COVALENT DRUG BINDING: a MECHANISTIC EXPLORATION to ENHANCE SAFETY and EFFICACY CHAN CHUN YIP (B.Sc. Pharm (Hons.), NUS) a THES
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COVALENT DRUG BINDING: A MECHANISTIC EXPLORATION TO ENHANCE SAFETY AND EFFICACY CHAN CHUN YIP (B.Sc. Pharm (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2015 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _______________ Chan Chun Yip 18 November 2015 i Acknowledgments This thesis represents the collective contributions of numerous individuals to whom I would like to express my deepest gratitude. First and foremost, my heartfelt thanks to Prof Eric Chan, my doctoral advisor who has been a wonderful mentor since I was an Honors Year student. Thank you for taking a chance on someone rough around the edges, and investing time and effort in shaping and moulding me to be a better person both professionally and personally. You are a person who truly leads by example and clearly walks the talk. I am grateful that you are always challenging and inspiring me, and in the process, you have taught me that I have, in myself, the capacity to achieve greater heights. Thank you for trusting me, giving me the freedom and encouraging me to explore new ideas and research interests, and for providing all the necessary support to ensure the success of these endeavours. You are always ready to lend a listening ear despite your busy schedule, and I deeply appreciate all your advice and counsel. I continue to learn from you in so many ways, and I thank God for blessing me with you as my mentor and role model. I am deeply appreciative of my collaborators at the Singapore Eye Research Institute: Dr Zhou Lei and Ms Koh Siew Kwan, who believed in the potential of this project, and has tirelessly and generously provided invaluable support and technical expertise, without which our investigation on drug-induced protein glutathionylation would not have commenced and now come to fruition. I am very grateful for your strong commitment to this work and belief in its potential that helped sustain it through difficult times. My sincere gratitude to Dr Ong Pei Shi for partnering us in investigating the pharmacodynamic interaction between lapatinib and endoxifen. Thank you for personally teaching me the cell culture ii and molecular biology techniques needed for this study, and for tirelessly co- supervising 2 Honors Year students with us. I have learnt a great deal from your guidance in and out of the lab. To my collaborators at GlaxoSmithKline R&D Singapore: Dr Edward Browne, Dr Kishore Pasikanti and Ms Su Zhiting, my deepest gratitude for their unwavering support with investigating the pharmacokinetic interaction between lapatinib and endoxifen using the MDCKII- MDR1 model. A big thank you to Prof Christina Chai for hosting me in your lab to perform organic synthesis, for kindly treating me as part of your research group and for all the advice, support and encouragement. I am thankful to Dr Ngai Mun Hong, Yan Ting, Yanbo and Kuan Chieh for help with the synthetic work, and more memorably the camaraderie in and out of the Chai lab. My warmest gratitude also to Dr Vinita Uttamsingh and Dr Sophia Nguyen from Concert Pharmaceuticals for providing deuterated lapatinib analogues for our evaluation. My sincere thanks to past and present members of the Metabolic Profiling Research Group: Lee Sun for her guidance and friendship from my days as an Honours student; Alex, Davis, Devester, Kwok Foong, Preben, Peirong and Wanjing for their invaluable contributions as undergraduate students to the different parts of this work, making it greater than the sum of its individual parts; Hui Ting, Lee Cheng, Lian Yee, Yanjun, Gopal, Yee Min and Dorinda for providing me with their kind assistance for parts of my project; Liyan, Aneesh, Aparna, Yun Shan, Thiru, Kai Lun, Ronald, Gopal and Theebarina for making MPRG a wonderful and fun group, as well as Hai Ning for being a great friend and generously sharing resources; you are all part of precious memories and have left an indelible mark in my journey. A special thanks to Lian Yee for all the encouragement you have provided to me; your treasured friendship, cherished iii care and unflagging support through thick and thin made it possible to complete this journey, and I would not exchange it for anything in this world. I would like to convey my gratefulness to the Department of Pharmacy, NUS, for my education and I would like to thank in particular Dr Chew Eng Hui and Dr Ho Han Kiat for their sharp comments and suggestions which kept this project on the right track; Ms Ng Sek Eng, Ms Tan Bee Jen and Mr Sukaman for their excellent technical support and administrative efficiency. I would also like to acknowledge the financial support from NUS Pharmacy, GlaxoSmithKline, the President’s Graduate Fellowship and funding from the FYP research grants for making this project a reality. I would also like to remember the late Prof Sidney Nelson, who carried out pionerring investigations on acetaminophen toxicity and inspired us to investigate acetaminophen-induced protein glutathionylation. To my dad and mum: no words can adequately express my thanks and gratitude for all your love, care, concern and patience; supporting me through difficult times, and celebrating my achievements and successes; for your unwavering belief and unceasing encouragement; and being ever ready with a listening ear and a comforting shoulder. As difficult as it sometimes was for me during these eight years that I was away from home, it must have been harder for you; I am grateful for your trust and support which enabled me to pursue my dreams. Thank you for making countless trips between home and Singapore to visit me and keep me well-stocked with necessities, and never once griping or complaining. To the rest of my family and loved ones: I could not have completed this journey without you. Each of you gave of yourself in some way that made this possible. I thank God every day that I have you in my life. iv And finally, to my Lord and Saviour Jesus Christ: thank you for leading me through all these years, and for placing me at the right time and right place, with the right people, doing the right thing. Looking back, I can see Your hand in action guiding me and providing me with all that I need. Thank you for giving me a new perspective of Your creation as a scientist. Having glimpsed the marvelous intricacies of Your work in this thesis, I am utterly convinced that “… in Him all things were created: things in heaven and on earth, visible and invisible, whether thrones or powers or rulers or authorities; all things have been created through Him and for Him. He is before all things, and in Him all things hold together.” (Col 1:16-17, NIV). Glory be to Him. v Table of Contents DECLARATION ................................................................................................... I ACKNOWLEDGMENTS ................................................................................... II TABLE OF CONTENTS ................................................................................... VI SUMMARY ........................................................................................................... X PUBLICATIONS AND CONFERENCE ABSTRACTS ................................ XI LIST OF TABLES ........................................................................................... XIII LIST OF FIGURES ........................................................................................... XV LIST OF SUPPLEMENTARY TABLES ...................................................... XXI LIST OF SUPPLEMENTARY FIGURES ................................................... XXII LIST OF ABBREVIATIONS ...................................................................... XXIII CHAPTER 1 INTRODUCTION ....................................................................1 1.1 The Burden of Adverse Drug Reactions .....................................................1 1.2 Classification of ADRs ..................................................................................2 1.3 Xenobiotic Metabolism and Reactive Metabolite Formation ...................4 1.4 Covalent Drug Binding and Protein Adduct Formation ...........................5 1.5 Toxicological Significance of Covalent Binding .........................................6 1.5.1 Overt Consequences of Covalent Binding ........................................................... 6 1.5.1.1 Mechanism-based Inactivation ................................................................... 6 1.5.1.2 Type B idiosyncratic drug reactions ........................................................... 9 1.5.1.3 Type C dose-dependent toxicity ............................................................... 11 1.5.2 Covert Effects of Covalent Binding ................................................................... 12 1.5.3 Limitations of the Covalent Binding Hypothesis................................................ 15 1.6 Pharmacological Significance of Covalent Binding .................................16 1.7 Research Objectives and Significance .......................................................19 1.7.1 Research Question and Objectives .................................................................... 19 1.7.2 Thesis Outline .................................................................................................