Background: There are approximately 12 million statin users in the United Kingdom. Reports of statin intolerance occurs between 7 and 29% of users, manifesting as muscle ache, fatigue or more seriously, muscle breakdown leading to myopathy. Creatine phosphokinase (CK) levels are used as a biomarker of statin-induced muscle damage. Non-adherence or discontinuation of therapy is a common result of intolerance and can result in negative cardiovascular disease-related outcomes. Aim: This thesis attempts to identify trends in record-linked medical data in a Scottish Caucasian cohort (GoDARTS) that best represent statin intolerance in order to study associated genetic factors. Methods: Prescribing trends such as switching or discontinuation of statin therapy were examined, and thresholds created to select true cases of intolerance. Information on CK levels was gathered from medical records and appropriate test results were utilized. Genotypic data was gathered for the variants and genetic regions of interest using a variety of methods including chip-based genotyping followed by imputation, TAQMAN genotyping, and exome sequencing. Subsequently hypothesis-based association analyses were conducted, including linear and logistic regressions, followed by meta-analyses, regional GWAS followed by a regional meta –analysis. Results: The phenotypes of statin intolerance were validated both internally and externally. Previously reported missense variants in LILRB5 (Asp247Gly) and CKM (Glu83Gly) were replicated and shown to be associated with CK levels irrespective of statin usage in the GoDARTS cohort and the clinical trial setting (JUPITER). Further, the CKM variant was also associated with inducibility of CK at times of tissue injury. The Asp247 genotype in LILRB5 was associated with increased risk of statin intolerance, and was replicated in associations with non-compliance to statin therapy and the development of myalgia in the JUPITER trial. The association with myalgia showed a stratified effect based on therapy (statin or placebo), with those on placebo showing the genotype effect. Further, the variant was also associated with increased risk of statin-induced myositis, cases of which had been clinically adjudicated and exome sequenced for the PREDICTION-ADR consortium. Further exploration of the LILR gene region showed an association with variants in LILRB2 (His20Arg and Val235Met) which were in strong LD with each other but were not in linkage with the variant in LILRB5. Stratified analysis revealed that the risk for carriers of the LILRB2 variants was increased depending on the genotype carried at the LILRB5 variant. Conclusions: This study characterises novel genetic factors associated with statin intolerance impacting adherence. The findings point to the immunomodulatory effects of statins. The results suggest that true statin-induced myalgia and non-specific myalgia are distinct, with a possible role for the immune system in their development. The findings encourage further investigation into the immune-physiology of statin-induced muscle damage and identifies genetically susceptible groups who are more likely to be statin intolerant.

Familial hypercholesterolemias (FH) are inherited mutations that cause elevated total cholesterol and low-density lipoprotein cholesterol levels (LDL-C) which lead to premature coronary heart diseases. Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect the patient’s response to the drug. Single Nucleotide Morphism (SNP) mutations in the LDLR, apoB, LDRAP1, and PCSK9 genes are linked to familial hypercholesterolemia. The mutations in the LDLR gene are the most common while mutations in the apoB and PCSK9 genes are the least common in hypercholesterolemia patients. My research will study how pharmacogenetics can be used to diagnose and prescribe patients with FH who have apoB and PCSK9 double gene mutations. I will genotype and sequence the PCR amplified gene segments of the patients with FH to identify any of the 6 apoB SNPs and any of the 3 PCSK9 SNPs that are known. Then, I will provide 4 different treatments: placebo, antisense therapy (mipomersen), PCSK9 inhibitor (alirocumab), and a combination of mipomersen + alirocumab, and I will measure the LDL-C levels before and after a 12-week trial. I hypothesize that individuals with both apoB and PCSK9 gene mutations with the known SNPs that cause loss of function will be more responsive when given both treatments by observing a significant decrease in LDL-C levels.

Warfarin is one of the most widely used anticoagulants in the world. Treatment is complicated by a large inter-individual variation in the dose needed to reach adequate levels of anticoagulation i.e. INR 2.0 – 3.0. The objective of this thesis was to evaluate which factors, mainly genetic but also non-genetic, that affect the response to warfarin in terms of required maintenance dose, efficacy and safety with special focus on warfarin dose prediction. Through candidate gene and genome-wide studies, we have shown that the genes CYP2C9 and VKORC1 are the major determinants of warfarin maintenance dose. By combining the SNPs CYP2C9 *2, CYP2C9 *3 and VKORC1 rs9923231 with the clinical factors age, height, weight, ethnicity, amiodarone and use of inducers (carbamazepine, phenytoin or rifampicin) into a prediction model (the IWPC model) we can explain 43 % to 51 % of the variation in warfarin maintenance dose. Patients requiring doses < 29 mg/week and doses ≥ 49 mg/week benefitted the most from pharmacogenetic dosing. Further, we have shown that the difference across ethnicities in percent variance explained by VKORC1 was largely accounted for by the allele frequency of rs9923231. Other novel genes affecting maintenance dose (NEDD4 and DDHD1), as well as the replicated CYP4F2 gene, have small effects on dose predictions and are not likely to be cost-effective, unless inexpensive genotyping is available. Three types of prediction models for warfarin dosing exist: maintenance dose models, loading dose models and dose revision models. The combination of these three models is currently being used in the warfarin treatment arm of the European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) study. Other clinical trials aiming to prove the clinical validity and utility of pharmacogenetic dosing are also underway. The future of pharmacogenetic warfarin dosing relies on results from these ongoing studies, the availability of inexpensive genotyping and the cost-effectiveness of pharmacogenetic driven warfarin dosing compared with new oral anticoagulant drugs.

