Investigations into the therapeutic potential of a bacterial cocaine esterase for the treatment of cocaine toxicity and cocaine abuse by Remy Leigh Brim A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in The University of Michigan 2011 Doctoral Committee: Professor James H. Woods, Co-Chair Associate Professor Roger K. Sunahara, Co-Chair Professor Margaret E. Gnegy Professor Nicholas W. Lukacs Associate Professor John J. G. Tesmer © Remy Leigh Brim 2011 Dedication This thesis is to my parents, Barry and Claudia Brim, for their 25 years of love support and sacrifice that made this work, and all my dreams, possible. ii Acknowledgements Thank you to both of my mentors Drs Jim Woods and Roger Sunahara. I have been privileged to work with two mentors who have given me so many opportunities to succeed. Your support and encouragement have always made the difference. I appreciate your trust in me to take this project to so many new places that only I wanted to go! I have learned not only how to conduct experiments, write papers and grants and give presentations, but more importantly how to be the confident and thoughtful scientist I have become. Thank you to my committee Drs. John Tesmer, Maragret Gnegy, and Nick Lukacs. It has been such a pleasure working with you to shape this project. I appreciate all of the feedback and suggestions over the years and I hope that you enjoy this final product! Dr. Kathleen Noon: Dr. Noon performed and developed all of the mass spectrometry assays presented in this thesis. These assays allowed us to detect the in vivo time course of cocaine hydrolysis by CocE, determine the effect of CocE on cocaine across species, and demonstrate that CocE can hydrolyze the cocaine metabolites norcocaine and cocaethylene. Her hard work and perseverance to set up and conduct these assays made these results possible. Thank you. Dr. Holden Ko: Dr. Ko designed the repeat dosing regimen in Rhesus monkeys that allowed me to compare the efficacy of CocE across species. I appreciate him allowing me to use blood samples from these animals to conduct my studies. Thank you. iii Thank you to the University of Michigan Comprehensive Cancer Center Tissue Core for their work paraffin embedding tissue and performing immunoperoxidase immunohistochemistry. Also thank you to the University of Michigan Microscopy and Image Analysis Laboratory for use of their microscopes and slide scanner to produce images of my immunohistochemisty slides. Thank you to my first mentors in graduate school: Drs. Emily Jutkiewicz, Cheryse Furman and Greg Collins. When I began graduate school you gave me the hands-on training to be able to function in the lab, and had the patience to teach me so many of the things I rely on every day. You gave me the encouragement I needed to gain confidence in myself as a scientist. I am so honored to now call you my peers. Joe Crossno and Kendall Carey….where would I be without you? You have kept the lab running and helped me navigate the world of scientific bureaucracy! Thank you for all the time you have spent keeping me “in compliance” and on track. I would like to acknowledge the incredible technical animal assistance that made this work possible. Davina Barron, Alyssa Cunningham and Adam Kynaston have provided invaluable animal services, including surgeries, and experiment monitoring. I have also had the privilege of working with Daniel Youngstrom, an intelligent and motivated undergraduate student who assisted me in completing toxicity and self-administration studies. Thank you! Thank you to the entire Woods lab past and present: Dr. Gail Winger, Drs. Chad and Tammy Wade Galuska, Dr. Chris Podlesnik, Dr. Corina Jimenez-Gomez, Dr. Micky iv Koffarnus, Jeremiah Bertz, and Nhu Truong. Your companionship has been amazing, and I couldn’t have asked for a better group of people to work with! Thank you Joey Nichols and Dr. Diwa Narasimhan for teaching me all things cocaine esterase. It has been quite a journey together on this project and being alongside both of you has been a great experience. Thank you for all of your help with experiments, writing, and editing, and especially the moral support! Diane Calinski and Gisselle Velez-Ruiz: It has been a true honor to work with two amazing women every day. Thank you for your companionship and support in every way imaginable. Through all the fun times, and all of the tough stuff, you made me look forward to coming into the lab every day: Thank you! Thank you to the entire Sunahara lab past and present: Dr. Matt Whorton, Dr. Adam Kuszak, Dr. Avi Bandyopadhya, Elin Edwald, Brian Devree, Jimmy Chan, and Faiza Haji-Abdi. Thank you for being such a supportive and collaborative group. Thanks to Dr. R. D. Davenport from the University of Michigan Hospital for providing the human