greece: a nationwide study from 2014

to 2017.

Hamel Mouna, Chatzipanagiotou Stylianos, Hadjadj Linda, Petinaki Efthimia, Papagianni Sophia,

Charalampaki Nikoletta, Tsiplakou Sophia, Papaioannou Vassiliki, Skarmoutsou Nikoletta,

Spiliopoulou Iris, Christofidou Myrto, Papamichalopoulos Nikolaos, Skalidis Tilemachos, Legakis

Nicholaos, Fountoulis Kimon, Perivolioti Efstathia, Kraniotaki Heleni, Bournia Maria, Ioannidis

Anastasios, Baron Sophie Alexandra, Rolain Jean-Marc.

Abstract

Introduction: In Greece, the spread of carbapenem-resistant Enterobacteriaceae in humans has led to

the reintroduction of colistin as a therapeutic agent. Unfortunately, resistance to colistin has emerged

with different mechanisms involved. The present work aims to determine the prevalence of carbapenem

and colistin resistance and the corresponding mechanisms in Klebsiella pneumoniae clinical isolates

from Greece. Methods: From 2014 to 2017, 288 carbapenem-resistant K. pneumoniae clinical strains

were collected among a collection of 973 isolates from eight different hospitals in Greece. Antibiotic

susceptibility testing was performed using three different methods. Screening of carbapenem and

colistin resistance genes was conducted using PCR amplification and sequencing. Results: among the

288 (29.6 %) carbapenem-resistant isolates, 213 (73.9%) were colistin-resistant (MIC>2 mg / L). The

KPC type was the most common carbapenemase gene (116; 40.3%), followed by VIM (41; 14.2%),

NDM (33; 11.5%) and OXA-48 (22; 7.6 %). Moreover, 44 (15.3%) strains co-produced two types of

carbapenemases. For colistin resistance, no mcr genes were detected, but rather mutations in

chromosomal genes were found. These included inactivation of the mgrB gene for 148 (69.5%),

including insertion sequences 94 (44.1%) as well as nonsense 4 (1.9%) and missense mutations 24

(11.3%). Moreover, PCR amplification of mgrB gene was negative for 26 (12.2%) strains. Finally, 65

(30.5%) colistin-resistant strains exhibited a wild-type mgrB, the mechanisms of which remain to be

elucidated. Conclusion: Our study shows that K. pneumoniae clinical strains in Greece are resistant to

both carbapenems and colistin which becomes endemic and likely chromosomally encoded.


51

Major discrepancy between factual antibiotic resistance and consumption in South of

France: analysis of 539,037 bacterial strains.

Ousmane Oumou Diallo, Sophie Alexandra Baron, Gregory Dubourg, Hervé Chaudet, Philippe Halfon, Sabine Camiade, Béatrice Comte, Stéphanie Joubert, Arnaud François, Philippe Seyral, François Parisot, Jean-Paul Casalta, Raymond Ruimy, Christophe Maruejouls, Jean-Christophe Achiardy, Sophie Burignat, Joseph Carvajal, Edouard Delaunay, Sandra Meyer, Pierre-Yves Levy, Patricia Roussellier, Patrick Brunet, Claude Bosi, Philippe Stolidi, Jean-Pierre Arzouni, Gisele Gay, Pierre Hance, Philippe Colson, Didier Raoult and Jean-Marc Rolain Abstract Introduction: The burden of antibiotic resistance is usually estimated by mathematical modeling, but this method is not directly factual. The objective of this work was to report the actual rate of antibiotic resistance (AR) of the bacteria most frequently isolated in human infectious diseases in our local epidemiological investigation network involving community acquired and hospital acquired infections to evaluate this problem factually. Methods: We conducted a retrospective study on antibiotic susceptibility of 539,107 strains

isolated in both hospital and private laboratories in the Provence-Alpes-Côte d'Azur (PACA)

region, France from January 2014 to January 2019. The resistance rate to key antibiotics as well

as the proportion of bacteria classified as Difficult-to-Treat (DTR) have been determined and

compared with the Mann-Whitney U test, the χ2 test or the Fisher’s exact test.

