Les cancers du foie et du pancréas sont classés parmi les cancers les plus fréquents et agressifs à travers le monde et présentent une résistance à la chimiothérapie. L'efficacité des médicaments anticancéreux est affectée par les activités des enzymes métaboliques, transporteurs membranaires et par l’environnement tumoral. Le but de notre thèse est 1) de développer un modèle cellulaire hépatique et caractériser les mécanismes sous-jacents de la modulation de l’expression et de la fonctionnalité des transporteurs membranaires et des enzymes clés qui régissent le métabolisme des médicaments et 2) d’évaluer in vitro, dans différents modèles cellulaires (hépatique et pancréatique) en 2D et 3D, l’effet apoptotique de médicaments anticancéreux associés à des polyphénols en vue d’optimiser leur activité. Dans une première partie, nous avons mis en place une nouvelle lignée cellulaire hépatique humaine dérivée des HepG2, stable, exprimant suffisamment et significativement les enzymes CYP450 et les transporteurs hépatiques (MRP2, MDR1 et OATP1B1). Ce modèle pourrait être un outil de choix pour des études précliniques de métabolisme et de prédiction d’hépatotoxicité. Dans une deuxième partie, nous avons pu voir que les cellules pancréatiques et hépatiques dans un environnement 3D sont plus prédictives d’une tumeur in vivo et peuvent être un modèle de choix pour des études pharmacologiques de criblage de nouveaux médicaments anticancéreux ou des stratégies de combinaisons de molécules (avec des PP). Ainsi, nous avons montré que la quercétine, dans les cellules 3D, était capable d’augmenter l’activité de la gemcitabine et de la doxorubicine, en augmentant le taux des cellules mortes jusqu’à 60 %, par modulation des protéines MDR1 et par diminution significative du facteur HIF-1 alpha dans les cellules cancéreuses. En conclusion, les polyphénols peuvent être des molécules d’intérêt en combinaison avec des médicaments anticancéreux pour diminuer la résistance à ces traitements et servir d’outil pharmacologique pour mieux comprendre les mécanismes de résistance des cellules tumorales. / Liver and pancreatic cancers are among the most common and aggressive cancers worldwide that are resistant to chemotherapy. The efficacy of anticancer drugs is affected by the activities of metabolic enzymes, transporters and the tumor environment. The aim of my thesis was based on to main objectives: 1) developement of a hepatic cellular model and characterize the underlying mechanisms of modulation of the expression and functionality of transporters and key enzymes involved in the regulation of drug metabolism 2) study the effect of new strategies in vitro by combining anti-cancer drugs with polyphenols in these processes in order to optimize their activities on different cellular models (hepatic and pancreatic) in 2D and 3D cultures. Our results showed that we have developed a new human hepatic cell line derived from HepG2 cells. The novel cell line is a good in vitro model with a capacity of predicting hepatotoxicity of novel drugs with significant differences for chromosomes 5, 17 and 20 and high expression level of CYP450 and transporters (MRP2, MDR1 and OATP1B1). Secondly, our results indicate that the combination of anticancer drugs and polyphenols increased the rate of apoptosis in cancer cells by up regulation of the expression levels of cleaved caspase-3 and the regulator of apoptosis p53. Moreover, our results demonstrated that polyphenols inhibit the efflux activity of MDR1. In addition, our results indicate that the combination of anti-cancer drugs and quercetin down regulated the expression of HIF-1α and increased the expression levels of the cleaved caspase-3 and p53 on human pancreatic and liver cell line cultured in 3D culture. In conclusion, polyphenols may be promising agents for novel combination therapy since they potentialize the cytotoxic activity of anticancer drugs to eradicate cancer and therefore the cellular resistance.

