Mother's milk provides multiple benefits to the offspring. However, xenobiotics transferred into breast milk may pose a risk to the nursing infant. The breast cancer resistance protein (BCRP) actively transports xenobiotics into breast milk. BCRP also transports nutrients, like riboflavin, and together with recently identified riboflavin transporters (RFT), may provide a mechanism for riboflavin secretion into breast milk. Expression of RFT in the mammary gland remained unknown. Our objective was to characterize Bcrp and Rft mRNA expression in the mammary gland of FVB/N mice, and investigate a strategy to decrease excretion of BCRP-transported xenobiotics into the milk using riboflavin intervention. Rft and Bcrp mRNA were upregulated in the mammary gland of lactating mice. An intravenous riboflavin administration significantly reduced the levels of BCRP-transported cimetidine in milk. This study demonstrates the use of riboflavin to exploit the function of mammary BCRP in order to reduce xenobiotic secretion into breast milk.

Indiana University-Purdue University Indianapolis (IUPUI) / Human BCRP is one of the major ATP-binding cassette transporters involved in the development of multidrug resistance in cancer chemotherapy. Overexpression of BCRP in the tumor cell plasma membrane and apical membrane of the gastrointestinal tract leads to decreased intracellular accumulation of various anticancer drugs as well as reduced drug bioavailability. BCRP has been shown to exist on the plasma membrane as higher forms of homo-oligomers. In addition, the oligomerization domain of BCRP has been mapped to the carboxyl-terminal TM5-ECL3-TM6 and this truncated domain, when co-expressed with the full-length BCRP, displays a dominant inhibitory activity on BCRP function. Thus, the oligomerization of BCRP could be a promising target in reversing multidrug resistance mediated by BCRP. To further dissect the oligomerization domains of human BCRP and test the hypothesis that TM5, ECL3, and TM6 each plays a role in BCRP oligomerization and function, we engineered a series of BCRP domain-swapping constructs with alterations at TM5-ECL3-TM6 and further generated HEK293 cells stably expressing wild-type or each domain-swapping construct of BCRP. Using co-immunoprecipitation and chemical cross-linking, we found that TM5, ECL3, and TM6 all appear to partially contribute to BCRP oligomerization, which are responsible for the formation of oligomeric BCRP. However, only TM5 appears to be a major contributor to the transport activity and drug resistance mediated by BCRP, while ECL3 or TM6 is insufficient for BCRP functions. Taken together, these findings suggest that homo-oligomeric human BCRP may be formed by the interactions among TM5, ECL3 and TM6, and TM5 is a crucial domain for BCRP functions and BCRP-mediated drug resistance. These findings may further be used to explore targets for therapeutic development to reverse BCRP-mediated drug resistance and increase the bioavailability of anti-cancer drugs for better treatment of multidrug resistant cancers.

L'objet de ce travail est de présenter de facon détaillée des méthodes de modélisation appliquées à l'analyse des mécanismes de reconnaissance moléculaire et à la conception de nouveaux composés bioactifs selon deux approches : la conception basée sur la structure des récepteurs et la conception basée sur la structure des ligands.<br />Dans le cadre du premier axe, la méthode de construction de protéines par homologie de Blundell, implémentée dans le module COMPOSER de SYBYL et la méthode d'amarrage de Morris, implémentée dans le logiciel AUTODOCK3, sont décrites et appliquées à la modélisation et à l'étude des mécanismes de reconnaissance moléculaire d'un antigène polysaccharidique de la bactérie Shigella flexneri 5a et de mimes peptidiques immunogènes par un anticorps humain protecteur : IgA I3.<br />Dans le cadre du second axe, l'analyse statistique de descripteurs de champs d'interaction moléculaire de type CoMSIA et les méthodes de validation des modèles qu'elle génère sont présentées et appliquées à l'étude des relations structure activité en trois dimensions d'une série de 27 analogues de flavonoïdes modulateurs du transporteur ABCG2 (BCRP), impliqué dans le mécanisme de résistance multiple aux anticancéreux que développent les cellules tumorales. La production de modèles statistiquement fiables et performants a permis de concevoir de nouveaux composés biologiquement actifs.

The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice. A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport. In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.

