INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas. / BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.

Le rein joue un rôle essentiel dans l’élimination des médicaments et de leurs métabolites, de ce fait il assure la défense de l'organisme contre de potentiels xénobiotiques toxiques. Particulièrement, les transporteurs des tubules proximaux rénaux qui ont un rôle dans la sécrétion tubulaire des médicaments. Ainsi, ils sont des déterminants important de la biodisponibilité des xénobiotiques dans l’organisme.Dans cette thèse nous nous sommes intéressés à l’implication des transporteurs rénaux humains hOAT1 et hOAT3 dans des interactions médicamenteuses moyennant des modèles in vitro. Après construction et validation des modèles d’études cellulaires HEK-hOAT1 et HEK-hOAT3, nous avons testé l’effet des inhibiteurs de la pompe à protons sur le transport du méthotrexate par les OATs ainsi que l’effet des antiviraux sur l’influx du tenofovir par ces mêmes transporteurs. Grâce à nos modèles cellulaires nous avons tenté d’expliquer in vitro de probables interactions médicamenteuses décrites en clinique. / The kidney plays an essential role in the elimination of drugs and their metabolites, thus it ensures the defense of the body against potential toxic xenobiotic. Particularly, the secretory transporters in the proximal tubule are major determinants of the disposition of xenobiotic in the body.In this thesis we investigated the involvement of human organic anions transporters hOAT1 and hOAT3 in drug drug interactions through study on in vitro cell models. After construction and validation of cells models studies HEK-hOAT1 and HEK-hOAT3, we tested the effect of proton pump inhibitors on methotrexate transport by OATs and the effect of antivirals on the influx of tenofovir by these two transporters. With our models we tried to explain in vitro probable drug interactions described in the clinic.

INTRODUÇÃO: A desregulação da via de sinalização JAK-STAT é uma característica marcante das neoplasias mieloproliferativas (NMPs), doenças clonais da célula tronco hematopoética, dentre as quais encontra-se a mielofibrose (MF). Diversos inibidores de JAK foram desenvolvidos para o tratamento da MF e encontram-se em diferentes fases de desenvolvimento clínico. Devido ao seu desenvolvimento recente, pouco se sabe a respeito do papel de transportadores de membrana na farmacocinética desses compostos. Essas proteínas realizam o influxo e efluxo celular de substratos endógenos e xenobióticos, e alterações na expressão desses transportadores podem influenciar a resposta a esses fármacos. OBJETIVO: Avaliar o efeito de um inibidor comercial da via JAK-STAT na expressão gênica dos transportadores de membrana ABCB1, ABCG2, SLC22A1 e SLCO1A2 em células HepG2, Caco-2 e HEL92.1.7. MÉTODOS: Linhagens de carcinoma hepatocelular (HepG2), adenocarcinoma colorretal (Caco-2) e eritroleucemia humana homozigotas para JAK2V617F (HEL92.1.7) foram cultivadas e tratadas o inibidor comercial da via JAK-STAT JAK Inhibitor I. Para determinar a concentração ideal para o tratamento com o inibidor, as células foram tratadas com diversas concentrações do inibidor de JAK por 24 horas e foram feitos testes de viabilidade celular e fragmentação do DNA. Com as condições de tratamento padronizadas, foi extraído o RNA total das células e sintetizado o cDNA, para análise das expressões de RNAm dos genes ABCB1, ABCG2, SLC22A1 e SLCO1A2 por PCR em tempo real. Foi também avaliada a expressão dos transportadores de efluxo ABCB1 e ABCG2 por citometria de fluxo, utilizando anticorpos primários direcionados a essas proteínas. RESULTADOS: Nas células HepG2, foi observado um aumento da expressão de RNAm de ABCB1 nas células tratadas com 4,00 µM do inibidor de JAK, quando comparado com o controle (células incubadas apenas com o veículo) (P=0,041). Não foi observada alteração da expressão de RNAm de ABCG2 e SLC22A1 com o tratamento com o inibidor de JAK nessa linhagem (P>0,05); a expressão de RNAm de SLCO1A2 não foi detectada nessa linhagem. Nas células Caco-2, a expressão de ABCB1, ABCG2, SLC22A1 e SLCO1A2 não se alterou com o tratamento com o inibidor de JAK nas concentrações utilizadas (0,25 µM a 1,00 µM) por 24 horas (P>0,05). Para as células HEL92.1.7, não foi observada diferença na expressão de RNAm de ABCB1, ABCG2 e SLC22A1 com o tratamento com 1,00 µM do inibidor de JAK por 24 horas em comparação ao controle (P>0,05); nessa linhagem, a expressão de RNAm de SLCO1A2 não foi detectada. A expressão proteica dos transportadores ABCB1 e ABCG2 não sofreu alteração com o tratamento com o inibidor de JAK nas condições utilizadas nas três