The dissertation presents GALAS models for the estimation of the acute toxicity towards two rodent species following different administration routes as well as for the prediction of CYP3A4 and CYP2D6 regioselectivity in the main metabolic reactions mediated by these enzymes (13 individual models in total). All these models feature the ability of the quantitative model Applicability Domain (AD) evaluation via the estimated prediction Reliability Indices (RI). I.e., the obtained models conform to one of the main requirements for the QSAR model acceptance as an alternative research method by the EU regulatory institutions. Evident correlation between prediction reliability and its accuracy allowed classifying each model result into one of several qualitative classes according to RI values. One possible way of utilizing such information, discussed in this study, is compound prioritization before experimental testing potentially resulting in reduction of the number of necessary measurements. As demonstrated the AD of the obtained GALAS models can be easily expanded to cover specific compound classes of researcher interest using ‘in-house’ databases of experimental data. This feature significantly improves the possibilities for the practical application of these models, based on public data, in industry. Especially given the fact that the described improvements in predictions following the addition of similar compounds was instant and required no rebuilding of the baseline models. / Disertacijoje pristatomi GALAS metodika paremti ūmaus toksiškumo dviems graužikų rūšims bei visai eilei skirtingų medžiagos patekimo į organizmą būdų ir CYP3A4 bei CYP2D6 fermentų regioselektyvumo pagrindinėse jų katalizuojamose metabolizmo reakcijose prognozavimo modeliai (iš viso 13 individualių modelių). Visi minimi modeliai kokybiškai išsiskiria iš anksčiau publikuotų savo analogų dėl kiekybinio jų pritaikomumo srities įvertinimo galimybės, kurią suteikia apskaičiuojamos prognozės patikimumo indekso (RI) reikšmės. Tokia savybė yra vienas pagrindinių reikalavimų vertinant bet kokio modelio galimybes tapti ES oficialiai pripažintu alternatyviu tyrimo metodu. Aiški prognozių kokybės priklausomybė nuo jų patikimumo išraiškos taip pat suteikia galimybę modelio rezultatus suskaidyti į kokybines klases pagal apskaičiuotąsias RI reikšmes. Vienas iš tokios informacijos panaudojimo būdų siūlomų disertacijoje yra junginių prioritetizavimas prieš bet kokius eksperimentinius matavimus ir netgi pastarųjų skaičiaus potencialus sumažinimas. Disertacijoje taip pat išnagrinėta galimybė greitai bei efektyviai apmokyti gautuosius GALAS modelius naujais eksperimentiniais duomenimis, išplečiant jų pritaikomumo sritį. Ši esminė savybė radikaliai padidina nagrinėjamųjų modelių, paremtų viešai prieinamų duomenų rinkiniais, realaus praktinio panaudojimo farmacijos pramonėje galimybes.

The cytochromes P450 (CYPs) are a superfamily of biological catalysts that are ubiquitous throughout the biological domain. CYPs are heme-b containing monooxygenases which oxidize substrates with the help of accessory redox partners. CYP substrates include endogenous compounds required for many biological functions and homeostasis, such as steroids, as well as the majority of clinically used drugs and environmental xenobiotics. The majority of studies that have been performed to date are on P450cam (CYP101) from Pseudomonas putida. Of the numerous reactions catalyzed by CYPs, unactivated carbon-carbon bond cleavage, is one of particular versatility. Being unique in their catalytic mechanisms, the C-C bond cleaving enzymes and in particular CYP51 from Mycobacterium tuberculosis are though to be capable of utilizing multiple reactive oxygen intermediates. During the process of C-C bond cleavage, CYP51 catalyzes two classical hydroxylation reactions. The final reaction culminates in an enigmatic third step which cleaves a C-C bond, liberates formate, and installs a 14,15 double bond within its steroid substrate. The mechanism of CYP51s final step is still unclear and the exact activated oxygen species has yet to be observed. CYP51 is also distinct from most CYPs owing to the fact that the acid functionality of the conserved active site “acid-alcohol pair” found in most CYPs, is replaced by a histidine. This study aimed to trap and characterize dioxygen reactive intermediates, and elucidate the role of the unique acid-alcohol pair in the formation and stabilization of these intermediates. This study demonstrates our success in generating, stabilizing, and spectroscopically characterizing reactive dioxygen intermediates in Mtb CYP51. As the life-time of the oxyferrous intermediate in Mtb CYP51 is extremely short at ambient temperatures, this work has shown the laboratory’s expertise in being able to generate reduced oxyferrous intermediates at cryogenic temperatures. These intermediates have only been generated in a handful of cytochromes P450 and as such this work adds critical information to the small body of work currently reported.

