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Replacement therapy with concentrates of factor VIII or IX remains the gold standard for severe haemophilia management. The recent development of clotting factor products with extended half-life, widely available on the market since 2 years, facilitates adherence, improves considerably the patients' quality of life, and simplifies the management of breakthrough bleedings or surgery. These molecules have also brought to the limelight the concepts of optimization and personalization of anti-haemophilic prophylaxis. Pharmacokinetics (PK) is one of the tools that can help haematologists to adapt in a more objective and precise manner the prophylaxis regimen to each individual patient's specific needs. For many years, clinicians at haemophilia centres have been using some simple PK parameters, such as recovery and residual level. However, recently, they have been confronted with an important number of new PK parameters they were not familiar with, but that can be used to improve patient management. Due to the accumulation of PK data and their relative complexity, it is now necessary to analyse the relevance of the different PK parameters relative to haemophilia specificities, and also to know their limits to better use them.

International audience; ABC transporters, such as P-gp and BCRP, are involved in rivaroxaban pharmacokinetics and can lead to drug-drug interactions (DDIs). Investigations of the victim role for rivaroxaban and transporter-mediated DDI are commonly performed using in vitro models. However, interpretation of rivaroxaban efflux transport and DDI studies in cell models may be influenced by P-gp and BCRP transporter abundance. This study aimed to develop an LC-MS/MS quantification method for assessing the relationship between transporter expression and functionality in Caco-2ATCC, Caco-2ECACC, MDCK-MDR1, MDCK-BCRP cell models. First, the relative and absolute quantities of the transporters were determined by LC-MS/MS. P-gp and BCRP expression was then confirmed by western blotting and immunofluorescence staining. Finally, P-gp and BCRP functional activities and half-inhibitory concentrations (IC50s) of two specific inhibitors (verapamil and ko143) were determined by bidirectional transport experiments. P-gp and BCRP protein expression was detected at the cell membrane and was greater in the respective transfected models. Efflux ratios were correlated with P-gp and BCRP quantities. The lowest IC50s were obtained in the MDCK-MDR1 and MDCK-BCRP models for verapamil and ko143, respectively. In conclusion, this study demonstrated that LC-MS/MS can accurately quantify P-gp and BCRP efflux transporters and thereby improve the interpretation of transport data and in vitro-in vivo correlations.

The preclinical evaluation of nasally administered drug candidates requires screening studies based on in vitro models of the nasal mucosa. The aim of this study was to evaluate the morpho-functional characteristics of the 3D MucilAir™ nasal model with a pharmacological focus on [ATP]-binding cassette (ABC) efflux transporters. We initially performed a phenotypic characterization of the MucilAir™ model and assessed its barrier properties by immunofluorescence (IF), protein mass spectrometry and examination of histological sections. We then focused on the functional expression of the ABC transporters P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)1, MRP2 and breast cancer resistance protein (BCRP) in bidirectional transport experiments. The MucilAir™ model comprises a tight, polarized, pseudo-stratified nasal epithelium composed of fully differentiated ciliated, goblet and basal cells. These ABC transporters were all expressed by the cell membranes. P-gp and BCRP were both functional and capable of actively effluxing substrates. The MucilAir™ model could consequently represent a potent tool for evaluating the interaction of nasally administered drugs with ABC transporters.

International audience; Endothelial cells are main components of the Blood-Brain Barrier (BBB) and form a tight monolayer that regulates the passage of molecules, with the ATP-Binding Cassette (ABC) transporters efflux pumps. We have developed a human in vitro model of HBEC-5i endothelial cells cultivated alone or with human astrocytes conditioned medium on insert. HBEC-5i cells showed a tight monolayer within 14 days, expressing ZO-1 and claudin 5, a low apparent permeability to small molecules, with a TEER stability during five days. The P-gp, BCRP, MRPs transporters were well expressed and functional. Accumulation and efflux ratio measurement with different ABC transporters substrates (Rhodamine 123, BCECF AM, Hoechst 33342) and inhibitors (verapamil, Ko143, probenecid and cyclosporin A) were conducted. At barrier level, the functionality of ABC transporters was three-fold enhanced in astrocyte conditioned medium. We validated our model by the transport of pharmacological substrates: caffeine, rivaroxaban, and methotrexate. The rivaroxaban and methotrexate were released with an efflux ratio >3 and were decreased by more than half with inhibitors. HBEC-5i model could be used as relevant tool in preclinical studies for assessing the permeability of therapeutic molecules to cross human BBB.

