Universidad Autónoma de Madrid Faculty of Medicine Department of Pharmacology and Therapeutics Evaluation of genetic polymorphisms associated with the metabolic effects of aripiprazole and olanzapine Doctoral Thesis Dóra Koller 2020 Universidad Autónoma de Madrid Facultad de Medicina Departamento de Farmacología y Terapéutica Dr. Francisco Abad Santos, Jefe del Servicio de Farmacología Clínica en el Hospital Universitario de La Princesa y Profesor Titular del Departamento de Farmacología y Terapéutica de la Facultad de Medicina de la Universidad Autónoma de Madrid, CERTIFICA: Que Doña Dora Koller, ha realizado la presente Tesis Doctoral “Evaluation of genetic polymorphisms associated with the metabolic effects of aripiprazole and olanzapine” con objeto de obtener el Grado de Doctor. Como director del trabajo hago constar que ha sido realizado con todas las garantías técnicas y metodológicas, y las conclusiones obtenidas son plenamente válidas, siendo considerado, por tanto, apto para ser presentado como Tesis Doctoral. En Madrid, a 10 de junio de 2020 Fdo. Dr. Francisco Abad Santos This thesis, submitted for the degree of Doctor of Philosophy (Ph.D.), has been elaborated in the Clinical Pharmacology Department, Instituto de Investigación Sanitaria of Hospital Universitario de La Princesa from June 2017 until June 2020 under the supervision of Dr. Francisco Abad Santos. This work was supported by the following grant: Horizon 2020 Marie Sklodowska-Curie Innovative Training Network 721236 grant to Dóra Koller and Francisco Abad-Santos. TABLE OF CONTENTS LIST OF TABLES .................................................................................................................................... 15 LIST OF FIGURES .................................................................................................................................. 17 ABBREVIATIONS ................................................................................................................................... 20 ABSTRACT ............................................................................................................................................... 27 I. INTRODUCTION ................................................................................................................................. 37 1. CLINICAL TRIALS ................................................................................................................................... 39 2. LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY ................................................................... 41 2.1. LIQUID CHROMATOGRAPHY ................................................................................................................. 41 Components and workflow of LC ................................................................................................. 41 Analytical column and mobile phase ............................................................................................. 43 Chromatographic separation .......................................................................................................... 43 2.2. MASS SPECTROMETRY .......................................................................................................................... 43 2.3. SAMPLE PREPARATION FOR LC-MS ..................................................................................................... 46 2.4. ANALYTICAL METHOD VALIDATION .................................................................................................... 47 Selectivity ...................................................................................................................................... 47 Linearity ......................................................................................................................................... 48 LOD and LLOQ ............................................................................................................................. 48 Accuracy and precision .................................................................................................................. 48 Extraction recovery and matrix effects .......................................................................................... 48 Stability .......................................................................................................................................... 49 2.5. LC-MS IN CLINICAL PHARMACOKINETIC STUDIES ............................................................................... 49 3. PHARMACOGENETICS AND PHARMACOGENOMICS .............................................................................. 53 3.1. CONCEPT ............................................................................................................................................... 53 3.2. GENETIC POLYMORPHISMS ................................................................................................................... 54 3.3. PHARMACOGENES ................................................................................................................................. 56 Cytochrome P450 enzymes ........................................................................................................... 57 Drug transporters ........................................................................................................................... 59 3.4. CLINICAL PRACTICE GUIDELINES ........................................................................................................ 59 4. SCHIZOPHRENIA ..................................................................................................................................... 62 5. ANTIPSYCHOTIC TREATMENT ............................................................................................................... 63 5.1. ADVERSE DRUG REACTIONS ................................................................................................................. 65 5.2. OLANZAPINE ......................................................................................................................................... 68 9 Pharmacokinetics ........................................................................................................................... 68 Mechanism of action ...................................................................................................................... 69 5.3. ARIPIPRAZOLE ...................................................................................................................................... 71 Pharmacokinetics ........................................................................................................................... 71 Mechanism of action ...................................................................................................................... 72 5.4. ADVERSE DRUG REACTIONS TO ARIPIPRAZOLE AND OLANZAPINE ...................................................... 74 6. PUPILLOMETRY ...................................................................................................................................... 76 7. THE PHARMACOGENETICS OF ARIPIPRAZOLE AND OLANZAPINE ...................................................... 78 7.1. GENES RELATED TO PHARMACOKINETICS ............................................................................................ 78 7.2. GENES RELATED TO PHARMACODYNAMICS ......................................................................................... 82 II. OBJECTIVES ...................................................................................................................................... 87 III. MATERIALS AND METHODS ....................................................................................................... 91 1. CLINICAL TRIAL ..................................................................................................................................... 93 1.1. STUDY DESIGN ...................................................................................................................................... 93 1.2. STUDY POPULATION ............................................................................................................................. 93 1.3. PROCEDURES ........................................................................................................................................ 95 Sample collection for pharmacokinetic analysis ........................................................................... 95 Biochemical and haematological analyses .................................................................................... 98 Analysis of blood pressure and electrocardiogram ........................................................................ 98 Safety and tolerability assessments ................................................................................................ 99 Pupillometric measurements .......................................................................................................... 99 2. ANALYTICAL METHOD VALIDATION ................................................................................................... 100 2.1. CHEMICALS AND REAGENTS .............................................................................................................. 100 2.2. PREPARATION OF CALIBRATION STANDARDS AND QUALITY CONTROLS ........................................... 101 2.3. EQUIPMENT ........................................................................................................................................