Results: Of the 303 laboratories that make up our current monitoring network, 267 isolated a

total of 711,031 strains including 539,037 isolates from 15 frequently bacteria responsible for

human infections, for which antibiotic susceptibility results were available. Overall, we did not

observe any significant general increase or decrease in resistance for 5 years, including oxacillin

resistance in Staphylococcus aureus, carbapenem resistance in enterobacteria and

Pseudomonas aeruginosa and 3rd generation cephalosporin in Escherichia coli and Klebsiella

pneumoniae. However, we observed a significant decrease in imipenem resistance for

Acinetobacter baumannii from 2014 to 2018 (24.19% to 12.27%; p=0.005) and a significant

increase of ceftriaxone resistance in Enterobacter aerogenes (9.9% to 24,03%; p=0.001) and

Enterobacter cloacae (24,05% to 42,05%; p=0.004). Of these 539,037 isolates, 1,604 (0.3%)

had a DTR phenotype.

Conclusion: Over a 5-year period, we did not observe a burden of AR in our region despite a

high rate of antibiotic consumption in our country. These results highlight the need for

implementation of AR surveillance systems which use factual data but are access limited by the

General Data Protection Regulations legislation.


52

MMG

53

Cell nucleus mechanics and premature senescence

Cécile Jebane1, Alice-Anais Varlet2, Catherine Badens2, and Emmanuèle Helfer1

1Aix Marseille Univ, CNRS, CINaM, Marseille, France2Aix Marseille Univ, MMG, Inserm, Marseille, France

Abstract

This project aims to propose an integrated model to better understand a pathway

to premature senescence. Cellular senescence is a normal process de�ned as the pro-

gressive decline of all cellular functions ending with cell cycle arrest. When senescence is

accelerated, it induces premature ageing and pathologies such as metabolic syndrome or

cardiovascular diseases. It can a�ect parts of the organism or the whole body. In the most

severe case of Progeria, where the patient ages prematurely, a pathway to senescence is

related to a defect in the lamina. Lamina is a component of the nuclear envelope (NE)

which ensures the mechanical stability and rigidity of the nucleus and chromatin integrity.

NE and associated proteins play a major role in the cell response to various external envir-

onmental mechanical stimuli. Two hypotheses are evoked in laminopathies, genetic diseases

induced by lamina mutations. The �rst one, the mechanical hypothesis, proposes that

alterations of the NE protein network lead to increased nucleus susceptibility to mechanical

stress (mechanotransmission). The second, the gene expression hypothesis implies that

NE structural changes impact directly on gene transcription (mechanotransduction). In

this project, we investigate the relationship between lamina mutations, alteration of cell

mechanotransduction and severity of the disease, from nuclear to multi-cellular scale. We

propose an interdisciplinary approach combining cell biology, biochemistry, and physics.

Firstly, we will determine the mechanical and rheological properties of single cells. Secondly,

we will explore how shear �ow a�ects cell organisation and mechanotransduction process

in a 2D-layer of lamina-de�cient cells. Thirdly, we will investigate the e�ect of isotropic

pressure on the geometry and gene expression of 3D-aggregates. From our results, we aim

to develop a physical model of the senescence pathway.


54

VITROME

55

MASTER INGÉNIERIE DE LA SANTÉ

56

Projet Tuteuré

M1 MPS

2018-2019

Levothyrox® : questions et réponses

Laboratoire : Pharmacocinétique

Equipe : SMARTc

Tuteur : Dr Florence GATTACCECA Adresse : Faculté de Pharmacie N° de téléphone : +33 4 91 32 46 70 E-Mail : [email protected]

Sujet :

Le Levothyrox® est un médicament destiné aux patients qui souffrent de troubles de la thyroïde, glande qui régule de nombreuses fonctions du métabolisme. Il s’agit d’un traitement hormonal de substitution, consommé par près de 3 millions de personnes en France. La composition du médicament a changé fin mars 2017 à la demande de l’ANSM pour améliorer la stabilité et supprimer un excipient à effet notoire, le lactose. Depuis, des milliers de patients affirment souffrir d’effets secondaires nombreux, invalidants et graves. En cause : le changement de formulation. On se propose avec ce projet de rétablir la vérité sur un sujet de santé publique qui a provoqué une crise sanitaire et un véritable tollé.


