CARBOXYLESTERASE 1 GENETIC VARIABILITY, EXPRESSION AND POTENTIAL FOR DRUG-DRUG INTERACTIONS Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Teresa Sofía Sánchez Pascua September 2014 TABLE OF CONTENTS Abstract i Acknowledgements ii Abbreviations iii List of figures viii Table of single nucleotide polymorphisms ix Chapter 1: General Introduction.......................................................................... 1 1.1. Human metabolism ................................................................................................. 2 1.1.1. Drug metabolising enzymes ........................................................................ 2 1.1.2. Drug transporters ......................................................................................... 5 1.1.3. Drug interactions ......................................................................................... 7 1.1.4. Pharmacogenetics ........................................................................................ 9 1.2. Carboxylesterase 1 ................................................................................................ 12 1.2.1. Protein classification .................................................................................. 12 1.2.2. Tissue expression ....................................................................................... 15 1.2.3. Physiological roles ..................................................................................... 16 1.2.4. Protein structure ......................................................................................... 18 1.2.5. Enzymatic mechanism ............................................................................... 19 1.2.6. Substrate specificity ................................................................................... 20 1.2.7. Therapeutic compounds ............................................................................. 21 1.2.8. CES1 gene ................................................................................................. 22 1.2.9. Genetic variability ..................................................................................... 23 1.2.10. Physio-pathological conditions .................................................................. 25 1.2.11. Regulation .................................................................................................. 26 1.2.12. DDIs and CES1 ......................................................................................... 27 1.3. Aims of the thesis .................................................................................................. 29 Chapter 2: Methods ............................................................................................. 30 2.1. Introduction ........................................................................................................... 31 2.2. HPLC-UV method for the quantification of clopidogrel main circulating metabolite in human plasma ................................................................................. 32 2.2.1. Introduction ............................................................................................... 32 2.2.2. Materials and methods ............................................................................... 34 2.2.3. Results ....................................................................................................... 41 2.2.4. Discussion .................................................................................................. 47 2.3. Optimization and validation of allelic discrimination assays of CES1 gene genetic variants .................................................................................................................. 49 2.3.1. Introduction ............................................................................................... 49 2.3.2. Materials and methods ............................................................................... 51 2.3.3. Results of the assays validation ................................................................. 63 2.3.4. Discussion .................................................................................................. 67 Chapter 3: Effect of CES1, CYP2C9 and CYP2C19 genetic variability on clopidogrel antiplatelet efficacy .............................................................................. 69 3.1. Introduction ........................................................................................................... 70 3.2. Methods................................................................................................................. 76 3.2.1. Clinical study ............................................................................................. 76 3.2.2. Study population ........................................................................................ 77 3.2.3. Platelet function testing ............................................................................. 77 3.2.4. Genotyping ................................................................................................ 78 3.2.5. Determination of plasma metabolite concentrations ................................. 81 3.2.6. Data analysis .............................................................................................. 81 3.3. Results ................................................................................................................... 83 3.3.1. Patient demographics ................................................................................. 83 3.3.2. SNP frequencies and linkage disequilibrium ............................................. 85 3.3.3. Impact of demographic, clinical and genetic factors on platelet aggregation . .................................................................................................................. 88 3.3.4. Demographics and genetic variants impact on CLPM plasma concentration . .................................................................................................................. 92 3.4. Discussion ............................................................................................................. 96 Chapter 4: Effect of efavirenz and nevirapine on the disposition of antiplatelet agent clopidogrel in HIV positive subjects ...................................... 101 4.1. Introduction ......................................................................................................... 102 4.2. Methods............................................................................................................... 105 4.2.1. Study design ............................................................................................ 105 4.2.2. Determination of CLPM plasma concentration ....................................... 108 4.2.3. PK and statistical analysis ....................................................................... 108 4.3. Results ................................................................................................................. 108 4.3.1. Subject characteristics ............................................................................. 110 4.3.2. PK assessment and comparison to historical data ................................... 112 4.3.3. Differences between NVP and EFV treated subjects .............................. 115 4.4. Discussion ........................................................................................................... 114 Chapter 5: Impact of CES1 single nucleotide polymorphisms on isoniazid pharmacokinetics ................................................................................................... 121 5.1. Introduction ......................................................................................................... 122 5.2. Methods............................................................................................................... 125 5.2.1. Study design ............................................................................................ 125 5.2.2. DNA extraction and SNP genotyping ...................................................... 126 5.2.3. INH plasma quantitation and PK parameters calculation ........................ 128 5.2.4. Statistical analysis .................................................................................... 128 5.3. Results ................................................................................................................. 130 5.3.1. Subjects demographics ............................................................................ 130 5.3.2. Frequencies of SNPs ................................................................................ 132 5.3.3. Demographics impact on INH PK parameters ........................................ 134 5.3.4. Impact of genetic variants on INH PK parameters .................................. 138 5.3.5. Multifactorial models and the effect of factor interactions on PK parameters ............................................................................................................... 141 5.4. Discussion ........................................................................................................... 145 Chapter 6: Impact of rifampicin, rifabutin and rifapentine on CES1 and CES2 expression ..................................................................................................... 149 6.1. Introduction ........................................................................................................