La variabilité de la pharmacocinétique de la névirapine et de l’éfavirenz, deux médicamentsantirétroviraux inhibiteurs non nucléosidiques de la transcriptase inverse du VIH, a été étudiéechez des patients cambodgiens infectés par le VIH par une méthode de pharmacocinétique depopulation. Cent soixante dix patients traités par névirapine faisaient partie de la cohorteESTHER de l’hôpital Calmette de Phnom-Penh et 312 patients co-infectés par le VIH et latuberculose et traités par éfavirenz étaient inclus dans l’essai clinique CAMELIA (ANRS1295–CIPRA KH001) conduit au Cambodge. Les dosages plasmatiques de névirapine et d’éfavirenz ontété réalisés par des méthodes CLHP avec détection UV. Après 18 et 36 mois de traitement, lesconcentrations plasmatiques médianes de la névirapine sont de 5,7 μg/mL. Après 22 et 50semaines de traitement, les concentrations médianes d’éfavirenz sont de 2,7 μg/mL, quel’éfavirenz soit associé (22 semaines) ou non (50 semaines) à la rifampicine. Les clairancesapparentes estimées de la névirapine et de l’éfavirenz sont respectivement de 2,6 L/h et de 7,7L/h. Les variabilités intra et inter individuelles des clairances apparentes sont respectivement de17% et 28% pour la névirapine et 15% et 37% pour l’éfavirenz. Parmi les covariablesdémographiques, biologiques ou génétiques étudiées, seul le polymorphisme génétique duCYP2B6 G516T est significativement associé à la clairance apparente de ces deux médicaments.Ainsi la clairance apparente estimée de la névirapine est de 2,95 L/h, 2,62 L/h et 1,86 L/hrespectivement pour les génotypes CYP2B6 516GG, 516GT, et 516TT. La fréquence de l’allèlemutée T qui code pour une enzyme non fonctionnelle est de 34% dans cette population de 442patients d’Asie du Sud-Est. / HIV-infected patients by population method. 170 patients on nevirapine-based antiretroviraltherapy were from the ESTHER cohort of the Calmette hospital in Phnom-Penh. 312 patients onefavirenz-based therapy were included in the CAMELIA (ANRS1295–CIPRAKH001) clinical trialconducted in Cambodia. Plasma concentrations of nevirapine and efavirenz were measured byHPLC and UV detection. Median plasma concentrations of nevirapine and efavirenz were 5.7μg/mL and 2.7 μg/mL respectively. Apparent plasma clearances of nevirapine and efavirenz were2.6 L/h and 7.7 L/h respectively. Among demographic, clinical, biological or genetic covariates,genetic polymorphism of CYP2B6 G516T was the only one which was shown to affect theclearance of the 2 drugs. Frequency of the T allele was 34% in this population of South-East Asia.

The term "biologically active compounds" covers a wide range of substances originating from natural or synthetic origins. These compounds can enter the aquatic environment through wastewater treatment plants, manufactural discharge or they are naturally present in the aquatic plants or microorganisms. Their adverse effects on fish has been widely studied and accepted. This thesis is focussed on the adverse effect of several bioactive compounds (i.e. pharmaceuticals, phytochemicals, or complex mixture of pollutants) on fish using different experimental design as in vitro, in vivo and in situ. In the first part of this thesis, in vitro effects of pharmaceuticals and phytochemicals or their combinations on fish CYP system were investigated. Moreover, effects of standard carrier solvents used in enzyme activity assays were also investigated. An important finding in this study was the lack of effects of either dexamethasone, quercetin, or indole-3-carbinol on EROD activity; however, when these agents were combined, EROD activity was strongly inhibited. This demonstrates that combination of compounds might exert different effects than single compounds, and the effects of mixture compounds cannot be predicted from the effects of individual compounds. In the second part of the thesis, the effects of chronic exposures of rainbow trout to dexamethasone and clotrimazole were investigated. The results regarding dexamethasone showed similarities with in vitro studies, and both in vitro and in vivo exposure of dexamethasone did not alter the CYP enzyme activities. On the other hand, in vivo exposure of clotrimazole yielded conflicting results with findings from the in vitro studies. Clotrimazole induced EROD activity in chronically exposed rainbow trout, and BFCOD activity showed biphasic pattern in which it was inhibited at environmentally relevant concentrations and induced at high concentrations. Thus, the observed effects suggest that clotrimazole could negatively affect fish CYPs at environmentally relevant concentrations. However, in the environment, the effects of clotrimazole and dexamethasone on fish CYPs system might be modified because of unknown compounds in these mixtures. Therefore, further investigations were done to identify the effects of mixture compounds using an in situ model. The last part of the dissertation addresses the effects of cocktail PPCPs on common carp under natural conditions. In situ studies provide valuable information on both hepatic and intestinal CYP activities. Both EROD and BFCOD activities were affected by the PPCPs that are present in the exposed fish. Moreover, changes in intestinal CYP activities suggest that fish can ingest some of these contaminants through their feed. Therefore, the intestines might be responsible for elimination of some of these pollutants and act as the first barrier of pollutant entry in fish. Despite the extensive studies concerning aquatic pollution, further studies are necessary. Development of new pharmaceuticals, their occurrence in the aquatic environment, and their effects on non-target organisms should be continuously monitored.