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Projet Tuteuré

M1 MPS

2018-2019

Perturbateurs endocriniens exerçant leur action en agissant sur la méthylation des promoteurs de leurs gènes cibles

Laboratoire :IMBE

Equipe : Bio-indicateurs Environnement et Santé

Tuteur : VILLARD Pierre-Henri Adresse : Laboratoire Toxicologie UFR Pharmacie N° de téléphone : 0491835638 E-Mail : [email protected]

Sujet :

Les perturbateurs exercent leurs effets délétères le plus souvent par des mécanismes épigénétiques. Parmi ces mécanismes, figure la méthylation de l'ADN. L'étude de la méthylation des gènes peut être réalisée au moyen de la technique des puces à ADN qui permettent des définir l'intégralité des "CpG islands", mais cette technique est couteuse. Nous nous proposons d'utiliser l'Hydre comme espèce sentinelle et de déterminer dans cet organisme la méthylation totale de l'ADN au moyen de la technique ELISA. Pour ce faire, il faudra réaliser, au cours du projet tutoré, une étude bibliographique, pour lister les principaux perturbateurs endocriniens agissant sur la méthylation (PEMs) de l'ADN et leur taux dans l'eau douce de surface. Par la suite, des Hydres seront exposées à des doses environnementales de ces PEMs, les atteintes morphologiques, la reproduction asexuée et la méthylation totale de l'ADN seront évaluées.

Références bibliographiques :

Toxicol Lett. 2018 Oct 15;296:10-22. doi: 10.1016/j.toxlet.2018.07.011. Epub 2018 Jul 11. Endocrine Disruption: Current approaches for regulatory testing and assessment of plant protection products are fit for purpose. Day P, Green RM, Gross M, Weltje L, Wheeler JR.

Basic Clin Pharmacol Toxicol. 2018 Aug 23. doi: 10.1111/bcpt.13118. [Epub ahead of print] The developmental basis of disease: Update on environmental exposures and animal models. Heindel JJ

Environ Int. 2018 May;114:77-86. doi: 10.1016/j.envint.2018.02.014. Epub 2018 Feb 27. Epigenetics as a mechanism linking developmental exposures to long-term toxicity. Barouki R, Melén E, Herceg Z, Beckers J, Chen J, Karagas M, Puga A, Xia Y, Chadwick L, Yan W, Audouze K, Slama R, Heindel J, Grandjean P, Kawamoto T, Nohara K.

Basic Clin Pharmacol Toxicol. 2018 Jan;122(1):38-45. doi: 10.1111/bcpt.12878. Epub 2017 Sep 14. Understanding Epigenetic Effects of Endocrine Disrupting Chemicals: From Mechanisms to Novel Test Methods. Alavian-Ghavanini A, Rüegg J.


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Environ Toxicol Pharmacol. 2017 Apr;51:94-99. doi: 10.1016/j.etap.2017.02.004. Epub 2017 Feb 10. Environmental epigenomics: Current approaches to assess epigenetic effects of endocrine disrupting compounds (EDC's) on human health. Tapia-Orozco N, Santiago-Toledo G, Barrón V, Espinosa-García AM, García-García JA, García-Arrazola R.

Reprod Biomed Online. 2016 Dec;33(6):668-683. doi: 10.1016/j.rbmo.2016.09.006. Epub 2016 Sep 27. Oxidative stress and alterations in DNA methylation: two sides of the same coin in reproduction. Menezo YJ, Silvestris E, Dale B, Elder K.

Front Cell Dev Biol. 2015 Jun 18;3:37. doi: 10.3389/fcell.2015.00037. eCollection 2015. Endocrine disrupters: the new players able to affect the epigenome. Casati L, Sendra R, Sibilia V, Celotti F.

Semin Cell Dev Biol. 2015 Jul;43:66-75. doi: 10.1016/j.semcdb.2015.05.008. Epub 2015 May 28. Multigenerational and transgenerational effects of endocrine disrupting chemicals: A role for altered epigenetic regulation? Xin F, Susiarjo M, Bartolomei MS.

Water Res. 2016 Jun 1;96:62-73. doi: 10.1016/j.watres.2016.03.043. Epub 2016 Mar 21. Multi-scale biomarker evaluation of the toxicity of a commercial azo dye (Disperse Red 1) in an animal model, the freshwater cnidarian Hydra attenuata. de Jong L, Pech N, de Aragão Umbuzeiro G, Moreau X.


