ABSTRACT Title of Document: POLY (AMIDO AMINE) DENDRIMERS: TRANSEPITHELIAL TRANSPORT MECHANISMS AND APPLICATIONS IN ORAL DRUG DELIVERY Deborah Sweet Goldberg, Doctor of Philosophy, 2010 Directed By: Professor Hamidreza Ghandehari, Fischell Department of Bioengineering Small molecule chemotherapy drugs used in clinical practice are plagued by dose-limiting side effects due to off-target toxicities. In addition, because of their low water solubility and poor bioavailability, they must be administered intravenously, leading to high treatment costs and recurring hospital visits. There is a significant need for therapies that improve the bioavailability of chemotherapy agents and enhance specific drug release in the tumor environment. Dendrimers, a class of highly-branched, nanoscale polymers, share many characteristics with traditional polymeric carriers, including water solubility, high capacity of drug loading and improved biodistribution. Poly (amido amine) (PAMAM) dendrimers have shown promise as oral drug carriers due to their compact size, high surface charge density and permeation across the intestinal epithelial barrier. Attachment of chemotherapy drugs to PAMAM dendrimers has the potential to make them orally administrable and reduce off-target toxicities. In this dissertation we investigate the transport mechanisms of PAMAM dendrimers and their potential in oral drug delivery. We demonstrate that anionic G3.5 dendrimers are endocytosed by dynamin-dependent mechanisms and their transport is governed by clathrin-mediated pathways. We show that dendrimer cellular internalization may be a requisite step for tight junction opening. We also demonstrate that conjugation of small poly (ethylene glycol) chains to anionic dendrimers decreases their transport and tight junction opening due to reduction in surface charge, illustrating that small changes in surface chemistry can significantly impact transepithelial transport. Knowledge of transport mechanisms and the impact of surface chemistry will aid in rational design of dendrimer oral drug delivery systems. The potential of dendrimers as oral drug delivery carriers is demonstrated by the evaluation of G3.5 PAMAM dendrimer-SN38 conjugates for oral therapy of hepatic colorectal cancer metastases, a pathology present in over 50% of colorectal cancer cases that is responsible for two-thirds of deaths. Conjugation of SN38, a potent chemotherapy drug with poor solubility and low bioavailability, to PAMAM dendrimers via a glycine linker increased intestinal permeability, decreased intestinal toxicity and showed selective release in the presence of liver carboxylesterase, illustrating that PAMAM dendrimers have the potential to improve the oral bioavailability of potent anti-cancer therapeutics. POLY (AMIDO AMINE) DENDRIMERS: TRANSEPITHELIAL TRANSPORT MECHANISMS AND APPLICATIONS IN ORAL DRUG DELIVERY By Deborah Sweet Goldberg Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2010 Advisory Committee: Professor Hamidreza Ghandehari, Co-Chair Professor William Bentley, Co-Chair Professor Robert Briber Professor Silvia Muro Professor Peter Swaan © Copyright by Deborah Sweet Goldberg 2010 Dedication To my wonderful husband Hirsh, for his constant, unwavering love and support. ii Acknowledgements I have had the honor of working with many talented individuals during my PhD studies who have helped me reach this milestone in my life. I would first like to thank my advisor Dr. Hamid Ghandehari for his enduring commitment to my training as a scientist. Although Dr. Ghandehari moved to the University of Utah shortly after I began my research, he has always strived to maintain constant communication and provide advice on my project. I appreciate his attention to detail in his careful review of my abstracts, posters, presentations, publications and dissertation. Dr. Ghandehari has always inspired me to do my best work, and I know that the impact of his guidance will follow me into my future scientific endeavors. I would also like to thank Dr. Peter Swaan for his contributions to my PhD studies as both my co-advisor and local contact. Dr. Swaan has helped me feel at home at the University of Maryland, Baltimore. I sincerely appreciate the many research discussions that we had during group meetings and individual meetings. I am also thankful for the fresh perspective he offered on my publications and presentations. Dr. Swaan has taught me to think critically and continually question everything, a mindset that will serve me well in the future. I also wish to thank my committee members, Dr. Silvia Muro, Dr. Robert Briber and Dr. William Bentley for their helpful suggestions during my research proposal and committee meeting. Their insights have helped shape the trajectory of my project, and I am grateful for their guidance. In addition to my advisors and my committee members, two other professors have had a significant impact on my PhD studies. I will be forever indebted to Dr. iii Rohit Kolhatkar for his guidance during the first few years of my PhD studies. Rohit taught me experimental techniques and advised me on planning experiments, interpreting results and troubleshooting chemical synthesis strategies. After Rohit left to become an assistant professor at the University of Illinois, Chicago I realized that his guidance had taught me everything I needed to think like a scientist and successfully complete the remainder of my studies. I would also like to thank Dr. Anjan Nan for graciously allowing me to conduct my research in his laboratory space and use his equipment. Dr. Nan’s support of my long distance advisement by Dr. Ghandehari made it possible for me to continue my research on dendrimers. I wish to acknowledge all of my fellow lab members past and present in the Ghandehari, Swaan and Nan labs who have had an influence on my day-to-day life in the lab. Specifically, I would like to acknowledge Dr. Mark Borgman for his endless technical advice on experiments and his wonderful friendship. Even after he graduated, Mark was only a phone call or an email away, and has continued to be a great friend and knowledgeable research consultant. I would also like to thank Brittany Avaritt, Tatiana Claro da Silva and Paul Dowell for their friendship and support over the years. Finally, I would like to thank Carl the janitor who always managed to make me smile on his early morning rounds. During my PhD experience I was fortunate to complete a summer internship in the Department of Formulation Sciences at MedImmune LLC. Although this internship experience