UNIVERSITY OF ALBERTA CYTOCHROME P450 ENZYMES AND THE METABOLISM OF TRAZODONE AND NEFAZODONE SUSAN E. ROTZINGER O SUBMllTED TO THE FACULTY OF GFWDUATE STUDIES AND RESEARCH IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DIVISION OF NEUROSCIENCE - PSYCHIATRY EDMONTON, ALBERTA FALL, 1998 National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions ana Acquisitions et Bibliographie Services seMces bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON KIA ON4 ûttawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to ~ibliothequenationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la proprieté du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or oîherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation, ABSTRACT This thesis represents the results of an investigation of the involvement of the cytochrome P450 (CYP) enzymes in the metabolism of two related antidepressants. trazodone and nefazodone. Several converging lines of in vitro experimentation were used to identify the enzymes involved in the metabolism of NEF to its active metabolites hydroxy-nefazodone (OH-NEF), triamledione (TD), and m-chlorophenylpiperazine (mCPP). The metabolism of trazodone to the active metabolite mCPP was also investigated, as was the metabolism of mCPP to its major metabolite hydroxy-mCPP (OH-mCPP). The identification of the major enzymes involved in a particular metabolic pathway was accomplished in vitro using human liver microsomes and cells expressing human CYP enzymes. Novel analytical methods were developed for the detection of trazodone, NEF, and their metabolites using high pressure liquid chromatography (HPLC) and gas chromatography (GC). The major findings of the present thesis were that CYP3A4 is an important enzyme in the metabolism of NEF to OH-NEF and mCPP, and of OH-NEF to TD and mCPP. Furthemore, CYP3A4 was found to be important in the metabolism of trazodone to mCPP, and CYP2D6 was found to be important for the rnetabolism of mCPP to OH-mCPP. These findings have important implications for predicting metabolic dmg-drug interactions involving trazodone and nefazodone. This thesis is dedicated to my parents, Marcel Rotzinger and Beverley TaIlon, for the support and encouragement they have provided unconditionally throughout my life, and to Bryan, for teaching me life's tmly important lessons. ACKNOWLEDGEMENTS First and foremost, I rnust acknowledge the exceptional leadership of Dr. Glen Baker, whose hard work, integrity, and dedication to excellence serves as an example of how research should be conducted. 1 thank him for providing me with the opportunity to conduct this research in an atmosphere fosterhg learning and growth, and for the guidance and encouragement necessary to make me believe that this thesis could one day become a reality. I also wish to thank Drs. RT Coutts, IL Martin, J Fang, and A Holt for their willingness to listen to my questions, and for their patience and enthusiasm in providing the answers. I am deeply indebted to Gai1 Rauw, for her limitless patience and intelligence, and to Jordyce Van Muyden for her exceptional technical and organizational support. The friends that I have made during the course of rny schooling have enhanced my education and my life in countless ways. I gratefully acknowledge the constant support and encouragement from rny family, as well as Bryan's patience, understanding, and insight, which has helped me inestimably. Finally, 1 wish to thank the Medical Research Council of Canada and the University of Alberta (Walter Johns Graduate Fellowship) for the financial support which made this research possible. TABLE OF CONTENTS Page CHAPTER 1 Introduction ............................................................. -1 1. i INTRODUCTION .................. .,. ...................................... 2 1.2 LITERATURE REVIEW ............................................................ II 1-2- 1 Drug Metabolism ............................................................. II 1.2.1 .1 General Principles...................................................... 11 The Cytochrome P450 enzymes .................................. -12 Classification and nomenclature......................... -12 Structure, function and mechanism of action ......... -13 Factors affecting dnrg metabolism....................... 15 Inhibition ............................................... 15 Induction.............................................. -16 Genetics................................................ 17 Consequences of altered enzyme levels.................................. 20 Important CYPs in human drug metabolism ........................22 CYP3A4 ................................................................. -22 CYP2D6 .................................................................. -24 Drug metaboiism in psychiatry............................................... 25 Pharmacokinetic and pharrnacodynarnic drug interactions ......... -28 Methods for studying drug interactions.................................... 30 In vitro ......................................................................31 ln vivo .................................................................... -35 . * 1.2.1.7.3 Clmcal relevance ...................................................... 36 1.2.2 Antidepressant Drug Development.............................................. -37 1.2.2.1 History ............................................................................. -37 1.2.2.2 Rational drug design (RDD) and the 5-HT system.................... -40 1.2.2.3 5-HT, receptors as targets for rational drug design .................. -44 1.2.3 Trazodone ................................................................................ -45 1.2.3.1 Introduction.......... .... ................................................. -45 1.2.3.2 P harmacokinetics............................................................... 46 1.2.3.3 Metabolism....................................................................... 47 1.2.3.4 Dnig-drug interactions......................................................... 47 1.2.4 m-Chlorophenylpiperazine ......................................................... 49 1.2.4.1 Introduction..................................................................... -49 1.2.4.2 Pharmacokinetics. ............................................................... 51 1.2.4.3 Metabolism and drug interactions......................................... -52 1 .2.5 Nefazodone ............................................................................. -53 1. 2.5. 1 Introduction....................................................................... 53 1.2.5.2 Pharmacokinetics............................................................... 54 1.2.5.3 Metabolism...................................................................... -55 1.2.5.4 Dnig-drug interactions........................................................ -56 1.2.6 Relevant Analytical Techniques ................................................. -60 1.2. 6.1 Chromatograp hy ............................................................... -60 1.2.6.1.1 Gas Chrornatography................................................ -61 . 1.2.6. 1.1 .1 Pnnciples. ........................................................ 61 Instrumentation............................................... -62 .. Derivatizations............................................... -66 High PerffomanceLiquid Chromafography (HPLC)................... 67 .. Principles...................................................... -67 Instrumentation-............................................... 67 Mass Spectrometry........................................................... -69 1.2.7 Thesis Objectives and Rationale................................................ -70 MATERIALS.............................................................................. 72 Chemicals........................................................................ 72 Microsomal Products ........................................................ 73 Human liver microsomes............................................ 73 cDNA-expressed human ceil lines ................................ 74 Instrumentation and Apparatus ..........................................75 Gas C hrornatography ............................................... -75 Nitrogen-Phosphonis Detection ......................... -75 Electron- Capture De tection ............................... -75 High Performance Liquid C hromatography............................. -76 Mass Spectrometry........................................................... 77 S haker-mixer .................................................................. -77 Hot water bath................................................................. -77 Centrifuges ..................................................................... -78 Savant evaporator ............................................................