Philosophiae Doctor - PhD / Solute carrier transporters belonging to the major facilitator family of membrane transporter are increasingly being recognized as a possible mechanism to explain inter-individual variation in drug efficacy and response. Genetic factors are estimated to be responsible for approximately 15-30% of inter-individual variation in drug disposition and response. The aims of this study were to determine the minor allele frequencies of 78 previously identified single nucleotide polymorphisms in the pharmacogenetically relevant SLC22A1-3 and SLC47A1 genes in the indigenous African population of South Africa. Secondly, to determine whether allele and genotype frequencies for these SNP were different from that reported for other African, Caucasian, and Asian populations. Thirdly, to infer haplotypes from the genetic information which can potentially be used in future to design and interpret results of pharmacogenetics association studies involving these genes and their substrate drugs. Finally, to determine whether the Xhosa population harbour novel SNPs in the SLC22A2 gene, that encodes the kidney-specific hOCT2. SNaPshot™ multiplex single base minisequencing systems were developed and optimized for each of SLC22A1, SLC22A2, SLC22A3, and SLC47A1 covering the previously identified 78 SNPs. These systems were then used to genotype the alleles of 148 healthy Xhosa subjects residing in Cape Town, South Africa. In addition, the proximal promoter region and all 11 exons and flanking regions of the SLC22A2 gene of 96 of the participants were screened for novel SNPs by direct sequencing. The Xhosa subjects investigated lacked heterozygosity and were monomorphic for 91% of the SNPs screened. None of the SLC22A3 and SLC47A1 SNPs investigated was observed in this study. Sequencing of the SLC22A2 gene revealed 28 SNPs, including seven novel polymorphic sites, in the 96 Xhosa subjects that were screened. The minor allele frequencies of the seven previously identified variant SNPs observed in this study were different compared to that observed for American and European Caucasian, and Asian populations. Moreover, the allele frequencies for these SNPs differed amongst African populations themselves. Eight and seven haplotypes were inferred for SLC22A1 and SLC22A2, respectively, for the Xhosa population from the information gathered with SNaPshot™ genotyping. This study highlights the fact that African populations do not have the same allele frequencies for SNPs in pharmacogenetically relevant genes. Furthermore, the Xhosa and other African populations do not share all reduced function variants of the SLC22A1-3 and SLC47A1 genes with Caucasian and Asian populations. Moreover, this study has demonstrated that the Xhosa population harbours novel and rare genetic polymorphisms in the key pharmacogene SLC22A2. This study lays the foundation for the design and interpretation of future pharmacogenetic association studies between the variant alleles of the SLC22A1-3 and SLC47A1 genes in the Xhosa population and drug disposition and efficacy.

Indiana University-Purdue University Indianapolis (IUPUI) / Developmental changes in the liver significantly impact drug disposition. Due to the emergence of microRNAs as important regulators of drug disposition, we hypothesize that age-dependent change in microRNA expression and genetic variants in microRNA target sites contribute to variability in drug disposition. In human liver tissues, expression of 533 microRNAs and over 14,000 genes were measured. In all, 114 microRNAs were upregulated and 72 downregulated from fetal to pediatric, and 2 and 3, respectively, from pediatric to adult. Among these microRNAs, 99 microRNA-mRNA interactions were predicted or have previously been validated to target drug disposition genes and over 1,000 significant negative correlations were observed between miRNA-mRNA pairs. We validated these interactions using various cell culture models. Genetic variants in the promoter and coding regions of drug disposition genes have also been shown to alter enzyme expression and/or activity. However, these variants do not account for all variability in enzyme activity. Emerging evidence has shown that variants in the 3’UTR may explain variable drug response by altering microRNA regulation. Five 3’UTR variants were associated with significantly altered CYP2B6 activity in healthy human volunteers. The rs70950385 (AG>CA) variant was associated with decreased CYP2B6 activity among normal metabolizers. In vitro luciferase assays confirmed that the CA allele altered miR 1275 targeting of CYP2B6 mRNA. Due to the large number of 3’UTR variants predicted to alter microRNA regulation, a high-throughput method, PASSPORT-seq, was developed to test over 100 3’UTR variants simultaneously in different cell lines. Thirty-eight variants resulted in FDR-significant altered expression between wild-type and variant sequences. Our data suggest a mechanism for the marked changes in hepatic gene expression between the fetal and pediatric developmental periods, support a role for these age dependent microRNAs in regulating drug disposition, and provide strong evidence that 3’UTR variants are also an important source of variability in drug disposition.