Background: Patients treated for pulmonary arterial hypertension (PAH) frequently receive vitamin K antagonists (VKAs) for PAH or validated indications (such as atrial fibrillation or venous thromboembolism). In these latter indications, VKAs are challenged by direct oral anticoagulants (DOAs). Decreased dosage of DOAs has been proposed in patients at risk of bioaccumulation. Objectives: We aimed to evaluate the frequency of bioaccumulation risks in patients treated with PAH-targeted therapy, particularly regarding the presence of validated indications. Methods: We conducted a retrospective study in three different PAH referral centers. All patients receiving PAH-targeted therapy were classified according to demographics, prescription and indications of VKAs, and the presence of major bioaccumulation risk factors (renal failure, low body weight, strong P-glycoprotein or cytochrome P3A4 inhibitors). Results: Two hundred and thirty-nine of the 366 patients included received VKAs, 94 for validated indications. At least one major risk factor was found in 231 (63.1%) of the whole study population, and in 54 (57.4%) of the patients anticoagulated for a validated indication. No specific patient phenotype could be individualized. Conclusions: About 1 in 2 patients treated with PAH therapy has at least one of the three major risk factors for DOA bioaccumulation. DOAs in the PH setting could be associated with bioaccumulation and should be individualized, mainly in patients with confirmed indication.

International audience; Purpose Fondaparinux, a selective activator factor X (factor Xa) inhibitor, is effective and safe for preventing venous thromboembolism after major orthopaedic surgery (MOS) at the once-daily subcutaneous dose of 2.5 mg. As the drug is mainly eliminated by the kidneys, a reduced dosage (1.5 mg once daily) was developed for patients with renal impairment. Methods We studied the pharmacokinetics (PK) of this dosage regimen using data from a real-world cohort of 442 patients with renal impairment (creatinine clearance 20-50 ml/min) undergoing MOS. Data were analysed using NON-linear Mixed Effect Modelling software (NONMEM) software. Fondaparinux PK was modelled using a two-compartment model with first-order absorption. Results This analysis confirmed the relationship between renal function and fondaparinux PK profiles. The mean predicted steady-state area under the plasma concentration time curve, peak and trough plasma concentrations of fondaparinux were lower by 15.6 %, 13.0 % and 10.3 %, respectively, in patients with renal impairment treated with 1.5 mg compared with patients with normal renal function treated with 2.5 mg (p Conclusion Although administration of 1.5 mg fondaparinux in patients with renal impairment resulted in a predicted exposure slightly lower than that achieved with 2.5 mg in patients with normal renal function, fondaparinux 1.5 mg is a valuable thromboprophylactic option in MOS patients with renal impairment who are at risk of bleeding.

International audience; In a meta-analysis of case-control studies, Zhang et al. (2011) found an increased risk of venous thromboembolic events (VTE) in patients exposed to antipsychotics (OR=2.39 [1.71-3.35]). Our updated meta-analysis including the 2 available cohort studies, recognized as a more relevant type of observational study, showed a weaker, but still strong association (OR=1.84 [1.39; 2.44]). In view of the lack of data on the confirmed risk factors for VTE in existing studies, prospective studies including adjustment for these risk factors are warranted to confirm this association and to assess the benefit/risk ratio of antipsychotics in high-risk patients.

