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...

<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>

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. 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. 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.

Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registered drugs. Focus was set on the ATP-binding cassette (ABC) transport proteins expressed in the canalicular membrane of human hepatocytes. The inhibition of the ABC transport proteins multidrug-resistance associated protein 2 (MRP2/ABCC2) and bile salt export pump (BSEP/ABCB11) was experimentally investigated using membrane vesicles from cells overexpressing the investigated proteins and sandwich cultured human hepatocytes (SCHH). Several previously unknown inhibitors were identified for both of the proteins and predictive in silico models were developed. Furthermore, a clear association between BSEP inhibition and clinically reported drug induced liver injuries (DILI) was identified. For the first time, an in silico model that described combined inhibition of Pgp, MRP2 and breast cancer resistance protein (BCRP/ABCG2) was developed using a large, structurally diverse dataset. Lipophilic weak bases were more often found to be general ABC inhibitors in comparison to other drugs. In early drug discovery, in silico models can be used as predictive filters in the drug candidate selection process and membrane vesicles as a first experimental screening tool to investigate protein interactions. In summary, the present work has led to an increased understanding of molecular properties important in ABC inhibition as well as the potential influence of ABC proteins in adverse drug reactions. A number of previously unknown ABC inhibitors were identified and predictive computational models were developed.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Dominika Bezděková Supervisor: doc. PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: IN VITRO STUDY OF DRUG-DRUG INTERACTIONS OF HIV PROTEASE INHIBITOR DARUNAVIR ON EFFLUX ABC TRANSPORTERS Abstract: Darunavir is a drug used in the therapy of HIV belonging to the group of protease inhibitors. These protease inhibitors are used as a part of the combination antiretroviral therapy. For the increase of bioavailability, darunavir is always used in combination with ritonavir or cobicistat. As the CYP3A4 and ABCB1 (P-glycoprotein) transporter substrate, darunavir is a drug with a high potential to drug interactions. Considering the amount of adverse effects that can be caused by darunavir, it is necessary to know these drug interactions for the safety of therapy. Inhibition of the intestinal ABCB1 by the co-administrated drugs could also lead to the increased bioavailability of darunavir and to reduction of frequency of administration leading to a cheaper therapy. This thesis studies the drug-drug interactions of darunavir with in vitro methods using two cell lines - MDCKII and Caco-2 cells. The results from the transport of darunavir across the MDCKII cell monolayer indicates that darunavir is a ABCB1...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Halodová Supervisor: doc. PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: In vitro and ex vivo study of drug-drug interactions of antivirals on intestinal membrane transporters Tenofovir (TFV) is the first-line agent in the treatment of hepatitis B virus (HBV) infection for patients aged over 12 years and one of the first-line choices for the combination antiretroviral therapy (cART) of infections caused by human immunodeficiency virus (HIV). Two commercially available prodrugs have been developed for oral administration of TFV, tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide fumarate (TAF). These prodrugs increase TFV membrane permeability and oral bioavailability. One of the factors that can affect the bioavailability of orally administrated drugs is active transport mediated by efflux transporters, mainly by P-glycoprotein (ABCB1, P-gp) and Breast cancer resistance protein (ABCG2, BCRP). It has been already proved that TDF and TAF are substrates of both of these transporters. The goal of this diploma thesis was to use in vitro and ex vivo models of intestinal barrier to assess the impact of the efflux transporters on TDF and TAF transport in the intestine and on their...