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L’échec thérapeutique des maladies cérébrales est lié, entre autres, à la présence de barrières entre le sang et le Système Nerveux Central (SNC), en particulier la Barrière Hémato-Encéphalique (BHE). La BHE est une structure neuro-vasculaire localisée au niveau des MicroVaisseaux Cérébraux (MVC) limitant l’entrée des molécules thérapeutiques dans le cerveau. Ce rôle barrière est dû à plusieurs facteurs, dont principalement, l’existence du côté luminal et/ou abluminal de la BHE de plusieurs transporteurs d’efflux, dont les transporteurs de type ABC (ATP Binding Casette) et SLC (SoLute Carrier) et qui sont à l’origine des phénomènes de résistance aux médicaments. Les études de recherche actuelles visent à identifier les voies de signalisation régulant l’activité de ces protéines d’efflux afin d’optimiser la pharmacothérapie cérébrale. Mais la majorité de ces études sont effectuées chez l’adulte. Très peu de données existent chez l’enfant.Cette étude a été réalisé dans la perspective de 1) Etudier l’ontogenèse des transporteurs ABC et SLC de la BHE au cours de la maturation cérébrale, 2) Elucider le rôle fonctionnel de quatre transporteurs d’efflux ((P-glycoproteine (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters) dans le cerveau des enfants et 3) Elucider les mécanismes qui régulent leur expression fonctionnelle dans des conditions normales et pathologiques, notamment inflammatoires, parce que des modifications dans les composantes structurales et fonctionnelles de l'unité neurovasculaire ont été rapportées dans une longue liste de pathologies du SNC chez les enfants et les adultes. Nos résultats ont montré l’existence de différences fonctionnelles, en terme de passage de molécules, entre la BHE pédiatrique et celle adulte. De plus, cette étude a mis en évidence une régulation différentielle liée à l'âge des transporteurs d'efflux de médicaments de la barrière dans des conditions normales et inflammatoires.Ces résultats fournissent des preuves sur l’intérêt de prendre en compte les propriétés spécifiques de la BHE pédiatrique et la distinguer de la BHE adulte lors des définitions des stratégies thérapeutiques destinées à traiter les maladies cérébrales chez les enfants. / One major reason of CNS pharmacotherapy’s impediment is the existence of “barriers” between blood and CNS, especially the Blood-Brain Barrier (BBB), a neurovascular structure localized at the level of brain microvasculature. Main factors responsible for this barrier function are drug efflux transporters type ABC (ATP-Binding Cassette) and SLC (SoLute Carrier) expressed at BBB level and known to be at the origin of multi-drug resistance phenomenon. Recent researches aim at unraveling the signaling mechanisms regulating these transporters in order to modulate their activity and improve pharmacotherapy in brain diseases. For years, these transporters have been studied in adult organism. But, there is a wide spread belief that the BBB in embryo, fetus, new born and infant is “immature”, implying caution in giving drugs to infants. However, current knowledge on the functional status of the BBB in immature organism remains very limited.This study was performed in the aim of understanding: 1) The ontogenesis of ABC and SLC transporters during brain maturation, 2) the functional role of four BBB drug efflux transporters (P-glycoprotein (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters) in children’s brain, and 3) the mechanisms that regulate their functional expression under normal and pathological conditions, mostly under inflammatory conditions, because indeed alterations in structural and functional components of the BBB have been reported in a long list of CNS pathologies in adults. Our results showed changing properties of the BBB during ontogenesis, as well as an age-related differential regulation of BBB drug efflux transporters under normal and inflammatory conditions.These findings highlight the importance of considering an age-related response of CNS to drugs and of taking into account the specific properties of juvenile BBB during definition of therapeutic strategies designed to treat childhood brain diseases, and this in the clinical perspective of developing new drugs with enhanced efficacy in children’s CNS.

