Development of Polymeric Micelles for Site Specific Delivery of Nonsteroidal Anti-Inflammatory Drugs: Effect on the Cardiovascular Risk in an Experimental Model of Arthritis by Hanan Mustafa Al Lawati A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences Faculty of Pharmacy and Pharmaceutical Sciences University of Alberta © Hanan Mustafa Al Lawati, 2019 Abstract Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX) enzymes, are among the most widely used medications worldwide with proven efficacy in controlling inflammation and pain associated various disease conditions. The chronic systemic use of most NSAIDs, however, is known to be associated with an increased risk of cardiovascular (CV) events, including myocardial infarction and stroke. Cardiovascular complications associated with the use of NSAIDs are believed to be partly linked to the extent of their exposure to the cardiac tissue among other factors. Research in the field of nanomedicine has shown a remarkable progress in the design of targeted drug delivery systems which can improve drug disposition towards the diseased sites. A systematic search of the scientific literature revealed that reports on the use of such advanced systems for the delivery of NSAIDs that show evidence of improved cardiac safety are lacking. Therefore, in this research work we considered designing nano-formulations for the delivery of diclofenac as a model NSAID for its known serious CV toxicity. The aim was to improve diclofenac pharmacokinetics and biodistribution whereby reducing its accumulation and toxicity in the cardiac tissue. We hypothesize that reduced diclofenac cardiac exposure will lower its CV side effects. Towards this goal, different polymeric micellar formulations based on a shell of poly(ethylene oxide) (PEO) and various hydrophobic polyesters as the hydrophobic core were tried for the encapsulation of diclofenac or its ethyl ester prodrug. The micellar formulations were characterized for size, size distribution, morphology, and in vitro as well as ex vivo drug release. ii The results pointed to the superiority of polymeric micelles with a core of poly(-caprolactone) (PCL) or poly(α-benzyl carboxylate --caprolactone) (PBCL) encapsulating diclofenac ethyl ester as the optimal formulations. In the next step, the biodistribution of the optimal polymeric micellar formulations of diclofenac was investigated in vivo in healthy rats following intravenous (iv) and/or intraperitoneal (ip) administration and compared to that of the free diclofenac. The intravenous administration of the polymeric micellar formulation resulted in prolonged diclofenac systemic circulation and reduced its accumulation in the heart and kidneys compared to the free drug. The polymeric micellar formulations of diclofenac administered through ip route proved to be equally bioavailable as the iv administration. In the following step, multiple dose administration of polymeric micellar formulation of diclofenac in an experimental model of arthritis in rats was employed to assess the anti-inflammatory activity and the safety of the micellar formulation over free drug. To assess the cardiac safety of polymeric micellar formulation versus free drug, the levels of cardioprotective versus cardiotoxic metabolites of arachidonic acid (ArA) was measured in plasma, heart and kidneys following drug administration to these rats with adjuvant arthritis. The results showed that the micellar formulation provided similarly effective therapy for the management of inflammation when compared to the free drug. On the other hand, administration of diclofenac as polymeric micellar formulation reduced the ratio of cardiotoxic over cardioprotective metabolites of ArA in the heart and plasma when compared to the free drug administration. These results provide the first evidence for a potential role in the tissue disposition of diclofenac on its cardiac-safety. It also points to the potential of developed formulations for effective and safe delivery of NSAIDs. iii Preface The following papers are published or considered for publication in refereed journals and are part of the work presented in this thesis: Section 1.5 of chapter 1 is considered for publication as Al-Lawati H., Binkhathlan Z., Lavasanifar A., Nanomedicine for the effective and safe delivery of non-steroidal anti-inflammatory drugs: a review of preclinical research. H. Al Lawati is the lead author and was responsible for identifying and screening relevant reports, analysing the reports, data collection, and composition of the manuscript. Dr. Z. Binkhathlan reviewed the included reports and helped with the article revision. Dr. A. Lavasanifar was the supervisory author and was involved in formulation of