International audience; A pharmacokinetic-pharmacodynamic (PK-PD) drug-drug interaction between acenocoumarol and amoxicillin + clavulanic acid antibiotic was assessed in eight healthy volunteers, using a population PK-PD) model. Each subject received at day 1 a single dose of 8 mg of acenocoumarol. Then 1 g of amoxicillin + 250 mg of clavulanic acid was given from days 3 to 9. On day 8, each subject received a single dose of 8 mg of acenocoumarol concomitantly with the antibiotic combination. Eleven blood samples were taken during 48 h following each acenocoumarol administration. Acenocoumarol plasma concentrations and prothrombin time were measured at each sampling time. We first identified the structural PK model by pooling data from this trial with individual data from other acenocoumarol PK trials. An indirect response model was used to fit PD data. Models were built using a non-linear mixed effect modelling approach with nonmem software. Covariates were tested on PK and PD parameters, including antibiotic treatment. Acenocoumarol PK data were fitted by a two-compartment, first-order input model with log normal inter-individual variability. Weight and antibiotic treatment were found to improve significantly the fit of PK data with a 15% decrease in acenocoumarol clearance with concomitant antibiotics (P < 0.05). An indirect response model was successfully applied to the PK-PD data of acenocoumarol. No covariate, including antibiotic treatment effect, significantly affected PT. Drug-drug interaction was demonstrated at the PK level, without any PD corollary.

Contexte et objectifs de l’étude : le rituximab est un anticorps monoclonal chimérique anti CD20 utilisé depuis plus de 20 ans dans le traitement de diverses pathologies d’onco-hématologie. Ses caractéristiques pharmacocinétiques atypiques, l’absence de fenêtre thérapeutique et la forte variabilité dans la réponse thérapeutique rendent nécessaires la poursuite d’études pour mieux caractériser les relations pharmacocinétiques et pharmacocinétiques/pharmacodynamiques. Afin de répondre aux attentes des hématologues de l’AP-HM, il a été décidé de mettre en place le suivi thérapeutique pharmacologique du rituximab. Le travail présenté ici répond à deux objectifs : d’une part le développement et la validation d’une méthode de dosage du rituximab, d’autre part la réalisation de simulations de profils pharmacocinétiques typiques du rituximab et l’identification des paramètres pharmacocinétiques individuels des patients du service d’hématologie par approche bayésienne à partir de modèles publiés dans la littérature.Matériel et méthodes : la technique choisie pour la méthode de dosage est la chromatographie liquide couplée à la spectrométrie de masse en tandem. La préparation des échantillons consiste en une extraction en phase solide avec enrichissement en immunoglobulines G. La sélection des peptides protéotypiques du rituximab est réalisée en amont par la société Promise Advanced Proteomics. Il faut ensuite sélectionner des transitions de quantification et de qualification. La validation de la méthode est basée sur les recommandations de l’Agence Européenne des Médicaments. Deux modèles ont été sélectionnés dans la littérature afin de réaliser des simulations de profils pharmacocinétiques avec une fonction du package mlxR du logiciel R, Simulx. Ces deux modèles se distinguent par une description différente de l’élimination spécifique du rituximab : soit une élimination médiée par la cible soit une clairance variant avec le temps. Les valeurs publiées des paramètres PK de population de chaque étude ont été utilisées pour faire ces simulations.Résultats et discussion : la transition de quantification et les deux transitions de qualification sélectionnées appartiennent au même peptide protéotypique. Le gradient utilisé au cours de l’analyse chromatographique a été optimisé, ainsi que les valeurs de la tension de cône et de l’énergie de collision. L’ensemble des critères de performance a été validé. Des échantillons prélevés chez des patients du service d’hématologie de l’hôpital de la Conception (AP-HM, Marseille) ont été dosés afin de tester la méthode de dosage avec des échantillons cliniques. Concernant les simulations, malgré des choix de modélisation de l’élimination différents, les profils pharmacocinétiques obtenus sont semblables. Les deux modèles permettent ensuite, par approche bayésienne de déterminer les paramètres pharmacocinétiques de chacun des patients prélevés en hématologie.Conclusion et perspectives : la chromatographie liquide couplée à la spectrométrie de masse en tandem est une méthode robuste et spécifique qui offre l’avantage de pouvoir doser plusieurs mAb en simultané. La capacité des deux modèles sélectionnés à estimer les paramètres PK individuels doit être testée sur un plus grand nombre de patients : en fonction des résultats, il sera décidé de les utiliser en routine ou de développer un nouveau modèle. Le développement du suivi thérapeutique pharmacologique des anticorps monoclonaux se justifie par l’aspiration à une médecine personnalisée mais aussi par des considérations pharmaco-économiques.

