ROLE OF MEMBRANE TRANSPORTERS IN DRUG DELIVERY, DRUG DISPOSITION AND DRUG-DRUG INTERACTIONS A DISSERTATION IN Pharmaceutical Sciences and Chemistry Presented to the Faculty of University of Missouri-Kansas City in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY By VARUN KHURANA B. Pharmacy, Guru Gobind Singh Indraprastha University, India, 2008 Kansas City, Missouri 2014 © 2013 VARUN KHURANA ALL RIGHTS RESERVED ROLE OF MEMBRANE TRANSPORTERS IN DRUG DELIVERY, DRUG DISPOSITION AND DRUG-DRUG INTERACTIONS Varun Khurana, Candidate for the Doctor of Philosophy Degree University of Missouri-Kansas City, 2014 ABSTRACT Tissues such as liver, kidney, brain and intestine expresses membrane transporters which play a vital role in drug absorption, distribution, metabolism and excretion. Understanding of functionality and molecular expression of drug transporters can prove to be of utmost importance in drug delivery or drug design by targeting specific transporter proteins. It’s a well-known fact that drug transporters play an important role in governing drug disposition which act as potential piece of information during the drug discovery and development process. By exploring the transporter functionality chances of delivering a therapeutic agent to the target organ enhances. Transporter targeted drug delivery helps in improving the bioavailability, controlling the elimination process and also avoid distribution to non-specific organs, hence diminishes the odds of toxic adverse effects. It is always suitable to choose a potential molecule which may or may not interact with the membrane transporters, depending on whether such an interaction is of any use or not. Activity of individual transport process can be examined by exploring the expression system of transporters. Therapeutic efficacy many important drugs, directly or indirectly, get affected due to genetic polymorphisms and drug-drug interactions involving membrane transporters which ultimately effects the pharmacokinetics of a drug molecule. During the drug discovery and development process, knowledge about the contribution of these transporters iii towards interindividual differences by regulating drug absorption, distribution, metabolism and excretion will act as an important tool. The objective of this dissertation project was to understand the role of hepatic uptake transporters (OATP-1B1 and -1B3) in governing the disposition of tyrosine kinase inhibitors (TKIs). Since selected TKIs are the substrates and/or inhibitors of OATP-1B1 and -1B3 expressed in hepatic tissue, these compounds can be regarded as molecular targets for transporter mediated drug-drug interactions (DDIs). Any alteration in the function of these hepatic OATPs might account for the pharmacokinetic variability of TKIs. These finding also provide the basis of further pre-clinical and clinical studies investigating the transporter based DDI potential of TKIs. As a secondary aim of this investigation we developed novel pentablock (PB) copolymer nanoparticles of pazopanib for treatment of ocular neovascularization. Our results indicated that PB copolymer based drug delivery systems can serve as a platform technology for the development of sustained release therapy along with evasion of drug efflux for the treatment of ocular neovascularization. This drug delivery system can also be utilized for other chronic diseases as well. We also investigated the presence of ascorbic acid-specific transport system and delineate the functional and molecular aspects of vitamin C transporter (SVCT2) in ocular and breast cancer cells. SVCT2 system can be targeted for the design of ascorbic acid prodrugs or for NPs surface modified with ascorbic acid to achieve enhanced permeability for highly potent but poorly bioavailable drugs by evading drug efflux in the treatment of cancer and ocular diseases. Interaction of TKIs with hepatic OATP-1B1 and -1B3 delineates the role of hepatic uptake transporters in drug disposition and drug-drug interactions. These OATP transporters in conjunction with the efflux proteins (P-gp, MRP and BCRP) may eventually decide on the overall iv flux/loss of the therapeutic agents within the hepatic tissue. Similary, functionality of membrane tranporters have been exploited and examined in terms of drug delivery. Pazopanib encapsulated nanoparticles of novel pentablock polymers were successful in bypass drug efflux mediated via efflux proteins. Also, influx transporters (vitamin C transporter, SVCT2) on ocular cell lines can be further utilized as a potential target for enhancing absorption and permeability of AA- conjugated drugs or drug delivery systems by evading drug efflux. v APPROVAL PAGE The faculty listed below, appointed by the Dean of the School of Graduate Studies have examined a dissertation titled ‘Role of Membrane Transporters in Drug Delivery, Drug Disposition And Drug-Drug Interactions’, presented by Varun Khurana, candidate for the Doctor of Philosophy degree, and certify that in their opinion it is worthy of acceptance. Supervisory Committee Ashim K. Mitra, Ph.D., Committee Chair Department of Pharmaceutical Sciences Kun Cheng, Ph.D. Department of Pharmaceutical Sciences Betty Herndon, Ph.D. Department of Basic Medical Science Karen B. Williams, Ph.D. Department of Medical Bioinformatics James R. Durig, Ph.D. Department of Chemistry vi TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii LIST OF ILLUSTRATIONS ......................................................................................................... ix LIST OF TABLES ....................................................................................................................... xvi ACKNOWLEDGEMENTS ....................................................................................................... xviii THE CONVOLUTIONS INVOLVED IN HEPATIC DRUG TRANSPORT: FOCUS ON MEMBRANE TRANSPORTERS .................................................................................................. 1 Rationale...................................................................................................................................... 1 Drug or Membrane Transporters of the Hepatic Basolateral Membrane .................................... 4 Drug or Membrane Transporters of the Hepatic Apical (Canalicular) Membrane ................... 17 Drug Interactions Involved In Hepatic Transport ..................................................................... 26 Future Of Hepatobiliary Drug Transport Research ................................................................... 33 ROLE OF OATP-1B1 AND/OR OATP-1B3 IN HEPATIC DISPOSITION OF TYROSINE KINASE INHIBITORS ................................................................................................................ 35 Rationale.................................................................................................................................... 35 Materials And Methods ............................................................................................................. 37 Results ....................................................................................................................................... 40 Discussion ................................................................................................................................. 56 Conclusion ................................................................................................................................. 63 INHIBITION OF OATP-1B1 AND OATP-1B3 BY TYROSINE KINASE INHIBITORS ....... 64 Rationale.................................................................................................................................... 64 Materials And Methods ............................................................................................................. 66 Results ....................................................................................................................................... 70 Discussion ................................................................................................................................. 80 Conclusion ................................................................................................................................. 86 THE ROLE OF TRANSPORTERS AND EFFLUX SYSTEM IN DRUG DELIVERY ............ 87 Rationale.................................................................................................................................... 87 ABC Transporters ..................................................................................................................... 89 Strategies To Overcome Active Efflux ................................................................................... 106 Conclusion ............................................................................................................................... 128 NOVEL PENTABLOCK COPOLYMER BASED NANOPARTICLES CONTAINING PAZOPANIB: A POTENTIAL THERAPY FOR OCULAR NEOVASCULARIZATION ..... 129 vii Rationale.................................................................................................................................. 129 Material And Methods ............................................................................................................ 132 Results And Discussion ..........................................................................................................