Cadmium is present in food and groundwater. Tobacco smoking and occupational exposure are also major sources for cadmium. Cadmium is primarily accumulated in liver, a major organ metabolizing exogenous chemicals. Chemical metabolism may cause detoxification, but it can also cause bio-activation resulting in liver damage. Cytochrome P450s (CYP) are major liver metabolism enzymes, and cadmium chloride (CdCl2) can induce CYP2A5 in mice. We examined the effect of CYP2A5 on CdCl2-induced liver injury using CYP2A5-knockout (cyp2a5-/-) mice. The cyp2a5-/- mice and their control WT mice were injected CdCl2 intraperitoneally at 5 mg/kg body weight, respectively, to induce liver injury. The control group of cyp2a5-/- mice and WT mice were injected saline at the same volume. Twenty-four hours later, all the mice were sacrificed. As indicated by biochemical assays and pathological evaluation, CdCl2-treated WT mice exhibited more severe liver injury than CdCl2-treated cyp2a5-/- mice, suggesting that CYP2A5 contributes to Cd-induced liver injury.

<p>CYP2C9 is one of our major drug metabolising enzymes and belongs to the cytochrome P450 (CYP) super family. The aim of this thesis was to gain an understanding of the quantitative structure–activity relationships (QSAR) of CYP2C9 substrates and inhibitors. This information will be useful in predicting drug metabolism and the potential for drug–drug interactions. To achieve this, a well characterised data set of structurally diverse, competitive CYP2C9 inhibitors was identified in our laboratory. Several computational methodologies, many based on GRID molecular interaction fields, were applied or developed in order to handle issues such as compound alignment and bioactive conformer selection. First, a traditional 3D QSAR was carried out in GOLPE, generating a predictive model. In this model the selection of a bioactive conformer and alignment was based on docking in a homology model of CYP2C9. Secondly, we introduced the concept of alignment independent descriptors from ALMOND. These descriptors were used to generate quantitatively and qualitatively predictive models. We subsequently derived conformation independent descriptors from molecular interaction fields calculated in FlexGRID. This enabled the derivation of 3D QSAR models without taking into account the selection of an alignment or a bioactive conformer. A subsequent programming effort enabled the conversion of this model back to 3D aligned pharmacophores. Similar alignment independent descriptors were also used in the development of the software MetaSite® that predicts the site of metabolism for CYP2C9 ligands. Finally, as crystal information on this isoform emerged, the performance of molecular dynamics simulations and homology models and the flexibility of the protein were evaluated using statistical analyses.</p><p>These modelling efforts have resulted in detailed knowledge of the structural characteristics in ligand interactions with the cytochrome P450 2C9 isoform.</p>