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Projet Tuteuré

M1 MPS

2018-2019

Synthèse et design de molecules à large spectre dirigées contre les Picornavirus d’intérêt

Laboratoire : ICR – UMR 7273 Equipe : PCR (P. VANELLE) Tuteur : Manon ROCHE Adresse : Faculté de pharmacie, 27 Bd Jean Moulin, 13385 Marseille CEDEX 05 N° de téléphone : 04 91 83 55 25 E-Mail : [email protected]

Les Picornaviridae sont composés de 5 genres de virus : les enterovirus (EV), les rhinovirus (RV), les hepatovirus, cardiovirus et aphtovirus. Les EV et RV possèdent une morphologie proche notamment au niveau de la capside virale. Les enterovirus se découpent en plusieurs groupes (les poliovirus, les coxsackievirus A et B, les echovirus ; les rhinovirus en trois groupes distincts A, B et C. Les enterovirus sont impliqués dans des pathologies à sémiologie et clinique très variables allant de troubles bénins comme le syndrome « pied main bouche » à des pathologies mettant en jeu le pronostic vital à l’instar des méningites, endocardites et sepsis chez les nouveau-nés. Depuis quelques années, l’industrie pharmaceutique s’intéresse de plus en plus au genre des entérovirus mais également des rhinovirus, depuis que des travaux de recherche ont démontré leur association à des pathologies plus sévères comme l’asthme, la BPCO ou la mucoviscidose. De nombreuses molécules ont été développées pour cibler les protéines structurales et non structurales afin d’inhiber la réplication de ces virus. La protéine VP1 de la capside est une excellente cible pour bloquer l’infection de la cellule hôte. En effet, cette protéine possède une structure relativement identique pour toutes les espèces de RV/EV notamment au niveau de la région liante à la cellule hôte. De ce fait cette protéine est un très bon candidat pour le développement d’un inhibiteur à large spectre EV/RV. Trois molécules ont été évaluées dans ce cadre comme capsid binder (pirodavir, pleconaril et vapendavir) ; le vapendavir (Aviragen therapeutics) est en ce moment en évaluation clinique dans un essai randomisé de phase II. A ce jour, le manque de traitement efficace ainsi que la difficulté de produire un vaccin au vu du nombre de sérotypes qui existent, justifient l’intérêt pour le développement d’inhibiteurs dirigés contre ces virus. Le but de ce projet est de déterminer une stratégie multidisciplinaire afin de développer de nouveaux inhibiteurs à large spectre en incluant le groupe C des RV récemment décrit grâce aux nouvelles méthodes de biologie moléculaire et les EV possédant un intérêt majeur en clinique (EV-A71, D68 et coxsackievirus de groupe B). Cette stratégie sera déduite d’une revue de la littérature sur le sujet.

Références bibliographiques : 1) M. Roche, C. Lacroix, O. Khoumeri, D. Franco, J. Neyts, T. Terme, P. Leyssen, P. Vanelle, Eur. J. Med.Chem., 2014, 76, 445-459 DOI: 10.1016/j.ejmech.2014.01.034 2) C. Lacroix, J. Querol-Audí, M. Roche, D.Franco, M. Froeyen, P. Guerra, T. Terme, P. Vanelle, N. Verdaguer, J. Neyts, P. Leyssen, J. Antimicrob. Chem.,2014, 69, 2723-2732. DOI:10.1093/jac/dku200 3) L. Da Costa, M. Roche, E. Scheers, A. Coluccia, J. Neyts, T.Terme, P. Leyssen, R. Silvestri, P. Vanelle, Eur. J. Med. Chem., 2016, 115, 453-462.DOI:10.1016/j.ejmech.2016.03.049 4) L. Da Costa, E. Scheers, A. Coluccia, A. Rosetti, M. Roche, J. Neyts, T.Terme, R. Cirilli, C. Mirabelli, R. Silvestri, P. Vanelle. Eur. J. Med. Chem., 2017, 140, 528-541https://doi.org/10.1016/j.ejmech.2017.09.036. 5) L. Da Costa, E. Scheers, A. Coluccia, A. Casulli, M. Roche, CDi Giorgio, J. Neyts, T. Terme, R. Cirilli, G La Regina, R. Silvestri, C. Mirabelli, P. Vanelle. J. Med. Chem. 2018,61, 18, 8402-8416


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mailto:[email protected]

https://doi.org/10.1016/j.ejmech.2014.01.034

https://doi.org/10.1093/jac/dku200



Projet Tuteuré

M1 MPS

2018-2019

La résistance aux antibiotiques : les pompes d’efflux, nouvelles cibles thérapeutiques ?