Indiana University-Purdue University Indianapolis (IUPUI) / My first aim identified enrollment patterns and variables that predict enrollment in a diverse underserved population and evaluated barriers to enrollment. We analyzed data from the INGENIOUS, (Indiana GENomics Implementation and Opportunity for the UnderServed), pharmacogenomics implementation clinical trial conducted at a community hospital for underserved subjects and a statewide healthcare system. Our main finding revealed, African-Americans were less likely to refuse the study than non-Hispanic Whites (Safety net, OR =0.68, p<0.002; Academic hospital, OR=0.64, p<0.001), using a logistic regression model. The most frequent barriers to enrollment included not being interested, being too busy, transportation, and illness in African-American and non-Hispanic White subjects. In conclusion, improving research awareness, widening the inclusion criteria, and hiring recruiters who represent potential enrollees, should improve enrollment in African-Americans and other diverse populations. My 2nd research aim evaluated racial/ethnic differences in pharmacokinetics, safety, efficacy, and pharmacogenetics in 213 new molecular entities (NMEs). The current approved drug label for NMEs between 2014 to 2018 was updated in the FDA database. A qualitative analysis revealed ~ 9% (n=20/213) of NMEs reported racial/ethnic differences in the approved product label for PK, safety, efficacy, and/or pharmacogenetics. In conclusion, evaluating racial/ethnic differences in drug exposure and response early in the drug development program is essential to providing recommendations for different racial/ethnic subpopulations. My final aim 3, identified genetic biomarkers of Taxane Induced Peripheral Neuropathy (TIPN) in African-American breast cancer patients. We used an innovative computational tool, ALDY, to identify genetic variants in CYP2C8, CYP3A4, and CYP3A5 in 207 breast cancer subjects. TaqMan SNP genotyping for SNP, rs776746 (T>C) was performed in 160 subjects. Subjects were collapsed into three metabolizer groups; normal, intermediate, and poor metabolizer to test the association of peripheral neuropathy, dose reductions and CYP2C8/CYP3A5 metabolizer status. A logistic regression revealed CYP2C8 metabolizer status is associated with grades 3-4 peripheral neuropathy (p=0.04, OR= 2.21). CYP2C8*2 was modestly associated with dose reductions. In conclusion, evaluating pharmacogenetic and pharmacokinetic studies of paclitaxel and CYP2C8 is important. These studies may lead to clinical actionable prescribing of paclitaxel and improve the tolerance and efficacy in African-American breast cancer patients. / 2022-02-16

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Background and Objectives: Pharmacogenetics has the potential to maximize drug efficacy and minimize adverse effects of cardiovascular disease (CVD) but its translation into clinical practice been slow. However, recent advancements in genotyping and statistical methodologies have now provided robust evidence in the support of personalized medicine. This thesis addresses how the advancements in pharmacogenetics may help to gain novel insights into existing drug targets, inform and guide clinical decision-making and validate potential disease target pathways. Methods: This was achieved by exploring whether the COX-2 genetic variant (rs20417) is associated with a decreased risk of CVD outcomes, assessing whether bile acid sequestrants (BAS) are associated with a reduced the risk of coronary artery disease (CAD) using the principles of Mendelian Randomization and investigating whether genetic variants associated with dysglycaemia are associated with an increased risk of CAD. Results: We demonstrated that COX-2 carrier status was associated with a decreased risk of major cardiovascular outcomes. Furthermore, we also showed that BAS appear to be associated with a reduced risk of CAD and genetic variants associated with HbA1c and diabetes were associated with an increased risk of CAD. Conclusions: The convergence of technological and statistical advancements in pharmacogenetics have led to a more high-quality and cost-effective means of assessing the effect of CVD therapeutic agents. / Thesis / Doctor of Philosophy (PhD)