linhagens celulares estudadas (P>0,05). CONCLUSÕES: Apenas as células HepG2 apresentaram um aumento da expressão de RNAm do transportador de efluxo ABCB1 em concentrações elevadas do inibidor de JAK, sugerindo que os inibidores de JAK podem modular a expressão do gene desse transportador no fígado. O tratamento com o inibidor da via JAK-STAT não foi associado com alterações na expressão proteica de ABCB1 e ABCG2 em todas as células estudadas. / BACKGROUND: JAK-STAT pathway signaling disregulation is a hallmark of myeloproliferative neoplasms (MPN), hematopoietic stem cell clonal diseases, among which is myelofibrosis (MF). Several JAK inhibitors have been developed for MF treatment and are found in different stages of clinical development. Because the recent development of these compounds, the role of drug transporters in their pharmacokinetics is poorly understood. These proteins perform celular influx and effux of endogenous substrates and xenobiotics, and changes in the expression of these drugs transporters may affect the response to these drugs. AIM: To evaluate the effect of a JAK-STAT pathway commercial inhibitor in gene expression of drug transporters ABCB1, ABCG2, SLC22A1 and SLCO1A2 in HepG2, Caco-2 and HEL92.1.7 cells. METHODS: Hepatocellular carcinoma cell line HepG2, colorectal adenocarcinoma cell line Caco-2 and human erythroleukemia homozygous JAK2V617F cell line HEL92.1.7 were grown and treated with the JAK-STAT pathway inhibitor JAK Inhibitor I. In order to determine the optimal concentration for treatment with the inhibitor, cells were treated with several concentrations of JAK inhibitor by 24 hours, and cell viability and DNA fragmentation tests were performed. Once the treatment conditions were standardized, total RNA were obtained from the cells, and cDNA was synthesized in order to evaluate the mRNA expression of ABCB1, ABCG2, SLC22A1 and SLCO1A2 genes, performed by real time PCR. We also evaluate the expression of drug efflux transporters ABCB1 and ABCG2 by flow cytometry, using primary antibodies directed to these proteins. RESULTS: In HepG2 cells, it was observed an increase in ABCB1 mRNA expression in cells treated with 4,00 µM of JAK inhibitor, when compared with controls (cells exposed only to the vehicle) (P=0.041). There was no change in ABCB2 and SLC22A1 mRNA expression with the treatment with JAK inhibitor in this cell line (P>0.05); SLCO1A2 mRNA was not detected in this cell line. In Caco-2 cells, ABCB1, ABCG2, SLC22A1 and SLCO1A2 mRNA expression did not change with treatment with the JAK inhibitor at the concentrations used (0.25 µM to 1.00 µM) by 24 hours (P>0.05). In HEL92.1.7 cells, it was not observed differences in ABCB1, ABCG2 and SLC22A1 mRNA expression with the treatment with 1 µM of JAK inhibitor by 24 hours when compared with controls (P>0.05); in this cell line, SLCO1A2 mRNA was not detected. Protein expression of ABCB1 and ABCG2 drug transporters has not changed with treatment with the JAK inhibitor under the conditions used in the three cell lines studied. CONCLUSIONS: Only HepG2 cells presented an increase in mRNA expression of drug efflux transporter ABCB1 in presence of high levels of JAK inhibitor, suggesting that JAK inhibitors could modulate this transporter gene expression in liver. Treatment with JAK-STAT pathway inhibitor was not associated with changes in ABCB1 and ABCG2 protein expression in all cell lines studied.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Křečková Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Role of drug transporters in placental transfer of entecavir Entecavir (ETV), an analogue of guanosine, is a highly efficient anti-hepatitis B antiviral drug. It is the first-line therapy for both adults and children. Its use in pregnancy is limited due to a number of factors, including lack of data on placental pharmacokinetics. The placental transition of drugs is frequently controlled by drug transporters. ATP-binding (ABC) transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) or multidrug resistance-associated protein 2 (MRP2) localized in the apical membrane of syncytiotrophoblast and pumping their substrates in the feto-maternal direction belong to most significant determinants of placental pharmacokinetics. Moreover placental transport of nucleoside-derived drugs can be affected by the activity of nucleoside transporters (NTs); equilibrative nucleoside transporters (ENTs) mediate facilitated diffussion, while the concentrative nucleoside transporters (CNTs) control active influx of their substrates. The aim of the diploma thesis was to describe the role of P-gp, BCRP, MRP2 and NTs (ENTs and...