Laboratoire : UMR_MD1

Equipe :MCT :Membranes et cibles thérapeutiques

Tuteur : Dr Sandrine ALIBERT Adresse : Faculté de Pharmacie N° de téléphone : 04 91 83 55 38 E-Mail : [email protected]

Sujet :

Depuis plus d'un demi-siècle, les antibiotiques ont été considérés comme le « remède miracle » pour traiter les maladies infectieuses. Déjà en 1945, après la découverte de la pénicilline, Alexander Fleming indiquait que les bactéries pourraient devenir résistantes à cette classe pionnière de médicaments antimicrobiens. En 2014, l'OMS signale [1] des niveaux inquiétants de résistance bactérienne dans le monde entier impliquant l'échec de nombreux traitements antibiotiques disponibles. En effet, la grande utilisation (humaine et vétérinaire) de nouveaux médicaments antibactériens a fortement contribué à l'émergence de mécanismes de résistance mis en place par les bactéries pour s’en défendre. La résistance bactérienne a de graves conséquences sur les patients mais aussi sur les dépenses de santé. L'urgence s’intensifie par le manque de nouvelles thérapeutiques pour le traitement d’infections véhiculées plus particulièrement par les bactéries Gram-négatives (par exemple, les bactéries intestinales telles qu’Escherichia coli et Klebsiella, et les bactéries opportunistes comme Pseudomonas et Acinetobacter, appartenant au groupe ESKAPE [2,3]). Quand cette résistance est attribuée à la présence de mécanismes de résistance multiples et différents exprimés dans la même souche et actifs contre diverses familles d'antibiotiques, on parle alors de MultiDrug Resistance (MDR). En effet, la survie des agents pathogènes dépend non seulement des enzymes qui inactivent les médicaments spécifiques, mais aussi de l'efflux (transport actif) qui les exporte vers le milieu extrabactérien. L'expression des pompes d'efflux contribue à une diminution importante des concentrations intracellulaires de diverses classes d'antibiotiques et, par conséquent, à leur activité réduite dans les souches MDR Gram-négatives [4]. L'efflux affecte également les désinfectants, les antiseptiques et les conservateurs qui sont couramment utilisés [5, 6]. Cette polysélectivité des pompes d’efflux rend difficile la compréhension de leur mécanisme d’action. Cependant, les pompes d'efflux représentent une cible attractive afin de rétablir une concentration intracellulaire en agents antibactériens efficaces en inhibant leur fonctionnalité. C’est la raison pour laquelle on se propose dans ce projet de faire une étude exhaustive des caractéristiques physicochimiques et structurales des substrats des pompes d’efflux de la famille RND chez les bactéries Gram-négative d’intérêt clinique afin de valider ces pompes comme cibles thérapeutiques.

Références bibliographiques :

[1] Antimicrobial Resistance: Global Report on Surveillance 2014. WHO. 2014. Available from:http://www.who.int/drugresistance/docu ments/surveillancereport/en/[2] Boucher HW, Talbot GH, Bradley JS, et al. Bad bugs, no drugs: no ESKAPE! An update fromthe infectious diseases society of America. Clin Infect Dis. 2009;48:1–12.[3] Rice LB. Progress and challenges in implementing the research on ESKAPE pathogens. InfectControl Hosp Epidemiol. 2010;31(Suppl 1):S7–S10.[4] Nikaido H, Pagès J-M. Broad-specificity efflux pumps and their role in multidrug resistance ofGram-negative bacteria. FEMS Microbiol Rev. 2012;36:340–363.[5] Tumah HN. Bacterial biocide resistance. J Chemother. 2009;21:5–15.[6] Hegstad K, Langsrud S, Lunestad BT, et al. Does the wide use of quaternary ammoniumcompounds enhance the selection and spread of antimicrobiol resistance and thus threaten ourhealth ? Microb Drug Resist. 2010;16:91–104.


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ÈME JOURNÉE RECHERCHE PHARMACIE 24 octobre 2019 · Heidi Kidron initially studied biochemistry,...

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Transcript of ÈME JOURNÉE RECHERCHE PHARMACIE 24 octobre 2019 · Heidi Kidron initially studied biochemistry,...