concepts, analysis and composition of manuscript. Chapter 2 has been published as Al-Lawati H., Vakili M. R., Jamali F., Lavasanifar A., Polymeric micelles for the delivery of diclofenac and its ethyl ester derivative, Pharmaceutical Nanotechnology 2016; 4(2): 109 – 119. H. Al Lawati is the lead author and was responsible for data collection and analysis as well as for manuscript composition. Dr. M. R. Vakili helped with study design. Dr. F. Jamali and Dr. A. Lavasanifar were the supervisory authors and were involved in formulation of concept, analysis and composition of manuscript. Chapter 3 has been published as Al-Lawati H., Vakili M. R., Lavasanifar A., Ahmed S., Jamali F., Delivery and biodistribution of traceable polymeric micellar diclofenac in the rat, Journal of Pharmaceutical Sciences (in press, appeared online). H. Al Lawati is the lead author and was responsible for data collection and analysis as well as the manuscript composition. Dr. M. R. Vakili helped with study design. S. Ahmed helped with some data acquisition. Dr. A. Lavasanifar and iv Dr. F. Jamali were the supervisory authors and were involved in formulation of concept, analysis and composition of manuscript. Chapter 4 is to be submitted for publication as Al-Lawati H., Vakili M. R., Lavasanifar A., Ahmed S., Jamali F., Pharmacokinetics and pharmacodynamics of traceable polymeric micellar diclofenac in experimental arthritis. H. Al Lawati is the lead author and was responsible for data collection and analysis as well as the manuscript composition. Dr. M. R. Vakili helped with study design. S. Ahmed helped with some data acquisition. Dr. A. Lavasanifar and Dr. F. Jamali were the supervisory authors and were involved in formulation of concept, analysis and composition of manuscript. The supplementary chapter is part of a manuscript to be submitted for publication as Binkhathlan Z., Ali R., Qamar W., Al-Lawati H., and Lavasanifar A. Pharmacokinetic and tissue distribution study of orally administered cyclosporine A-loaded poly(ethylene oxide)-block-poly(ε- caprolactone) micelles versus Sandimmune® in rats. H. Al Lawati was completely responsible for the in vitro and in vivo experiments presented in this thesis in the supplementary chapter. She wrote the methods and results sections related to these experiments in the manuscript and helped with the article revision. Dr. Z. Binkhathlan, Dr. R. Ali, and Dr. W. Qamar were responsible for the other data collection and analysis as well as the manuscript composition. Dr. A. Lavasanifar was the supervisory author and was involved in formulation of concept, analysis and composition of manuscript. In addition, the following refereed journal papers, report research work that was carried out by H. Al Lawati in parallel to the work presented in this thesis and is indirectly related to the thesis: v • Al Lawati H. and Jamali F. Onset of action and efficacy of ibuprofen liquigel as compared to solid tablets: a systematic review and meta-analysis. J Pharm Pharm Sci 2016; 19 (3): 301-311. • Al-Lawati H. Aliabadi H. M., Makhmalzadeh B. S., Lavasanifar, A., Nanomedicine for immunosuppressive therapy: achievements in pre-clinical and clinical research. Expert Opin Drug Deliv. 2018 Apr; 15(4):397-418. vi Dedication To my beloved parents Mustafa and Batool for their endless love and support vii Acknowledgments I would like to express my sincere gratitude and appreciation to my supervisor Dr. Fakhreddin Jamali. His encouragement, ideas, continuous help and guidance, interesting courses, and many valuable discussions made this dissertation and my degree possible. I have been fortunate to have had him as my advisor during my studies at the University of Alberta. I would like to also extend my appreciation to my co-supervisor Dr. Afsaneh Lavasanifar for introducing me to the world of nanomedicine and for the many valuable ideas, discussions, suggestions, and for her continuous support and encouragement which made this work possible. I enjoyed working with both Dr. Fakhreddin Jamali and Dr. Afsaneh Lavasanifar on this project. I would like to thank Dr. Raimer Löbenberg and the other members of my thesis committee, Dr. Michael Doschak and Dr. Hasan Uludag, for their valuable comments and suggestions which better shaped this work. Special thanks go to the members of the Dr. Jamali and Dr. Lavasanifar labs, in particular Dr. Mohamed R. Vakili, Dr. Waheed Asghar, Igor Paiva, Nasim Ghasemi, Dr. Forough Sanaee and Surur Ahmed, for their encouragement and help from time to time. I wish them and all my fellow graduate students all the best. A special thanks go to the faculty and staff of the Faculty of Pharmacy and Pharmaceutical Sciences. Above all, I would like to thank my parents