No description available.

The liver is the major detoxifying organ, clearing the blood from drugs and other xenobiotics. The extent of hepatic clearance (CL) determines drug exposure and hence, the efficacy and toxicity associated with exposure. Drug-drug interactions (DDIs) that alter the hepatic CL may cause more or less severe outcomes, such as adverse drug reactions. Accurate predictions of drug CL and DDI risk from in vitro data are therefore crucial in drug development. Liver CL depends on several factors including the activities of transporters involved in the hepatic uptake and efflux. The work in this thesis aimed at developing new in vitro and in silico methods to predict hepatic transporter-mediated CL and DDIs in vivo. Particular emphasis was placed on interactions involving the hepatic uptake transporters OATP1B1, OATP1B3, and OATP2B1. These transporters regulate the plasma concentration-time profiles of many drugs including statins. Inhibition of OATP-mediated transport by 225 structurally diverse drugs was investigated in vitro. Several novel inhibitors were identified. The data was used to develop in silico models that could predict OATP inhibitors from molecular structure. Models were developed for static and dynamic predictions of in vivo transporter-mediated drug CL and DDIs. These models rely on a combination of in vitro studies of transport function and mass spectrometry-based quantification of protein expression in the in vitro models and liver tissue. By providing estimations of transporter contributions to the overall hepatic uptake/efflux, the method is expected to improve predictions of transporter-mediated DDIs. Furthermore, proteins of importance for hepatic CL were quantified in liver tissue and isolated hepatocytes. The isolation of hepatocytes from liver tissue was found to be associated with oxidative stress and degradation of transporters and other proteins expressed in the plasma membrane. This has implications for the use of primary hepatocytes as an in vitro model of the liver. Nevertheless, by taking the altered transporter abundance into account using the method developed herein, transport function in hepatocyte experiments can be scaled to the in vivo situation. The concept of protein expression-dependent in vitro-in vivo extrapolations was illustrated using atorvastatin and pitavastatin as model drugs.

No description available.

Les transporteurs membranaires sont des éléments importants dans le devenir, l’efficacité, et la toxicité du médicament. Ils influencent la pharmacocinétique et la pharmacodynamie de ces derniers. Plusieurs interactions médicamenteuses observées cliniquement sont attribuables à la fois aux enzymes responsables du métabolisme des médicaments et aux transporteurs membranaires. Il est connu qu’une variabilité existe entre différents individus dans la réponse à un médicament et les polymorphismes génétiques retrouvés dans les gènes codant pour les transporteurs membranaires peuvent partiellement expliquer cette variabilité. OATP1A2 est un transporteur membranaire exprimé sur des organes importants, comme le cerveau et le rein. Plusieurs médicaments utilisés en clinique sont des substrats d’OATP1A2 et l’expression localisée de ce transporteur suggère un rôle important dans le devenir du médicament. Donc, mon projet de doctorat consistait à caractériser l’activité d’OATP1A2 en relation avec ses substrats et inhibiteurs, et de plus, à évaluer l’impact de différents variants génétiques d’OATP1A2 sur leur transport. Dans le premier article, la rosuvastatine a été utilisée comme substrat-type pour étudier le transport d’OATP1A2. Les expériences ont été menées en introduisant la rosuvastatine en compétition avec différent β-bloqueurs, une classe de médicaments rapportée dans la littérature comme substrats d’OATP1A2. Parmi les β-bloqueurs évalués, le carvédilol était l’inhibiteur le plus puissant. Dans la deuxième partie de l’étude, des médicaments ayant une structure similaire au carvédilol, tels que les antidépresseurs tricycliques, ont été évalués quant à leur potentiel d’inhibition sur OATP1A2. Une relation structure-activité a été définie à l’aide de ces données. Nous avons démontré que des composés tricycliques avec une courte chaîne aliphatique pouvaient inhiber OATP1A2. Dans le deuxième article, OATP1A2 a été étudié en considérant son expression et son rôle au sein de la barrière hémato-encéphalique (BHE). Des études précédentes ont