Biotransformation of the analgesic-antipyretic drugs metamizole and aminopyrine by genetically polymorphic enzymes [Elektronische Ressource] / von Salem Omran Ali Abdalla

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 vari ......................................................................................................... 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 V and K ....................................................................41 max M
3.3.1.2.3 Determination of the IC and K for the Inhibition ........................................................................42 50 i
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 V and K42 max M
3.3.2.2.3 IC and K for the Inhibition...........................................................................................................43 50 i
3.3.2 Predication of pharmacokinetic clearance.................................................................................... 43
ITable 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............................................................................................................................... 44
3.4.2.1 Limit of detection (limit of quantification).......................................