Yes / Personalized medicine through Pharmacogenomics: choosing the right drug, and the right dose, for the right patients based on patient’s genetic makeup-is gradually being realised in Western countries. Yet, the practice of pharmacogenomics in Asian countries lags behind that of the West, but the medical needs for pharmacogenomics are expected to surge as better patient care is demanded in Asia. As next-generation sequencing technology advances quickly, previous technical challenges for performing pharmacogenomic studies or practices in Asia have been mostly resolved. What is lacking in Asia is an effective model of community-wide pharmacogenomics. On the delivery front, pharmacists, the drug and dosing professionals, can potentially be the main healthcare providers for pharmacogenomic services in Asia. The first large “Genomics for Precision Drug Therapy in the Community Pharmacy” in Canada, which is close to its completion, has successfully identified community pharmacists as key contact professionals for smooth facilitation and implementation of pharmacogenomics for personalized medication. It is anticipated that Asian pharmacists, with appropriate training, can have the capacity to provide expert pharmacogenomic supports for both physicians and patients in Asia. / The School of Biomedical Sciences Start-up Fund, the Chinese University of Hong Kong, the General Research Fund (#17114814; #17121616), the Theme-based Research Scheme (T12-401/13-R), Research Grant Council, Hong Kong, as well as the Hong Kong Cancer Fund, Hong Kong.

<p>Genetically determined differences in drug metabolism and disposition and drug targets play a pivotal role in the interindividual variability in the clinical outcome of antipsychotic treatment. The aim of this thesis was to study the impact of polymorphisms in genes involved in the pharmacokinetics and pharmacodynamics of antipsychotics, with special focus on their extrapyramidal and metabolic adverse effects. </p><p>Polymorphisms in serotonin 2A and 2C receptor coding genes (<i>HTR2A</i> and <i>HTR2C</i>) were found to be associated with the risk to develop extrapyramidal side effects (EPS) in patients on short term perphenazine treatment. A further study in a larger group of patients on long term treatment with various classical antipsychotics confirmed the association between occurrence of EPS and <i>HTR2C</i> polymorphisms. In another study, dose corrected steady state serum clozapine and N-desmethylclozapine concentrations (C/D) and insulin elevation during clozapine therapy were found to correlate with <i>CYP1A2</i> but not with <i>CYP2D6</i> polymorphisms. Furthermore, <i>HTR2C</i> and <i>HTR2A</i> polymorphisms were found to have significant influences on BMI and C-peptide levels in patients treated with olanzapine and clozapine. Evaluation of the impact of polymorphisms in genes encoding CYP3A4, CYP3A5 and P-glycoprotein (<i>ABCB1</i>) in addition to CYP2D6 on the steady state plasma levels of risperidone, 9-hydroxyrisperidone and their active moiety revealed a significant influence of <i>ABCB1 </i>genotype on 9-hydroxyrisperidone and active moiety C/Ds, while <i>CYP2D6</i> genotype associated with risperidone C/Ds but not with 9-hydroxyrisperidone or active moiety C/D. </p><p>We have shown that polymorphisms in genes involved in the pharmacokinetics and the pharmacodynamics of antipsychotic drugs play a role in the occurrence of adverse effects, both EPS and metabolic disturbances, induced by antipsychotic treatment. Genotyping for <i>HTR2A</i>, <i>HTR2C</i>, <i>CYP1A2</i>, <i>CYP2D6</i> and <i>ABCB1</i> polymorphisms may therefore potentially provide useful information to identify patients at higher risk to develop EPS or metabolic adverse during schizophrenia treatment with antipsychotic drugs.</p>