démontré qu’OATP1A2 est exprimé sur la membrane luminale des cellules endothéliales formant la BHE. Nos données démontrent que les triptans, une classe de médicaments couramment utilisées pour traiter la crise migraineuse, sont des substrats d’OATP1A2 et que les composés tricycliques identifiés comme inhibiteurs d’OATP1A2 dans nos études précédentes peuvent inhiber le transport des triptans par OATP1A2. Ces résultats sont importants puisque: 1) il a été suggéré que les triptans peuvent agir au niveau du système nerveux central en se liant aux récepteurs trouvés sur les neurones centraux; 2) comme les triptans sont des molécules hydrophiles, un mécanisme de transport facilité est nécessaire pour qu’ils pénètrent la BHE et OATP1A2 pourrait être l’élément clé; 3) l’inhibition d’OATP1A2 par les composés tricycliques pourrait limiter l’accès des triptans à leur site d’action. Le troisième article caractérise l’activité associée à deux variants génétiques d’OATP1A2 (OATP1A2*2 et *3). Leur capacité à transporter les triptans et leur potentiel d’inhibition par les médicaments tricycliques ont été évalués. Des résultats supplémentaires caractérisant OATP1A2, mais sans liens directs avec les trois articles, seront présentés en annexe. Dans l’ensemble, les résultats présentés dans cette thèse servent à caractériser le transporteur membranaire OATP1A2 en relation avec ses substrats et inhibiteurs, et en fonction de ses variants génétiques. / Drug transporters are important determinants in drug disposition, efficacy, and toxicity. They influence the pharmacokinetics and pharmacodynamics of drugs. Several clinically-observed drug-drug interactions are mediated through drug metabolizing enzymes and drug transporters. It is well known that there is an interindividual variability in the response to medications and polymorphisms found in genes encoding for drug transporters partially account for it. OATP1A2 is a membrane drug transporter expressed on important organs, such as the brain and the kidney. A wide spectrum of drugs used in the clinic are substrates of OATP1A2. Its localisation suggests an essential role in drug disposition. Thus, my PhD project consisted of characterizing the activity of OATP1A2 in regards to its substrates, inhibitors, and different protein variants due to genetic polymorphisms. In the first article, rosuvastatin was used as the probe substrate to study OATP1A2 transport activity. Experiments were conducted by putting rosuvastatin in competition with different β-blockers, a class of drugs known in the literature to be transported by OATP1A2. One of the drugs evaluated, carvedilol, inhibited OATP1A2 with much more potency than the others. In the second part of the study, drugs with a structure similar to carvedilol, such as tricyclic antidepressants, were tested for their potential to inhibit OATP1A2. A structure-activity relationship was defined using the data. It was demonstrated that drugs composed of a tricyclic ring with a short aliphatic amine chain were potent OATP1A2 inhibitors. In the second article presented, OATP1A2 was studied in the context of its localization at the blood-brain barrier (BBB). OATP1A2 expression at the luminal membrane of the endothelial cells making up the BBB was demonstrated in the literature. Our article showed that triptans, a class of commonly used anti-migraine drugs, were OATP1A2 substrates. The tricyclic drugs previously evaluated were shown to potently inhibit triptan transport through OATP1A2. These findings are important for three reasons: 1) it has been postulated that triptans may act at the central nervous system by binding to receptors found on central neurons; 2) as triptans are hydrophilic molecules, a facilitated transport mechanism is required for them to penetrate the BBB and OATP1A2 may be the key player; and 3) the inhibition of OATP1A2 by the tricyclic drugs may limit the entrance of triptans to their site of action. The third article characterized the transport activity of two OATP1A2 protein variants (OATP1A2*2 and *3). Their capacities to transport triptans and their potential of being inhibited by tricyclic drugs were evaluated. Additional data characterizing OATP1A2 but considered out of the scope of the three articles will be presented in appendices. In overall, the central theme of this thesis looks into the characterization of the OATP1A2 membrane drug transporter in regards to its substrates, inhibitors, and proteins variants.