Le cancer du sein de phénotype « Basal-like » triple négatif (BLTN) est particulièrement agressif et de mauvais pronostic. Il est insensible aux traitements hormonaux laissant pour seule stratégie de traitement la chimiothérapie conventionnelle. De ce fait, de nouvelles thérapeutiques ciblées sont en développement, tels que les inhibiteurs de la Poly-ADP-Ribose-Polymerase (PARP). Dans ce contexte, nos travaux de recherche ont été orientés sur l’optimisation du traitement des cancers du sein BLTN en modélisant l’action d’un anti-PARP modèle, l’Olaparib sur la lignée SUM1315 de phénotype BLTN. Dans un premier temps, l’étude de la coexpression de la BCRP et de la P-gp, deux protéines « Multidrug Resistance » (MDR) majeures en présence de 50 µM d’Olaparib® a montré une induction de leurs expressions chez les cellules SUM1315, avec une réponse de type relais. La BCRP établirait une première ligne de défense cellulaire et son action serait ensuite relayée par la P-gp durant 24h de traitement. Ce mécanisme est en corrélation avec la concentration intracellulaire d’Olaparib mesurée par HPLC. L’ensemble de nos résultats suggère qu’il serait possible de contourner le mécanisme de résistance induit par les protéines MDR si une concentration stable en Olaparib est maintenue dans les cellules à long terme. Nous avons ensuite étudié la potentialisation de l’action de l’Olaparib en le combinant avec un traitement par radiothérapie à basse et haute énergie, sur la viabilité des cellules de la lignée SUM1315. La comparaison des résultats avec un traitement Olaparib seul ou irradiation seule et ceux des traitements combinés Olaparib/radiothérapie a alors mis en évidence un effet synergique des deux traitements sur la viabilité cellulaire. L’effet synergique de cette combinaison fonctionne même avec de faibles doses d’Olaparib. De cette manière, il serait possible de réduire les doses d’anti-PARP utilisées tout en gardant les bénéfices du traitement. Enfin, nous avons développé deux techniques de cultures cellulaires en trois dimensions (i) « hanging drop » et (ii) « liquid overlay », permettant de mimer plus fidèlement les conditions des tumeurs in vivo. L’observation en microscopie électronique à transmission et à balayage des sphéroïdes obtenus par ces deux techniques a permis de démontrer l’intégrité des cellules au sein des sphéroïdes ainsi que la formation de jonctions cellulaires. Cependant, les sphéroïdes obtenus en « liquid overlay » ont montré une meilleure intégrité ultra-structurale. / « Triple Negative Basal-Like » (BLTN) breast cancer is particularly aggressive and of poor prognosis. It is insensitive to hormone-targeted therapies leaving conventional chemotherapy as the only treatment strategy. Therefore, new promising targeted therapies are being developed, such as Poly-ADP-Ribose-Polymerase inhibitors (anti-PARPs). In this context, our research has been directed towards optimizing the treatment of BLTN breast cancer by modelling the action of an anti-PARP model, Olaparib®, on BLTN cell line SUM1315. Firstly, the study of the co-expression of BCRP and P-gp, two major “Multidrug Resistance” proteins (MDR) in the presence of 50 µM Olaparib® showed an induction of their expression in SUM1315 cells, with a relay-type response. BCRP would establish a first line of cellular defense and its action would then be taken over by P-gp, for 24h of treatment. This mechanism is correlated with the intracellular concentration of Olaparib® measured by HPLC. All of our results suggest that it would be possible to circumvent the induced MDR resistance mechanism if a stable concentration of Olaparib® is maintained in cells in the long term. Secondly, we studied the potentiation of the action of Olaparib® combining it with low and high-energy radiations on the viability of SUM1315 cells. Comparison of the results with single Olaparib®, single irradiation, or the combination of Olaparib®/radiotherapy then demonstrated a synergistic effect of the two treatments when delivered concomitantly, on cell viability. The synergistic effect of this combination works even with low doses of Olaparib®. In this way it would be possible to reduce the anti-PARP doses while maintaining the benefits of this treatment. Finally, we have developed two techniques of cell culture in three dimensions: (i) "hanging drop" and (ii) "liquid overlay", in order to mimic more accurately the conditions of tumours in vivo. Observations of spheroids obtained by these two techniques by transmission and scanning electron microscopy demonstrated the integrity of cells within as well as the formation of cell junctions. However, the spheroids obtained by "liquid overlay" showed better ultra-structural integrity.