As enzimas CYP450 são as principais enzimas metabolizadoras de fármacos. Elas são codificadas por genes que apresentam polimorfismos gênicos que lhes confere características fenotípicas diversas. Estes fenótipos são: Metabolizadores Lentos ou PM, Metabolizadores Normais ou EM, Metabolizadores Intermediários ou IM e Metabolizadores Ultra-rápidos ou UM. A Farmacogenética é a ciência que estuda estas variações gênicas e sua relação com a resposta terapêutica no organismo. Entre as enzimas CYP450 se encontram as enzimas CYP2D6, que são responsáveis pelo metabolismo de 25% dos fármacos clinicamente prescritos. O objetivo principal deste estudo foi a identificação dos polimorfismos mais importantes deste gene: CYP2D6*1, CYP2D6*2, CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*6, CYP2D6*10, CYP2D6*17 e CYP2D6*41 pelos métodos de Genotipagem (PCR Tetra Primer e Seqüenciamento) e Fenotipagem (analisado pelo Índice Metabólico) em 75 voluntários sadios da região de Campinas. Para a caracterização da Fenotipagem foi usada a substância teste Dextrometorfano (DM). Esta foi monitorada por espectrometria de massa mediante a determinação da concentração do seu principal metabólito o Dextrorfano (DX), que foi extraída das amostras de urina. Os resultados foram comparados entre estas duas metodologias e apresentaram alta correlação. Os resultados obtidos são a identificação das freqüências dos alelos *1, *3 e *4 pelo método PCR Tetra Primer (30.66%, 1.3% e 14%, respectivamente). O método de seqüenciamento detectou também outros alelos que não foram detectados pela PCR Tetra Primer. A avaliação do número de cópias do gene CYP2D6 também foi avaliada, detectando em um voluntário 3 cópias do gene CYP2D6, característica de metabolizadores Ultra-rápidos. Podemos afirmar que os métodos usados forneceram perfis dos polimorfismos de maneira rápida e prática. / The CYP450 enzymes are the major drug metabolizing enzymes. They are encoded by genes that show genetic polymorphisms which gives them several phenotypic characteristics. These phenotypes are Poor Metabolizers or PM, or Extensive Metabolizers or EM, Intermediate Metabolizers or IM, and finally, Ultra-rapid Metabolizers or UM. Pharmacogenetics is the science that studies these genetic variations and its relationship to therapeutic response in the body. One of CYP450 enzymes is CYP2D6 enzyme, which are responsible for the metabolism of 25% of clinically prescribed drugs. The main objective of this study was to identify the most important polymorphisms of this gene: CYP2D6 * 1, CYP2D6 * 2, CYP2D6 * 3, CYP2D6 * 4, CYP2D6 * 5, CYP2D6 * 6, CYP2D6 * 10, CYP2D6 * 17 and CYP2D6 * 41 by genotyping methods (PCR Tetra Primer and Sequencing) and phenotyping (by metabolic rate monitoring) in 75 healthy volunteers in Campinas region.To characterize the phenotyping was used to test substance Dextromethorphan (DM). This was monitored by mass spectrometry by determining the concentration of its major metabolite the Dextrorphan (DX), which was extracted from urine samples. The results were compared between these two methods and showed high correlation. We can obtain the identification of allelic frequencies of alleles * 1, * 3 and * 4 by Tetra Primer PCR (30.66%, 1.3% and 14% respectively). The sequencing method has also detected other alleles that were not detected by PCR Tetra Primer. The assessment of the number of copies of the CYP2D6 gene was also assessed. This method detected a volunteer which carrying three copies of CYP2D6 gene, characteristic of Ultra-rapid metabolizers. We can say that the methods used in this study provide polymorphism profiles quickly and conveniently.