La bioéquivalence, une mesure de substitution de l'innocuité et de l'efficacité à différents stades du processus de développement des médicaments, est tout particulièrement importante lors du développement d'un médicament générique. Entre autres critères, la bioéquivalence garantit que les médicaments génériques sont équivalents aux produits innovateurs ou de références approuvés en termes d’efficacité clinique et d’innocuité tout en contournant le long cours et le coût élevé des essais chez les animaux et des essais cliniques chez les patients exigés pour les médicaments innovants. Malgré les avancées dans le développement d'approches robustes au cours des dernières décennies, la pratique actuelle de la bioéquivalence fait toujours l'objet de controverses. Le but de cette thèse est d'explorer certaines de ces controverses et de les aborder en proposant des approches nouvelles et alternatives. L'une des questions les plus controversées dans la pratique actuelle de la bioéquivalence est l'extrapolation des résultats d'études de bioéquivalence d'une population à une autre. La majorité des études de bioéquivalence portant sur des formes pharmaceutiques orales efficaces par voie systémique reposent sur les critères de pharmacocinétique obtenus chez des sujets sains, alors que la population cible est constituée de patients. Ceci est basé sur l'hypothèse que si deux produits sont bioéquivalents dans une population, ils devraient l'être dans une autre. L'extrapolation des résultats des études de bioéquivalence ne se limite pas à celle des sujets sains aux patients. Depuis 2007, une proportion croissante d'études de bioéquivalence pharmacocinétique portant sur des soumissions génériques nord-américaines ou européennes a été réalisée auprès de populations géographiques/ethniques autres que celles visées, en raison du coût moins élevé de ces études en dehors de l'Amérique du Nord et de l'Europe. Dans le premier volet de cette thèse, nous avons examiné si les résultats de la bioéquivalence obtenus dans une population géographique ou ethnique pouvaient être extrapolés à une autre. À cette fin, nous avons extrait les résultats des études de bioéquivalence pharmacocinétique disponibles publiquement et provenant de soumissions génériques à Santé Canada et à la Food and Drug Administration des États-Unis. Pour dix médicaments différents, nous avons calculé l'effet d’un repas normalisé sur le produit de référence et comparé les résultats obtenus chez deux populations ethniques, les indiens et les nord-américains. Cette approche novatrice est basée sur le raisonnement suivant: si l'effet d’un repas sur le produit de référence est le même chez les populations indienne et nord-américaine, le produit générique et sa référence qui se sont révélés bioéquivalents dans la population indienne devraient également l'être dans la population nord-américaine. Pour 90% des médicaments à l'étude, une différence statistiquement significative a été détectée entre les deux populations après un repas. Pour 30% de ces médicaments, la différence s'est révélée d'une pertinence clinique possible. Les résultats de cette étude ont mis en évidence que l’extrapolation des résultats de bioéquivalence d’une population à l’autre devrait possiblement être reconsidérée pour certains médicaments. Les défis dans le contexte de la bioéquivalence ne se limitent pas toujours aux études pivots où la performance d’un produit générique est comparée à celle de la référence. En effet, une étude pilote peut être menée afin d’établir un protocole d’étude approprié pour cette étude pivot. Par conséquent, les résultats inexacts provenant d'une étude pilote, tels qu'une estimation imprécise du moment ou de la durée d’administration optimale de la dose lors de la comparaison du produit testé par rapport à la référence, pourront affecter négativement les résultats de l’étude de bioéquivalence. Ceci est particulièrement crucial pour les produits indiqués pour un usage topique dermatologique dont les corticostéroïdes constituent un cas d’espèce. En effet, leur bioéquivalence est démontrée par une mesure pharmacodynamique, le blanchiment cutané, à différents temps après application topique. L’intensité du blanchiment est comparée entre le produit générique et le produit de référence à une durée d’administration spécifique d’une dose donnée, la DD50, soit la durée associée à 50% de l’effet maximal observé. Par conséquent, cette durée d’administration de la dose doit d’abord être déterminée dans le cadre d’une étude pilote. L’agence réglementaire américaine recommande l’utilisation d’une approche populationnelle basée sur la modélisation non linéaire à effets mixtes pour l'estimation de la DD50 et ce, quelle que soit la méthode d'analyse. Étant donné qu’il existe différents types de méthodes d’analyse non linéaire à effets