CYP2C9 is one of our major drug metabolising enzymes and belongs to the cytochrome P450 (CYP) super family. The aim of this thesis was to gain an understanding of the quantitative structure–activity relationships (QSAR) of CYP2C9 substrates and inhibitors. This information will be useful in predicting drug metabolism and the potential for drug–drug interactions. To achieve this, a well characterised data set of structurally diverse, competitive CYP2C9 inhibitors was identified in our laboratory. Several computational methodologies, many based on GRID molecular interaction fields, were applied or developed in order to handle issues such as compound alignment and bioactive conformer selection. First, a traditional 3D QSAR was carried out in GOLPE, generating a predictive model. In this model the selection of a bioactive conformer and alignment was based on docking in a homology model of CYP2C9. Secondly, we introduced the concept of alignment independent descriptors from ALMOND. These descriptors were used to generate quantitatively and qualitatively predictive models. We subsequently derived conformation independent descriptors from molecular interaction fields calculated in FlexGRID. This enabled the derivation of 3D QSAR models without taking into account the selection of an alignment or a bioactive conformer. A subsequent programming effort enabled the conversion of this model back to 3D aligned pharmacophores. Similar alignment independent descriptors were also used in the development of the software MetaSite® that predicts the site of metabolism for CYP2C9 ligands. Finally, as crystal information on this isoform emerged, the performance of molecular dynamics simulations and homology models and the flexibility of the protein were evaluated using statistical analyses. These modelling efforts have resulted in detailed knowledge of the structural characteristics in ligand interactions with the cytochrome P450 2C9 isoform.

No description available.

Ce projet de recherche comporte deux parties principales qui possèdent comme lien unificateur l’amélioration des méthodes et techniques utilisées actuellement pour évaluer aussi bien l’activité que l’expression des cytochromes P450 (CYP450s) et menant par la suite à leur application en clinique. Le premier volet de ce projet de recherche porte sur le développement de méthodes LC-MS/MS pour un cocktail de 7 substrats marqueurs des CYP450s. Notre objectif est de développer et valider une méthode LC-MS/MS spécifique et sensible permettant l’évaluation des activités des CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 et 2E1 suivant l’administration orale et à faible dose d’un cocktail de substrats marqueurs chez des patients et sujets sains. Les méthodes développées peuvent être utilisées pour évaluer les mécanismes de variabilité interindividuelle comme l’impact de polymorphismes génétiques, de facteurs environnementaux et de maladies dans le processus de métabolisme et d’élimination des médicaments, mais également pour investiguer les interactions médicamenteuses. Ce cocktail a été appliqué avec succès, dans un projet clinique portant sur l’évaluation des effets du diabète sur la capacité métabolique par les CYP450s. Le deuxième volet de ce projet de recherche vise à développer des méthodes analytiques par LC-HRMS afin de caractériser et quantifier les CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 et 4F2 dans l’intestin grêle humain. Notre hypothèse suggère que les CYP450s retrouvés le long de l’intestin grêle peuvent affecter significativement l’effet de premier passage de certains médicaments administrés par voie orale et influencer leurs concentrations plasmatiques et conséquemment, leurs effets pharmacologiques et/ou toxiques. Ma participation à ce projet a permis d’identifier des peptides protéotypiques par digestion in silico et in vitro et de développer des méthodes de quantification absolue par LC-HRMS. Ce projet est une première étape dans la caractérisation des CYP450s majeurs le long de l'intestin grêle. Il permettra de mieux comprendre les mécanismes de variabilité interindividuelle dans la réponse aux médicaments associés au processus d'absorption intestinal et de mieux prédire la variabilité dans la biodisponibilité des médicaments et de développer des modèles pharmacocinétiques plus complexes. / This research project is divided into two sections, both aiming at the development of sensitive and specific LC-MS methods to evaluate activity and expression of CYP450 and finally, looking at their clinical application. The first section of this research project focuses on the development of analytical methods by LC-MS/MS for a seven CYP450 probe-drug cocktail. Although these cocktails have shown value they also suffer from many limitations. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1 activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. These methods can be used to phenotype CYP450 activities, evaluate inter-individual variabilities, study the impact of pathological conditions on drug metabolism and elimination, and evaluate drug-drug interactions. Our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in type 2 diabetic patients. The second section of this project aims at the development of a LC-HRMS method for the characterization and absolute quantification of CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2 in the human small intestine. Our hypothesis suggests that CYP450 isoenzymes found along the small intestine can significantly affect the first-pass effect of certain drugs administered orally and thus influence their pharmacological and/or toxic effects. My participation in this project allowed to identify proteotypic peptides by in silico and in vitro digestion and to develop LC-HRMS methods allowing the absolute quantification of CYP450. This project is a first step in the characterization of the main CYP450 along the small intestine. This project will allow a better understanding of inter-individual variability in drug response associated with intestinal absorption of drugs, a better prediction of variability in drug bioavailability and to develop more complex pharmacokinetic models.