Lee, Chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 124-145). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 槪要 --- p.v / Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of pharmacogenetics --- p.2 / Chapter 1.2 --- Clinical importance of nitrogen oxidations --- p.8 / Chapter 1.3 --- Characteristics of trimethylamine (TMA) --- p.9 / Chapter 1.4 --- Genetic polymorphism of trimethylamine N-oxidation --- p.12 / Chapter 1.5 --- Enzyme systems mediating trimethylamine N-oxidation --- p.15 / Chapter 1.6 --- Aims and objectives --- p.19 / Chapter Chapter 2 --- Development of an Analytical Method for Trimethylamine & Trimethylamine N-Oxide --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials --- p.26 / Chapter 2.3 --- Methods --- p.27 / Chapter 2.3.1 --- Preparation of stock solutions --- p.27 / Chapter 2.3.2 --- Preparation of GLC glass column --- p.28 / Chapter 2.3.3 --- Optimizing GLC conditions for the separation of trimethylamine --- p.29 / Chapter 2.3.4 --- Sample pretreatment for GLC analysis --- p.29 / Chapter 2.3.5 --- Construction of calibration curve --- p.31 / Chapter 2.3.6 --- Determination of the required amounts of titanium (III) chloride for trimethylamine N-oxide reduction --- p.31 / Chapter 2.3.7 --- Intra- and inter-assay variations --- p.31 / Chapter 2.3.8 --- "Equations used in the determinations of free TMA, total TMA, and percentage TMA excreted as TMAO" --- p.32 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- GLC chromatogram --- p.33 / Chapter 2.3.2 --- Construction of calibration curve --- p.33 / Chapter 2.3.3 --- Determination of the required amounts of titanium (III) chloride for trimethylamine N-oxide reduction --- p.33 / Chapter 2.3.4 --- Intra- and inter-assay variations --- p.37 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter 3 --- Trimethylamine N-Oxidation in Chinese --- p.41 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Experimental protocols --- p.43 / Chapter 3.2.1 --- Whole day urine collections --- p.43 / Chapter 3.2.2 --- Spot urine collections --- p.44 / Chapter 3.3 --- Results --- p.44 / Chapter 3.3.1 --- Whole day urine collections --- p.44 / Chapter 3.3.2 --- Spot urine collections --- p.46 / Chapter 3.3.3 --- Comparison between whole day urine collections and spot urine collections protocols --- p.50 / Chapter 3.3.4 --- Comparison between smokers and non-smokers --- p.52 / Chapter 3.3.5 --- Comparison the results obtained in between four individual periods of whole day urine collections --- p.52 / Chapter 3.4 --- Discussion --- p.56 / Chapter Chapter 4 --- Effect of Age and Diet on Trimethylamine N-oxidation --- p.64 / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.2 --- Experimental protocols --- p.73 / Chapter 4.2.1 --- Effect of age on TMA N-oxidation --- p.73 / Chapter 4.2.2 --- Effect of diet on TMA N-oxidation --- p.73 / Chapter 4.2.3 --- Effects of control diet on TMA N-oxidation --- p.75 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1 --- Effect of age on TMA N-oxidation --- p.76 / Chapter 4.3.2 --- Effect of diet on TMA N-oxidation --- p.80 / Chapter 4.3.3 --- Effects of control diet on TMA N-oxidation --- p.83 / Chapter 4.4 --- Discussion --- p.89 / Chapter Chapter 5 --- Effect of Disease on Trimethylamine N-Oxidation --- p.101 / Chapter 5.1 --- Introduction --- p.102 / Chapter 5.2 --- Experimental protocols --- p.108 / Chapter 5.3 --- Results --- p.109 / Chapter 5.4 --- Discussion --- p.116 / Chapter Chapter 6 --- General Discussion --- p.119 / References --- p.124 / Appendices / Chapter Appendix A / Chapter A.1 --- Sample record sheet for food intake and activity (English) --- p.A-l / Chapter A.2 --- Sample record sheet for food intake and activity (Chinese) --- p.A-3 / Chapter Appendix B --- Food intake and activity record for volunteer Ain controlled diet experiment --- p.B-1