Biotransformation of the Analgesic-Antipyretic Drugs Metamizole and Aminopyrine by Genetically Polymorphic Enzymes Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften ( Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Salem Omran Ali Abdalla aus Sokna, Libyen 1. Referent: Professor Dr. Ingo Rustenbeck 2. Referent: Professor Dr. Jürgen Brockmöller eingereicht am: 29. Juni 2007 mündliche Prüfung (Disputation) am: 27. September 2007 Druckjahr 2007 The work described here was performed in the period from July 2002 to April 2007 at the Department of Clinical Pharmacology, Georg-August University, Göttingen To my parents And my children Omran and Raian Table of Contents TABLE OF CONTENTS TABLE OF CONTENTS.......................................................................................................................................I LIST OF ABBREVIATIONS............................................................................................................................ III 1 INTRODUCTION ....................................................................................................................................... 1 1.1 DRUG METABOLISM .............................................................................................................................. 1 1.1.1 Specific reactions in drugs metabolism ........................................................................................... 1 1.2 CYTOCHROME P450 ENZYMES.............................................................................................................. 5 1.2.1 Discovery and Background.............................................................................................................. 5 1.2.2 Function........................................................................................................................................... 6 1.2.3 Evolution.......................................................................................................................................... 8 1.2.4 Classification................................................................................................................................... 8 1.3 CLINICAL RELEVANCE OF GENETIC POLYMORPHISMS IN DRUG METABOLISM ...................................... 10 1.4 GENETIC VARIABILITY ........................................................................................................................ 12 1.4.1 Genetic variability in drug metabolism ......................................................................................... 13 1.4.2 CYP2D6 genetic variability........................................................................................................... 13 1.4.3 CYP2C19 genetic variability ......................................................................................................... 15 1.4.4 CYP1A2 genetic variability ........................................................................................................... 16 1.5 INVESTIGATED KNOWN AND PRESUMED SUBSTRATES OF CYTOCHROME P450 ENZYMES..................... 19 1.5.1 Analgesic-antipyretic drugs........................................................................................................... 19 1.5.2 Mechanism of action of NSAIDs.................................................................................................... 19 1.5.3 Metamizole .................................................................................................................................... 20 1.5.4 Aminopyrine .................................................................................................................................. 22 2 AIMS OF THE STUDY............................................................................................................................. 25 3 MATERIALS AND METHODS .............................................................................................................. 27 3.1 MATERIALS......................................................................................................................................... 27 3.1.1 Instruments .................................................................................................................................... 27 3.1.2 Consumable materials ................................................................................................................... 28 3.1.3 Chemicals ...................................................................................................................................... 29 3.1.4 Kits/Reagents................................................................................................................................. 30 3.1.5 Solvents.......................................................................................................................................... 30 3.1.6 Drug metabolizing enzymes........................................................................................................... 31 3.2 METHODS............................................................................................................................................ 32 3.2.1 In-vitro metabolism........................................................................................................................ 32 3.2.1.1 Human and rat liver samples................................................................................................................32 3.2.1.1.1 Preparation of Human liver microsomes.........................................................................................32 3.2.1.1.2 Protein quantification......................................................................................................................33 3.2.2 In-vitro incubation......................................................................................................................... 33 3.2.2.1 Metamizole...........................................................................................................................................33 3.2.2.1.1 Determination of inhibition characteristics. ....................................................................................35 3.2.2.1.2 Incubations with heterologously expressed isolated human CYP450s............................................36 3.2.2.2 Aminopyrine ........................................................................................................................................37 3.2.2.2.1 Determination of inhibition characteristics .....................................................................................37 3.2.3 HPLC analysis and chromatographic conditions.......................................................................... 39 3.2.3.1 Metamizole...........................................................................................................................................39 3.2.3.2 Aminopyrine ........................................................................................................................................39 3.3 DATA ANALYSIS.................................................................................................................................. 40 3.3.1 Software......................................................................................................................................... 40 3.3.1.1 Calculations and estimation of enzyme kinetic parameters..................................................................41 3.3.1.2 Metamizole...........................................................................................................................................41 3.3.1.2.1 Calculation of metamizole concentrations from the HPLC chromatograms ...................................41 3.3.1.2.2 Calculation of enzyme kinetic constants Vmax and KM ....................................................................41 3.3.1.2.3 Determination of the IC50 and Ki for the Inhibition ........................................................................42 3.3.1.3 Aminopyrine ........................................................................................................................................42 3.3.1.3.1 Calculation of 4-DMAA concentrations from the HPLC analyses .................................................42 3.3.1.3.2 Calculation of enzyme kinetic constants Vmax and KM ....................................................................42 3.3.2.2.3 IC50 and Ki for the Inhibition...........................................................................................................43 3.3.2 Predication of pharmacokinetic clearance.................................................................................... 43 I Table of Contents 3.4 METHOD VALIDATION......................................................................................................................... 44 3.4.1 Incubation...................................................................................................................................... 44 3.4.1.1 Solubility..............................................................................................................................................44 3.4.1.2 Standard curves....................................................................................................................................44 3.4.2 HPLC analysis..............................................................................................................................