plasma that allowed me to conduct many in vitro studies. Thank you to the University of Michigan faculty who know it takes a village to produce a Ph.D. and have contributed to my success: Dr. Helen Baghdoyan, Dr. Asim Beg, Dr. Jill Becker, Dr. Thomas Carey, Dr. Edward Domino, Dr. Paul Hollenberg, Dr. Lori Isom, Dr. Jeffery Martens, Dr. Rick Neubig, Dr. Yoichi Osawa, and Dr. John Traynor. v Thank you finally to my friends and family who have supported me through this process. I am so blessed to have the support system that I do, and could not have made it through without your love and encouragement. vi Table of Contents Dedication ......................................................................................................................... ii Acknowledgements .......................................................................................................... iii List of Figures .................................................................................................................. viii List of Tables ..................................................................................................................... x List of Abbreviations ......................................................................................................... xi Abstract ........................................................................................................................... xii Chapter 1. Introduction ............................................................................................................ 1 2. A Thermally Stable Form of Bacterial Cocaine Esterase: A Potential Therapeutic Agent for Treatment of Cocaine Abuse ........................................................................... 28 3. The Fate of Bacterial Cocaine Esterase in Sprague Dawley Rats: An in vivo Study of CocE-Mediated Cocaine Hydrolysis, CocE Pharmacokinetics, and CocE Elimination ........................................................................................................................................ 55 4. The Capacity of Bacterial Cocaine Esterase to Hydrolyze Cocaine’s Metabolites and the Alteration of the Esterase’s Hydrolytic Activity by Commonly Co-Administered Drugs ............................................................................................................................. 100 5. General Discussion and Conclusions ................................................................ 129 Appendix ....................................................................................................................... 149 vii List of Figures Figure 1.1 Chemical structure of cocaine .......................................................................... 2 Figure 1.2: Crystal structure of bacterial Cocaine Esterase ............................................ 15 Figure 1.3: Comparison of bacterial Cocaine Esterase to human butyrylcholinesterase 16 Figure 1.4: In vitro half-life of CocE compared to in vivo duration of action in mice. ....... 18 Figure 2.1: In vitro stability of KQ-CocE ......................................................................... 47 Figure 2.2: Crystal structure of KQ-CocE ........................................................................ 48 Figure 2.3: In vivo potency and duration of action of KQ-CocE ..................................... 49 Figure 2.4: KQ-CocE protection against cocaine-reinforced operant responding in Sprague Dawley rats ....................................................................................................... 50 Figure 2.5: In vivo CocE plasma half-life ........................................................................ 51 Figure 3.1: In vitro and in vivo cocaine hydrolysis by RQ-CocE..................................... 89 Figure 3.2: RQ-CocE hydrolysis of high-dose cocaine across species .......................... 90 Figure 3.3: Elimination of RQ-CocE in the serum of Sprague Dawley rats .................... 91 Figure 3.4: Kinetics of CocE in serum and urine of Sprague Dawley rats...................... 92 Figure 3.5: Comparison of Western Blot and radiography assessments of [35S]-RQ- CocE ............................................................................................................................... 93 Figure 3.6: Immunohistochemical analysis of RQ-CocE distribution in perfused organs from Sprague Dawley rats ............................................................................................... 94 Figure 3.7: Immunohistochemical analysis of RQ-CocE accumulation the renal papilla 95 viii Figure 3.8: Radiography and imunohistochemistry of organs one day post-CocE injection. .........................................................................................................................