Les pyréthrinoïdes et les organophosphorés sont des pesticides très utilisés, à l’origine d’une imprégnation forte de la population, exposée à ces contaminants principalement via l’alimentation. De plus en plus d’études scientifiques suggèrent des liens entre l’exposition à ces composés et des maladies chroniques ou des troubles du développement de l’enfant. Paradoxalement, leur devenir biologique chez l’homme est mal connu. Certaines études suggèrent que ces insecticides sont susceptibles d’intéragir avec les transporteurs membranaires ABC et SLC, protéines localisées au niveau d’interfaces hémato-tissulaires qui prennent en charge de nombreux substrats endogènes, médicaments et contaminants de l’environnement. L’objectif de notre étude a été de caractériser les effets d’insecticides des familles des pyréthrinoïdes et des organophosphorés sur l’activité de nombreux transporteurs ABC et SLC prenant en charge des médicaments (P-gp, BCRP, MRPs, OATP-1B1,-2B1,-1B3, OCT1-3, OAT1, OAT3, MATE1 et MATE2K) par une approche in vitro. Nous nous sommes également attachés à caractériser par des expérimentations in vitro et in silico, les mécanismes des interactions et les éléments structuraux des pesticides à l’origine de ces effets. Nous avons montré que de nombreux organophosphorés et pyréthrinoïdes étaient capables d’inhiber des transporteurs d’efflux (MRP, BCRP, P-gp) et d’influx (OATP1B1, OAT3, MATE1, OCT1-2) et de stimuler l’activité de certains OATPs. Les pesticides testés inhibaient très fortement l’activité des transporteurs de cations (OCT1 et OCT2) et ont pu bloquer le transport de catécholamines médiés par ces protéines. Une approche qSAR a permis de définir des paramètres physicochimiques associés aux effets modulateurs des pesticides et une approche d’amarrage moléculaire (docking) a mise en évidence les sites de liaisons de la P-gp impliquées dans ces interactions. Les conséquences des modulations de l’activité des transporteurs, en termes d’effets toxiques et d’interactions médicamenteuses, restent à définir pour les populations exposées à de fortes doses de pesticides. Toutefois, la contribution des interactions observées aux effets toxiques de ces insecticides est peu probable car nécessitant des concentrations nettement supérieures à celles atteintes dans le cadre d’une exposition environnementale de la population générale. / The general population is chronically exposed to pyrethroids and organophosphorus insecticides, mainly through alimentation. Several epidemiological studies have found an association between non-occupational exposure to these pesticides and chronic diseases and developmental disorders. Paradoxically, their biological fate in humans is poorly understood. Some studies suggest that these insecticides could interact with ABC and SLC membrane transporters. These membrane proteins, located at blood-tissue interfaces (liver, kidney, intestine ...), handle many endogenous substrates, drugs and pollutants. The objective of our study was to characterize, using an in vitro approach, the effects of pyrethroid and organophosphorus insecticides on the activity of numerous ABC and SLC human drug-transporters (P-gp, BCRP, MRPs, OATP-1B1, -2B1, -1B3, OCT1-3, OAT1, OAT3, MATE1 and MATE2K). We have also tried to analyze the mechanisms of interactions and the structural requirements for insecticides-mediated modulation of drug transporters activities using in vitro and in silico approach. We have shown that many organophosphorus and pyrethroids are able to inhibit ABC (MRP, BCRP, P-gp) and SLC (OATP1B1, OAT3, MATE1, OCT1-2) transporters and can stimulate the activity of some OATPs. Moreover, the tested pesticides inhibited very strongly the activity of OCT1 and OCT2 and blocked catecholamine transport mediated by these transporters. A qSAR approach allowed to define physicochemical parameters associated with the modulating effects of pesticides and a molecular docking approach revealed the P-gp binding sites involved in these interactions. The consequences of transporter activitie modulation, in terms of toxic effects and drug interactions, remain to be defined for populations exposed to high doses of pesticides, occurring notably in response to poisoning. However the alterations of these transporter activities by insecticides are unlikely to contribute to organophosphorus or pyrethroids toxicities of chronic low-dose exposure.

<p>Transport into and across the cells of the human body is a prerequisite for the pharmacological action of drugs. Passive membrane permeability and active transport mechanisms are major determinants of the intestinal absorption of drugs, as well as of the distribution to target tissues and the subsequent metabolism and excretion from the body. In this thesis, the role of ATP-binding cassette (ABC) transporters and passive permeability on drug absorption and disposition was investigated. Particular emphasis was placed on defining the molecular properties important for these transport mechanisms. </p><p>The influence of different transport pathways on predictions of intestinal drug absorption was investigated using experimental models of different complexity. Experimental models that include the paracellular pathway gave improved predictions of intestinal drug absorption, especially for incompletely absorbed drugs. Further, the inhibition of the ABC transporters breast cancer resistance protein (BCRP/ABCG2) and multidrug-resistance associated protein 2 (MRP2/ABCC2) was experimentally investigated using structurally diverse datasets that were representative of orally administered drugs. A large number of previously unknown inhibitors were identified among registered drugs, but their clinical relevance for drug-drug interactions and drug-induced toxicity remains to be determined. The majority of the inhibitors affected all three major ABC transporters BCRP, MRP2 and P-glycoprotein (P gp/ABCB1), and these multi-specific inhibitors were found to be enriched in highly lipophilic weak bases. </p><p>To summarize, the present work has led to an increased knowledge of the molecular features of importance for ABC transporter inhibition and passive membrane permeability. Previously unknown ABC transporter inhibitors were identified and predictive computational models were developed for the different drug transport mechanisms. These could be valuable tools to assist in the prioritization of experimental efforts in early drug discovery.</p>