INHALABLE FORMULATIONS OF PROSTAGLANDIN E1 (PGE1) FOR SUSTAINED VASODILATION IN PULMONARY ARTERIAL HYPERTENSION (PAH) by VIVEK GUPTA, B.Pharm A DISSERTATION IN PHARMACEUTICAL SCIENCES Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Advisory Committee Fakhrul Ahsan (Chairperson) Young Min Kwon Xinli Liu Reza Mehvar Thomas Thekkumkara Accepted Thomas A. Pressley, Ph.D. Interim Dean of the Graduate School of Biomedical Sciences Texas Tech University Health Sciences Center December 2010 Copyright 2010, Vivek Gupta Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 ACKNOWLEDGEMENTS I would like to take this opportunity to express my deepest gratitude and heartfelt thanks to my doctoral advisor Dr. Fakhrul Ahsan for his incredible mentorship, support and for being there at each and every step for me. I am totally blessed to be a part of his lab. He has been an ideal mentor for his excellent guidance, caring, patience; for providing me with an excellent research atmosphere and for allowing me to work independently. I am thankful to him for his continuous input throughout the project. It was only his perseverance that I could be able to learn the process of grant writing and write and publish manuscripts. I feel greatly indebted to Dr. Ahsan for providing me with an opportunity to work under his able guidance. I am grateful to all my committee members, Dr. Young Min Kwon, Dr. Xinli Liu, Dr. Reza Mehvar, and Dr. Thomas Thekkumkara for their constant guidance and input and for bringing out the best in me. I sincerely thank each one of them for all the time, suggestions and guidance they have given me for the completion of this doctoral project. It is a pleasure to thank those who have helped me immensely in making this dissertation work possible. It is an honor for me to me to show my gratitude to our collaborator, Dr. Eva Nozik-Grayck at University of Colorado at Denver. I deeply appreciate her for helping me with establishing the critical PAH animal model in our lab. A special mention goes to Dr. Amit Rawat, a friend and former colleague. He has been a constant source of knowledge throughout this project. He is the one who trained me in the basics of controlled and novel drug delivery systems. I would like to specially thank two of my former colleagues Dr. Shuhua Bai and Dr. Chandan Thomas for introducing me to basics of research in our lab and for their inputs during my PhD. I would also like to thank my current colleagues Nilesh Gupta, Kamrun Nahar, and Brijesh Patel, who ii Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 have been instrumental in helping me with some critical experiments, and also in routine lab maintenance chores. I would like to take this opportunity to thank people who have helped me in a number of ways toward the completion of my PhD. Dr. Imam Shaik, Dr. Sunny Guin, Dr. Ridhi Parasrampuria, Kaushik Shah, Mohd. Shahriarul Absar. Their help and guidance has been instrumental in completing this dissertation work. Further I would like to thank all the other faculty members, postdocs, technicians, graduate students, the office and research staff for all their support and help during my PhD program. My parents; Dr. Satish Chandra Gupta and Sushma Rani Gupta, have always been my inspiration and I would like to express my deepest thanks and sincere love and gratitude towards them for all the support and love they have showered on me all these years. A very special thanks goes to my brother Vikas Kumar Gupta and sister-in-law Richa Gupta for being guiding forces in my life, and for playing an important role in shaping me as an individual. I would also take this opportunity to acknowledge all my immediate family members, and all my friends for all their support and love. Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the project. iii Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………………ii ABSTRACT………...........................................................................................................vi LIST OF TABLES……………………………………………………………………………..viii LIST OF FIGURES……………………………………………………………………………...ix LIST OF ABBREVIATIONS……………………………………………………………………xi CHAPTERS 1. INTRODUCTION………………………………………………………………………..1 References…………………………………………………………………….23 2. FEASIBILITY STUDY OF AEROSOLIZED PROSTAGLANDIN E1 MICROSPHERES AS A NON-INVASIVE THERAPY FOR PULMONARY ARTERIAL HYPERTENSION………………………………………………………..39 Introduction…………………………………………………………………….39 Materials and Methods……………………………………………………….41 Results and Discussion………………………………………………………49 References…………………………………………………………………….61 3. PEI-MODIFIED PLGA MICROSPHERES OF