Aldehyde Oxidase Drug Metabolism: Evaluation of Drug Interaction Potential and Allometric Scaling Methods to Predict Human Pharmacokinetics By Rachel Denise Crouch Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmacology December, 2016 Nashville, Tennessee Approved: J. Scott Daniels, Ph.D. Joey V. Barnett, Ph.D. Colleen M. Niswender, Ph.D. C. David Weaver, Ph.D. Neil Osheroff, Ph.D. Wendell S. Akers, Pharm.D., Ph.D. For Mom, Dad, and Robby Colossians 3:17 ii ACKNOWLEDGEMENTS The research described herein was supported by the NIGMS Vanderbilt Training Program in Pharmacological Sciences, the PhRMA Pre-Doctoral Fellowship Program in Pharmacology/Toxicology, and the NIH Division of Loan Repayment Program in Clinical Research. The commitment of these organizations to the development of young scientists in biomedical, pharmaceutical, and translational research is vital to the discovery and advancement of disease diagnosis, treatment, and prevention, and I would like to thank them for the financial provisions they have provided to me throughout my Ph.D. training. In addition, I would like to thank the Lipscomb University College of Pharmacy/Vanderbilt University Department of Pharmacology Pharm.D./Ph.D. Degree Partnership Program for providing me the opportunity to train in an environment of exceptional scientists and a program dedicated to high-quality education. Most notably, I want to express my gratitude toward the individuals who created this program, Drs. Joey Barnett and Scott Akers, for the countless avenues of support they have provided to me. I would not be in the position to present this work today without their support and encouragement—no one, perhaps, is owed more credit for me taking the step toward this achievement than Dr. Akers. Likewise, several prior teachers and mentors at Lipscomb have contributed to my interest in science and my ultimate decision to pursue a career in research. From my undergraduate professors and mentors Drs. Kent Clinger and Ronnie Boone, to my pharmacy school professors and mentors Drs. Michael Fowler and iii Susan Mercer, I am thankful for the instruction, mentoring, and encouragement I received from these individuals. Likewise, I would not have made it through pharmacy school to reach this point without the support and encouragement of my pharmacy classmates Drs. Katie Black and Chris Stokes. In addition to Drs. Barnett and Akers, I am grateful for the others who have graciously served on my dissertation committee. Insightful suggestions from Dr. Colleen Niswender have been an essential contribution to improving the quality and focus of my studies, as have fresh perspectives and ideas from Dr. Neil Osheroff. I am also very appreciative of Dr. Dave Weaver for stepping in to serve as my co-chair alongside Dr. Niswender. Each member of my committee has been tremendously supportive and encouraging, and it has been a pleasure to work with them and gain from their expertise. I would also like to take this time to thank Dr. Matthew Hutzler, who has generously served as an additional mentor to me and a critical contributor to completion of this work. In addition to my committee members, I am especially thankful for the opportunity to work with my mentor, Dr. Scott Daniels, who has been more than just a research advisor, but a life coach, in support of my research, my career aims, as well as my personal well-being. I am appreciative to Dr. Daniels for granting me the freedom to take ownership of my research and for always encouraging me to explore my own ideas. At the same time, I have been fortunate for the opportunity to learn from his expertise and build a foundation for my career. Dr. Daniels has always been my advocate, and I am eternally grateful for his dedicated commitment to my iv success. It has truly been a privilege to train with Dr. Daniels and likewise to have gained a lifelong mentor, colleague, and friend. As for the DMPK lab members, past and present, I am thankful for their friendship and for all that they have graciously taught me. I am especially grateful to Dr. Tom Bridges, who I could always count on to take time to answer a question, Frank Byers for that all he taught and assisted me with and, especially, for making the lab a fun place to be, Jay Foster for always being willing to help and for keeping me entertained in the lab, and Dr. Annie Blobaum and Dr. Chuck Locuson for all their assistance and encouragement. I undoubtedly owe my gratitude to Ryan Morrison for the hours of time he generously dedicated to teaching me everything I know about LC/MS/MS, among countless other concepts and techniques relating to DMPK. His instruction was essential to me reaching this point, as was his friendship. I also thank Sichen Chang, Katrina Brewerer, and several other past labmates for all they have done to aid me. Thanks as well to those in the Lindsely Lab, especially Craig Lindsley, who aided in facilitating the completion of my research. I am also particularly thankful to Matt Mulder for the many ways in which he has assisted me, as well as being a great desk neighbor, and Jeanette Bertron for being a fun, supportive, and encouraging friend. Thanks to everyone else in Cool Springs for all thier help, especially Nathan Kett for keeping the place up and running, as well as many others for creating a fun environment in which to work. There are many others who are owed my gratitude for various means of support in the Department of Pharmacology and VCNDD. I specifically want to thank Karen Gieg, Donna Johnson, and Kristin Riggs for all their v assistance, as well as Dr. Jeffrey Conn and his research group, many members of which have aided me in one way or another. Of course, I must also thank my friends and family, who are too many to name, for all the moral support provided to me over the past several years. I most certainly could not have made it through this challenging time without their support and encouragement. My Mom and Dad have always supported me in whatever I chose to pursue and always believed I could achieve anything I set out to do. I could never repay them for all they have done for me, and I am forever indebted to them for all their love and support. I also want to thank my brother Matt for his love and encouragement, as well as my mother and father-in-law, Bob and Martha, who have treated me like their own daughter. I lastly am infinitely grateful for my husband and best friend, Robby, who has supported me through eight years of pharmacy school and graduate school without complaint. He makes me laugh, makes me think, makes me strong, makes me happy, and I could not make it through life without him. Finally, for all these blessings I have received, I am eternally grateful to God, who already knows the answer before we even conceive the question. vi TABLE OF CONTENTS Page DEDICATION ............................................................................................................................................. ii ACKNOWLEDGEMENTS ...................................................................................................................... iii LIST OF TABLES ...................................................................................................................................xiii LIST OF FIGURES ............................................................................................................................... xvii LIST OF EQUATIONS .......................................................................................................................... xxii LIST OF ABBREVIATIONS .............................................................................................................. xxiii Chapter I. INTODUCTION TO ALDEHYDE OXIDASE ................................................................................. 1 Aldehyde Oxidase Structure .......................................................................................................... 1 Aldehyde Oxidase Substrate Specificity and Reactions ...................................................... 2 Aldehyde Oxidase Catalytic Mechanism ................................................................................... 3 Known Clinical Aldehyde Oxidase Substrates ........................................................................ 4 Known Clinical Aldehyde Oxidase Inhibitors ......................................................................... 7 Aldehyde Oxidase Expression ...................................................................................................... 8 Single human aldehyde oxidase isoform ........................................................................... 8 Species-specific aldehyde oxidase isoforms ..................................................................... 9 Age-dependent aldehyde oxidase expression ...............................................................11 Regulation of aldehyde oxidase expression ...................................................................11 Species-Specific Aldehyde Oxidase Activity ..........................................................................12 Sex Differences in Aldehyde Oxidase Activity ......................................................................13 Endogenous Aldehyde Oxidase Substrates and Physiological Relevance .................15 Human Aldehyde Oxidase Single Nucleotide Polymorphisms ......................................16