AN INVESTIGATION INTO THE ROLE OF GLUCURONIDATION ON THE DISPOSITION AND TOXICITY OF MYCOPHENOLIC ACID USING TARGETED QUANTITATIVE PROTEOMICS David Edward Harbourt A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Toxicology. Chapel Hill 2009 Approved by: Advisor and Committee Chair: Philip C. Smith, Ph.D. Reader: Michael J. DeVito, Ph.D. Reader: Gary L. Glish, Ph.D. Reader: Mary F. Paine, Ph.D. Reader: Joseph K. Ritter, Ph.D. i ABSTRACT DAVID HARBOURT: “An Investigation into the Role of Glucuronidation on the Disposition and Toxicity of Mycophenolic Acid using Targeted Quantitative Proteomics” (Under the direction of Dr. Philip C. Smith) The prodrug mycophenolate mofetil (MMF) is used clinically for prophylaxis of organ rejection in transplant patients. MMF is metabolized to the active metabolite mycophenolic acid (MPA). While proven useful in this setting, a significant fraction of patients receiving MMF chronically experience delayed- onset diarrhea, which limits the long term effectiveness of their treatment. MPA is eliminated primarily through glucuronidation by the action of UDP- glucuronosyltransferase (UGT) 1A enzymes within the liver and intestine. Glucuronides in the liver are excreted into the bile by the canalicular transporter multidrug resistance-associated protein 2 (MRP2) where they are subject to enterohepatic cycling and excretion through the urine as glucuronides. Glucuronidation results in the formation of the inactive phenolic glucuronide (MPAG) and the labile acyl glucuronide (acMPAG). While the formation of MPAG is the primary method of detoxification of MPA in vivo, studies have attempted to link formation of acyl glucuronides with adverse drug reactions (ADRs). While evidence has been inconclusive in directly linking acyl glucuronides with toxicity, some drugs forming acyl glucuronides have been removed from the marketplace due to ADRs. The overall hypothesis of this ii dissertation project is that variable glucuronidation formation and efflux within the liver and gastrointestinal tract results in differential MPAG formation rates and thus modulates MPA toxicity. The study of the relationships between metabolism and transport was aided by quantitative measurement of relevant enzymes in humans and animals. This methodology was used to establish assays to quantify precise differences in UGT1A enzymes between the tissues in rats and humans to understand the differences in metabolism and toxicity of MPA between species. This dissertation research examined the disposition of acMPAG and MPAG in relation to expression levels of Ugt1a enzymes and efflux transporters using targeted quantitative proteomics. By correlating glucuronide catalysis and efflux with absolute quantification of Ugts and transporters, we increased our understanding of relationships between glucuronide formation and disposition and UGT enzymes. This research helps explain some differences in metabolism and elimination observed between rats and humans administered MPA and these relationships may be applied to other xenobiotics with Phase II substrates. iii ACKNOWLEDGEMENTS I am forever indebted to my committee chair and advisor, Dr. Philip Smith for his guidance and mentorship over the course of my graduate work. His advice and thoughtful comments during my numerous inquiries throughout my experiments have taught me how to think critically during problem solving. My graduate work in Dr. Smith’s lab has shaped me into the scientist I am today and it is my hope that he can be proud of the work presented as it is a testament to his guidance and thoughtfulness as my mentor. I also have the deepest gratitude for my committee members, Drs. Mike DeVito, Gary Glish, Mary Paine and Joe Ritter for their thoughtful comments and advice over the course of my project. I would especially like to thank the members of the Glish and Ritter labs along with Dr. Peter Bullock and Saber Malecki of Panacos Pharmaceuticals for their assistance and camaraderie as this work would not have been possible without their help. I would like to thank my parents, Cyrus and Bonnie, and my sister Diana for providing me with a solid foundation and for always believing in me. The many lessons they have taught me beginning in my early childhood help shape the kind of person I hope to be through the duration of my adult life. I would also like to thank the members of the Cho lab as my daily interactions with them helped me through all of the ups and downs of graduate school. I would like to thank all of my friends and members of my extended family for their support during the course of my graduate education. My friends have always been supportive of me during the course of my studies and have helped me in many ways that are too numerous to list. I will always be grateful for all of my interactions with them because they formed many of my fondest memories from graduate school. Finally, I would like to express my sincerest gratitude to my wonderful girlfriend Kelly Ennis. The past two years that we have spent together have been the happiest of my life as she has always given me someone to talk to and has been the person that I can always lean on during difficult times. I will always cherish her love and company and meeting her during the course of my graduate studies has been one of the best things that has ever happened to me. iv Table of Contents List of Tables ...................................................................................................... vii List of Figures .......................................................................................................v List of Abbreviations............................................................................................viii CHAPTER 1: INTRODUCTION ............................................................................1 A. INTRODUCTION .......................................................................................2 B. UDP-GLUCURONOSYLTRANSFERASES ..................................................4 B.1. Background and Function.......................................................................4 B.2. Human UGTs .........................................................................................6 B.3. Rat Ugts .................................................................................................8 B.4. Detoxification and Toxicity associated with glucuronidation ...................9 C. ACYL GLUCURONIDES ............................................................................10 C.1. Background ..........................................................................................10 C.2. Acyl Migration of Ester Glucuronides ...................................................11 C.3. Potential Toxicity ..................................................................................12 D. QUANTITATIVE PROTEOMICS.................................................................14 D.1. Background ..........................................................................................14 D.2. Biological Applications .........................................................................16 E. MYCOPHENOLIC ACID .............................................................................17 E.1. Clinical Uses.........................................................................................17 E.2. Toxicity .................................................................................................18 v E.3. Rat and Human MPA Metabolism ........................................................19 F. RATIONALE FOR THE PROPOSED PROJECT .......................................20 G. REFERENCES ..........................................................................................27 CHAPTER 2: STABILITY OF MYCOPHENOLIC ACID ACYL GLUCURONIDE AND ITS POTENTIAL BIOLOGICAL CONSEQUENCES...................................39 A. INTRODUCTION ........................................................................................40 B. METHODS.................................................................................................42 C. RESULTS ...................................................................................................48 D. DISCUSSION ............................................................................................52 E. ACKNOWLEDGEMENTS ..........................................................................59 F. REFERENCES............................................................................................69 CHAPTER 3: QUANTIFICATION OF UDP-GLUCURONOSYLTRANSFERASE ENZYME EXPRESSION LEVELS WITHIN HUMAN LIVER, INTESTINAL AND KIDNEY MICROSOMES USING nanoLC TANDEM MASS SPECTROMETRY.75 A. INTRODUCTION ........................................................................................76 B. METHODS.................................................................................................80 C. RESULTS ...................................................................................................88 D. DISCUSSION .............................................................................................93 E. ACKNOWLEDGEMENTS ........................................................................100 F. REFERENCES.........................................................................................112 CHAPTER 4: QUANTITATIVE RELATIONSHIP BETWEEN RAT UDP- GLUCURONOSYLTRANSFERASE