was distinct from my graduate research, it gave me an appreciation of the pharmaceutical industry and taught me new ways of approaching problems, which positively impacted my graduate work after the internship. I would iv like to thank my supervisor Dr. Hasige Sathish and my formulation sub-group leader Dr. Ambarish Shah for their positive contributions to my PhD experience and for providing me with this unique opportunity. Several fellowships and grants during my graduate studies have helped to fund my research. I would like to thank the National Science Foundation for the Graduate Research Fellowship, which sponsored my first three years of graduate school. It was an honor to receive this fellowship and it had a positive impact on my graduate school career, providing me with freedom in project selection. I would also like to thank Dr. Robert Fischell for generously sponsoring the Fischell Fellowship in Bioengineering, which funded my final years in graduate school. Dr. Fischell’s commitment to fostering new Bioengineering research is admirable and his enthusiasm is contagious. I also acknowledge NIH R01 EB007470 for funding supplies for this research. I would like to recognize my friends and family, whose support and encouragement have been critical for the successful completion of my PhD studies. My parents, Shari and Rick Sweet, have always been my biggest cheerleaders, supporting all of my academic and personal pursuits. I truly appreciate their excitement about all of my accomplishments from elementary school until today and their constant encouragement to strive for my dreams. I would also like to thank my mother and father in-law, Karen and Larry Goldberg for their interest and support throughout my PhD studies. I would like to acknowledge the wonderful support of my friends Marie Jeng, Chloe Marin, Rifat Jafreen and Kevin Nelson among many others who have shown interest in my PhD progress over the years and offered v countless words of encouragement. I wish to express my gratitude to the instructors at Columbia Jazzercise for providing a fun and exhilarating stress release as well as all of my friends at Jazzercise for their constant interest and encouragement. Finally, I would like to thank my husband Hirsh who has been my greatest supporter through all of the ups and the downs of my PhD research. Hirsh has always offered emotional support and encouragement when I was frustrated with unsuccessful experiments and has unselfishly taken over household responsibilities when I had to work late at night or on the weekend to meet a deadline. I know that his love and unwavering support helped me push through the most difficult challenges of my PhD and I could not have done it without him. He truly deserves this degree as much as I do. vi Table of Contents Dedication................................................................................................................ii Acknowledgements.................................................................................................iii List of Tables..........................................................................................................xi List of Figures........................................................................................................xii Abbreviations........................................................................................................xv Chapter 1 : Introduction.........................................................................................1 1.1 Introduction....................................................................................................1 1.1.1 Polymer Therapeutics................................................................................1 1.1.2 Oral Drug Delivery...................................................................................2 1.1.3 Poly (amido amine) Dendrimers................................................................3 1.1.4 SN38.........................................................................................................4 1.2 Specific Aims..................................................................................................5 1.3 Scope and Organization...................................................................................6 Chapter 2 : Background..........................................................................................8 2.1 Introduction.....................................................................................................8 2.2 Polymeric Drug Delivery.................................................................................8 2.2.1 Therapeutic Advantages of Polymer-Drug Conjugates in Chemotherapy...9 2.2.2 Polymer-Drug Conjugates Currently in Clinical Trials............................12 2.3 Administration of Drugs via the Oral Route...................................................13 2.3.1 Physiology of the Gastrointestinal Tract..................................................14 2.3.1.1 Compartments and Functions...........................................................14 2.3.1.2 Intestinal Epithelial Barrier...............................................................17 2.3.1.3 Tight Junction Biology: Structure and Function................................18 2.3.2 Mechanisms of Transport Across the Intestinal Barrier...........................21 2.3.2.1 Paracellular Transport......................................................................21 2.3.2.2 Passive Diffusion.............................................................................23 2.3.2.3 Carrier-Mediated Transport..............................................................24 2.3.2.4 Endocytosis......................................................................................25 2.3.3 Physiochemical Properties that Govern Intestinal Absorption..................28 2.3.3.1 The Lipinski Rule of 5......................................................................28 2.3.3.2 The Biopharmaceutics Classification System (BCS).........................28 2.4 Models to Predict Oral Absorption and Oral Bioavailability...........................30 2.4.1 In Silico Models......................................................................................30 2.4.2 Parallel Artificial Membrane Permeability Assay....................................31 2.4.3 Caco-2 Monolayers.................................................................................33 2.4.4 Fast-Caco-2 Assay..................................................................................36 vii