mixtes, chaque commanditaire peut en choisir une différente. Dans le deuxième volet de cette thèse, nous avons examiné si les mêmes estimations de DD50 pouvaient être obtenues en utilisant différentes méthodes non linéaires à effets mixtes. À cette fin, nous avons ajusté les données de blanchiment de la peau d’onze études avec deux méthodes non linéaires à effets mixtes différentes : le maximum de vraisemblance avec maximisation de l’espérance (MLEM) et l'estimation conditionnelle de premier ordre (FOCE). Les résultats ont favorisé MLEM, compte tenu d’une meilleure puissance discriminative pour l’estimation de la DD50 de population et d’une meilleure minimisation de la variabilité interindividuelle. Bien que l'approche de la bioéquivalence fondée sur la pharmacocinétique ait contribuée de manière significative au développement de versions génériques de haute qualité des formes pharmaceutiques orales indiquées pour un effet systémique, la disponibilité de versions génériques pour les produits dermatologiques topiques demeure limitée et ce, par manque de méthodes acceptées par les agences réglementaires pour l'évaluation de la bioéquivalence de ces produits. Dans le troisième volet de cette thèse, une nouvelle approche pour l’évaluation de la bioéquivalence de formulations de crème topique d’acyclovir a été développée en utilisant une analyse basée sur un modèle de données d’exposition locales récupérées à partir d’échantillons de peau abrasée prélevés à une seule durée d’administration de la dose, la DD50 à l’aide de bandes adhésives. Un seul échantillonnage de peau effectué à la DD50 a non seulement assuré que les données pharmacocinétiques étaient recueillies à la durée d’administration de la dose ayant le meilleur pouvoir discriminant pour détecter une différence au niveau des formulations, mais a également permis de diminuer considérablement le nombre d'échantillons à analyser. Et surtout, cette nouvelle approche a permis de générer un profil pharmacocinétique au niveau même de la peau. Ce faisant, nous avons pu utiliser l'analyse compartimentale populationnelle et contourner les nombreuses hypothèses et calculs sophistiqués requis par les méthodes précédentes. Notre approche a également permis de générer de nouveaux paramètres pharmacocinétiques permettant de décrire la vitesse et le degré d’exposition cutanée pour l'évaluation de la biodisponibilité et de la bioéquivalence topiques. Finalement, cette méthode a le potentiel de discerner une formulation bioéquivalente d’une autre qui ne l’est pas. / Bioequivalence is a surrogate measure of safety and efficacy in different stages of drug development process with the most pronounced significance in the development of generic drugs. Bioequivalence, among other standards, ensures that generic drugs are equivalent to their approved innovator or reference products in terms of clinical efficacy and safety while circumventing the lengthy-time course and high cost of animal and clinical trials in patients required for innovator drugs. Despite the advancements in development of robust bioequivalence approaches over the past decades, there are still controversies in the current practice of bioequivalence. The aim of this thesis is to explore some of these controversies and address them by putting forward new and alternative approaches. One of the most controversial issues in the current practice of bioequivalence is the extrapolation of bioequivalence study results from one population to another. The majority of bioequivalence studies for systemic effective oral dosage forms are conducted based on pharmacokinetic endpoints in healthy volunteers whilst the targeted population is patients. This is based on the assumption that if two products are bioequivalent in one population, they should be bioequivalent in another one. The extrapolation of bioequivalence study results is not limited to that from healthy volunteers to patients. Since 2007, an ever-increasing proportion of pharmacokinetic bioequivalence studies for North American or European generic submissions have been performed in geographical/ethnic populations other than the intended ones, due to the lower cost of these studies outside North America and Europe. In the first part of this thesis, we investigated whether the bioequivalence results obtained in one geographical or ethnic population can be extrapolated to another one. To this purpose, we extracted pharmacokinetic bioequivalence studies results from generic submissions to Health Canada and the US Food and Drug Administration. We calculated food effect for ten different reference drug products and compared the results for each product between two ethnic populations, Indians and North Americans. This is based on the reasoning that if food effect is found to be the same between the Indian and North