PGE1 FOR NONINVASIVE TREATMENT OF PULMONARY ARTERIAL HYPERTENSION………………..73 Introduction…………………………………………………………………….73 Materials and Methods……………………………………………………….76 Results and Discussion………………………………………………………84 References…………………………………………………………………….95 4. INHALABLE PLGA MICROPARTICLES ENCAPSULATING PROSTAGLANDIN E1-HYDROXYPROPYL-β-CYCLODEXTRIN (PGE1-HPβCD) COMPLEX FOR PULMONARY ARTERIAL HYPERTENSION (PAH) TREATMENT…………...110 iv Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 Introduction…………………………………………………………………..110 Materials and Methods……………………………………………………...112 Results and Discussion……………………………………………………..122 References…………………………………………………………………...134 5. PLGA PARTICLES OF PGE1 AMELIORATES SYMPTOMS OF PAH IN A RAT MODEL FOLLOWING ACUTE AND CHRONIC ADMINISTRATION VIA THE PULMONARY ROUTE………………………………………………………………151 Introduction…………………………………………………………………..151 Materials and Methods……………………………………………………...153 Results and Discussion……………………………………………………..158 References…………………………………………………………………...163 6. CONCLUSIONS……………………………………………………………………...175 v Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 ABSTRACT Pulmonary arterial hypertension (PAH) is a rare and debilitating disorder of the pulmonary circulation that afflicts the lives of thousands Americans. The major clinical features of PAH include increase in mean pulmonary arterial pressure (MPAP), right ventricular enlargement, increased pulmonary vascular resistance and smooth muscle hypertrophy in pulmonary arterioles. Several anti-PAH therapies targeting various pathways involved in PAH progression have been approved by the FDA. However, many of the currently available anti-PAH drugs suffer from a number of limitations including short biological half-life, poor pulmonary selectivity and infection or hematoma due to administration via the intravenous or subcutaneous route. Prostaglandin E1 (PGE1) is a potent vasodilator with selectivity toward the pulmonary circulation when administered via the pulmonary route. However, PGE1 has a very short half-life of 5-10 minutes. This dissertation investigates the feasibility of polymeric microparticles of PGE1 for administration via the non-invasive pulmonary route in order to develop a formulation that would produce long-term and selective vasodilation of pulmonary arteries. Toward this end, first, we developed poly (lactide-co-glycolide) (PLGA) microparticles to investigate the suitability of the polymeric delivery system for PGE1. Second, with the aid of polyethyleneimine (PEI) as a porosigen, we developed large porous PLGA microparticles of PGE1 to accomplish improved drug entrapment and enhanced deep lung deposition. In the third set of studies, we have evaluated the efficacy of hydropropyl-β-cyclodextrin (HPβCD) as a complexing agent to increase hydrophilicity of PGE1 and increase its absorption across the respiratory epithelium. Finally, the formulations that showed the most favorable pharmacokinetic and deposition patterns were tested in a monocrotaline-induced rodent model of PAH. We used this animal model to study the efficacy of the formulations in reducing pulmonary arterial pressure vi Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 and ameliorating pathological conditions associated with PAH following acute and chronic administration of the formulations. Results from these studies suggest that PGE1 can be encapsulated in PLGA microparticles to modify its systemic availability and extend its half-life. The incorporation of PEI in microparticulate formulations resulted in porous microparticles that showed excellent drug entrapment and favorable aerodynamic properties for efficient deposition in the lungs. Further, PGE1-HPβCD encapsulated microparticles showed sustained release of PGE1 and remarkable increase in relative bioavailability. Hemodynamic studies in PAH-rats demonstrate that the optimized formulations were efficacious in providing a continual reduction in pulmonary arterial pressure and improvement in the morphometry of pulmonary vasculature. Overall, this study is first to show that PLGA-based particles of PGE1 is a feasible inhalable formulation that produces selective and prolonged vasodilation of pulmonary arteries and ameliorates morphological changes of the pulmonary vasculature associated with PAH. vii Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 LIST OF TABLES Table 1.1. Updated clinical classification of pulmonary hypertension (Dana Point, CA, 2008)……………………………………………………………………………….30 Table 1.2. Advantages and limitations of prostacyclin analogues for PAH treatment….31 Table 2.1. Composition and polydispersity