The replacement of VKAs by DOACs is increasing, due to less constraining use for the patient and lower intra- and interindividual variability. Contrary to VKAs, DOACs are substrates of efflux pumps. These transporters are involved in the pharmacokinetics of DOACs, particularly in their absorption and both biliary and renal elimination. This work aimed to evaluate in vitro the role of P-gp and BCRP transporters in the pharmacokinetic variability of rivaroxaban, a direct specific activated factor X inhibitor. The first study focused on the interaction between riociguat and two DOACs, apixaban and rivaroxaban. The IC50 estimate highlighted the ability of the transfected MDCK cell models to detect the inhibition of BCRP-mediated efflux of both DOACs by riociguat, although this inhibition was not sufficiently strong to be clinically relevant. The second study focused on factors that could affect the results of in vitro rivaroxaban transport studies, in particular the expression levels of ABC transporters. Mass spectrometry analysis revealed a higher expression of P-gp and BCRP in transfected MDCK cells but also a different expression of transporters between two Caco-2 models coming from a different cell bank. Rivaroxaban efflux ratios and IC50s of specific inhibitors were correlated with the quantity of transporters detected in the different cell models. The third study was based on the development of an in vitro mode] of hepatic transport using the HepaRG cell line. HepaRG cells had characteristics similar to in vivo hepatocytes, particularly expression of functional canalicular efflux transporters which are involved in the biliary secretion of rivaroxaban. Accumulation studies showed that rivaroxaban was a substrate of hepatic efflux transporters and that canalicular secretion decreased in the presence of P-gp and BCRP inhibitors. Finally, in vitro cell models highlight the involvement of two efflux transporters, P-gp and BCRP, at the intestinal, renal and hepatic levels in the disposition of rivaroxaban, and that this transport can contribute to the pharmacokinetic variabilitv of rivaroxaban.; La prescription des AOD en remplacement des AVK est de plus en plus importante, du fait d'une utilisation moins contraignante pour le patient et d'une plus faible variabilité intra- et interindividuelle. Contrairement aux AVK, les AOD sont des substrats des pompes d'efflux. Ces transporteurs sont impliqués dans la pharmacocinétique des AOD, particulièrement dans leur absorption et dans leur élimination biliaire et rénale. L'objectif de ce travail de thèse a été d'évaluer in vitro le rôle des transporteurs P-gp et BCRP dans la variabilité pharmacocinétique du rivaroxaban, un inhibiteur direct du facteur Xa, La première étude a été axée sur l'étude d'une interaction entre le riociguat et deux AOD, l'apixaban et le rivaroxaban. L'estimation des IC50 a révélé la capacité des modèles cellulaires MDCK transfectés à détecter une inhibition de l'efflux lié à BCRP des deux AOD par le riociguat, bien que cette inhibition ne soit pas assez puissante pour être cliniquement pertinente. La seconde étude était centrée sur l'étude des facteurs pouvant faire varier les résultats des études de transport du rivaroxaban obtenus in vitro, en particulier la quantité des transporteurs ABC. Un analyse en spectrométrie de masse a révélé une expression plus élevée de P-gp et BCRP dans les cellules MDCK transfectées mais aussi une différence d'expression des transporteurs entre deux lignées Caco-2 issues d'une banque cellulaire différente. Les ratios d'efflux du rivaroxaban et les IC50 des inhibiteurs spécifiques étaient corrélés à la quantité de transporteurs mesurée dans les différents modèles. La troisième étude était basée sur la mise en place d'un modèle d'étude in vitro du transport hépatique, grâce à la lignée HepaRG. Ces cellules présentent des caractéristiques semblables aux hépatocytes in vivo, en particulier l'expression de transporteurs d'efflux fonctionnels au pôle canaliculaire, impliqués dans l'efflux biliaire du rivaroxaban. Des études d'accumulation ont démontré une prise en charge du rivaroxaban par les transporteurs d'efflux hépatiques, ainsi qu'une diminution de l'efflux dans les canalicules biliaires en présence d'inhibiteurs de P-gp et BCRP. Finalement, les modèles cellulaires in vitro démontrent que le rivaroxaban est pris en charge par deux transporteurs d'efflux, P-gp et BCRP, au niveau intestinal, rénal et hépatique, et que ce transport peut contribuer à la variabilité pharmacocinétique du rivaroxaban.