American populations, then the generic product and its reference that were found to be bioequivalent in the Indian population should also be bioequivalent in North American population. For 90% of the study drugs, statistically significant difference was detected in the food effect between two populations. For 30% of these drugs, the difference was found to be of possible clinical relevance. The results of this study raised a flag for extrapolating the bioequivalence results from one population to another. Challenges in the context of bioequivalence are not always limited to the pivotal studies where the performance of a generic product is compared to that of Reference. Prior to pivotal bioequivalence studies, a pilot study may be conducted to establish an appropriate study design for the pivotal bioequivalence study. Therefore, inaccurate results from a pilot study, such as inaccurate estimation of time point or dose duration for comparison of test versus reference, can affect the bioequivalence outcomes adversely. An example to this case is the comparison of the extent of skin blanching, the pharmacological effect of generic versus reference products of topical dermatological corticosteroids at specific dose duration, DD50, where the effect is half maximal. This dose duration should initially be determined in a pilot study. The US FDA 1995 Guidance document recommends the use of non-linear mixed effect population modeling for the estimation of DD50, irrespective of the method of analysis. Given the availability of different types of non-linear mixed effect modeling methods, each sponsor could choose a different one. In the second part of this thesis we investigated whether the same DD50 estimates can be obtained when different non-linear mixed effect modeling methods are used. To this purpose, we fitted the skin blanching data from eleven studies with two different non-linear mixed effect modeling methods, the Maximum Likelihood Expectation Maximization (MLEM) and the First Order Conditional Estimation (FOCE). The results favored MLEM given its lower population DD50 estimates that would locate in a more discriminative portion of the Emax curve and better minimization of inter-individual variability. Although the pharmacokinetic-based bioequivalence approach has contributed significantly to the development of high-quality generic versions of systemic effective oral dosage form, the availability of generic versions of topical dermatological products remains constrained due to the limited methods accepted for bioequivalence evaluation of these products. In the third part of this thesis, a novel approach for the bioequivalence assessment of topical acyclovir cream formulations was developed based on the model-based analysis of local exposure data recovered from tape stripping of the skin at a single dose duration, DD50. Conducting the stripping procedure only at DD50 not only ensured that the PK data was collected at the dose duration that is most discriminative of formulation differences, but it also decreased the number of samples to be analyzed significantly. More importantly, our novel approach in generating the local PK profile in the skin (dermatopharmacokinetic profile) and the implementation of population compartmental analysis circumvented the numerous assumptions and sophisticated calculations that were inherent to previous methods, while yielding the PK parameters relevant for topical bioavailability and bioequivalence assessment (rate and extent of exposure to the skin). This method successfully concluded bioequivalence and its absence.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zdeněk Záboj Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of drugs interactions of antiviral drugs with intestinal transporters Sofosbuvir is an antiviral agent widely used in the treatment of chronic hepatitis C. This orally administered prodrug is a designed substrate of ATP-binding (ABC) efflux transporters, P- glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). ABCB1 and ABCG2 are important determinants of intestinal absorption and are the site of significant pharmacokinetic drug interactions, leading to changes in drug exposure. Pharmacokinetic drug interactions may be undesirable (increasing the toxicity of the treatment) or desirable (allowing dose reduction). Because sofosbuvir is often administered in combination regimens with other anti(retro)virotics, the aim of this thesis was to study the ability to enhance intestinal absorption of sofosbuvir. To study the pharmacokinetic drug interactions on ABCB1 and ABCG2, a widely established in vitro bi-directional transport method through a polarized monolayer formed by the Caco-2 cell line derived from colorectal cancer has been used. We analyzed the drug interactions of sofosbuvir on these efflux...