The blood-brain barrier (BBB) controls the passage of drugs, in part through the expression of the ATP Binding Cassette (ABC) transporters. In many brain diseases, the BBB is altered. Among them, intracerebral haemorrhages (ICH), which are an iatrogenic effect of anticoagulants. Clinical analyses show that patients with Direct Oral Anticoagulants (DOACs) treatment have less HIC than patients treated with the reference anticoagulants, Vitamin K Antagonist (VKA), without understanding the cellular mechanisms. One of the differences between DOACs and VKA lies in their pharmacokinetic profile, indeed, DOACs are substrates of ABC transporters unlike VKA. During HIC, thrombin, is activated and causes alterations in the BBB by the cleavage of the protease activated receptor (PAR).The objectives of this thesis work were first to set up an in vitro model of the BBB in order to study the passage of drugs and their interactions with ABC transporters. In a second step, the model is used to study the interactions of DOACs in pathological conditions.The model developed is based on the HBEC-5i cell line seldom described in the literature. The cells were cultured in monolayer on insert with conditioned medium from human astrocytes. It allows the study of the interaction of therapeutics with ABC transporters by measuring efflux ratios. The model has been validated by transport studies of pharmacological molecules. In order to meet our second objective, we compared the effect of thrombin exposure with or without pretreatment with anticoagulants (rivaroxaban, dabigatran, apixaban, warfarin and heparin sodium) on our model. DOACs limit the BBB damage induced by the thrombin unlike other anticoagulants. Our results showed that alteration of the BBB is mediated by the cleavage of the PAR-1 receptor by thrombin. This cleavage is not the same depending on the class of anticoagulants used, DOACs minimizing this cleavage. All this thesis work made it possible to provide the first cellular explanations of the opening mechanisms of the BBB following HIC under DOACs., La barrière hémato-encéphalique (BHE) contrôle le passage des médicaments, en partie par la présence d’ATP Binding Cassette (ABC) transporteurs. Dans de nombreuses pathologies cérébrales, la BHE est altérée. Parmi elles, les hémorragies intracérébrales (HIC), qui sont un effet iatrogène des anticoagulants. Des analyses cliniques montrent que les patients sous Anticoagulants Oraux Directs (AODs) présentent moins d’HIC que les patients traités avec les anticoagulants de référence, les anti-vitamine K (AVK), sans que les mécanismes cellulaires soient élucidés. Une des différences entre les AODs et les AVK résident dans leur profil pharmacocinétique, effectivement, les AODs sont des substrats des ABC transporteurs contrairement aux AVKs. Au cours des HIC, la thrombine est activée et entraine une altération de la BHE par clivage et des récepteurs protease activated receptor (PAR).Les objectifs de ce travail de thèse ont été de mettre en place un modèle in vitro de BHE afin d’étudier les interactions des médicaments avec les ABC transporteurs. Ensuite, le modèle est utilisé pour étudier les interactions des AODs en condition pathologique.Le modèle développé est basé sur la lignée HBEC-5i, peu décrite dans la littérature. Les cellules ont été cultivées en monocouche sur insert avec milieu conditionné issu d’astrocytes humains. Le modèle permet l’étude de l’interaction de thérapeutiques avec des ABC transporteurs par des mesures d’efflux ratios. Le modèle a été validé par des études de transport de molécules pharmacologiques. Ensuite, nous avons comparé, sur notre modèle, les effets de l’exposition à la thrombine avec ou sans prétraitement d’anticoagulants (rivaroxaban, dabigatran, apixaban, warfarine et héparine). Les AODs limitent l’ouverture de la BHE induite par la thrombine contrairement aux autres anticoagulants. Nos résultats ont montré que l’altération de la BHE est médiée par le clivage du récepteur PAR-1 par la thrombine. Ce clivage n’est pas le même en fonction de la classe d’anticoagulants utilisée, les AODs minimisant ce clivage. L’ensemble de ce travail de thèse a permis de donner des premières explications cellulaires quant aux mécanismes d’ouverture de la BHE consécutifs aux HIC sous AODs.