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. Polymorphisms in serotonin 2A and 2C receptor coding genes (HTR2A and HTR2C) 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 HTR2C 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 CYP1A2 but not with CYP2D6 polymorphisms. Furthermore, HTR2C and HTR2A 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 (ABCB1) in addition to CYP2D6 on the steady state plasma levels of risperidone, 9-hydroxyrisperidone and their active moiety revealed a significant influence of ABCB1 genotype on 9-hydroxyrisperidone and active moiety C/Ds, while CYP2D6 genotype associated with risperidone C/Ds but not with 9-hydroxyrisperidone or active moiety C/D. 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 HTR2A, HTR2C, CYP1A2, CYP2D6 and ABCB1 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.

Schizophrenia (SCZ) is a severe neuropsychiatric disorder with a genetic component. Most candidate gene association studies have given mixed results. We investigated the GABAA receptor gamma2 subunit gene GABRG2, the dopamine receptor gene DRD3, and the Brain-derived neurotrophic factor gene BDNF that is required for D3 expression by genotyping polymorphisms spanning and surrounding these genes for association with SCZ, as well as suicidal behaviour. We also examined the BDNF, DRD3, as well as the dopamine receptor gene DRD2 and Protein Kinase B gene AKT1 for association with Tardive Dyskinesia (TD), a potentially irreversible motor side effect of long-term antipsychotic medication. Our analysis included single-marker tests, haplotype tests, and gene-gene interactions. We found a haplotype in the 5’ region of GABRG2 to be associated with SCZ in both families and matched case-control samples. We also found two synonymous DRD2 polymorphisms, rs6275 (C939T) and rs6277 (C957T), and their haplotypes, as well as a polymorphism 5’ of DRD3, rs905568, to be associated with TD. Further, we reviewed two putative functional DRD2 polymorphisms, -141C Ins/Del and TaqIA, in TD and found TaqIA 3’ of the gene to be associated with TD in a meta-analysis. Lastly, we found a significant interaction between AKT1 rs3730358 and DRD2 C939T in TD. Though replication studies are required, these results contribute to the future development of genetic tests to assess for the risks of SCZ and TD, leading to better outcome for patients suffering from these debilitating conditions.

Schizophrenia (SCZ) is a severe neuropsychiatric disorder with a genetic component. Most candidate gene association studies have given mixed results. We investigated the GABAA receptor gamma2 subunit gene GABRG2, the dopamine receptor gene DRD3, and the Brain-derived neurotrophic factor gene BDNF that is required for D3 expression by genotyping polymorphisms spanning and surrounding these genes for association with SCZ, as well as suicidal behaviour. We also examined the BDNF, DRD3, as well as the dopamine receptor gene DRD2 and Protein Kinase B gene AKT1 for association with Tardive Dyskinesia (TD), a potentially irreversible motor side effect of long-term antipsychotic medication. Our analysis included single-marker tests, haplotype tests, and gene-gene interactions. We found a haplotype in the 5’ region of GABRG2 to be associated with SCZ in both families and matched case-control samples. We also found two synonymous DRD2 polymorphisms, rs6275 (C939T) and rs6277 (C957T), and their haplotypes, as well as a polymorphism 5’ of DRD3, rs905568, to be associated with TD. Further, we reviewed two putative functional DRD2 polymorphisms, -141C Ins/Del and TaqIA, in TD and found TaqIA 3’ of the gene to be associated with TD in a meta-analysis. Lastly, we found a significant interaction between AKT1 rs3730358 and DRD2 C939T in TD. Though replication studies are required, these results contribute to the future development of genetic tests to assess for the risks of SCZ and TD, leading to better outcome for patients suffering from these debilitating conditions.

Warfarin is one of the most widely used anticoagulants. Therapy is complicated by warfarin’s narrow therapeutic range and pronounced variability in individual dose requirements. Although warfarin therapy is uncommon in children, it is crucial for children with certain congenital or acquired heart diseases. Treatment in children is especially difficult due to the lack of i) a decision support tool for efficient and consistent dose adjustments, and ii) a flexible warfarin formulation for accurate and reproducible dosing. The overall aim of this thesis was to develop a PKPD-based pharmacometric model for warfarin that describes the dose-response relationship over time, and to identify important predictors that influence individual dose requirements both in adults and children. Special emphasis was placed on investigating the contribution of genetic factors to the observed variability. A clinically useful pharmacometric model for warfarin has been developed using NONMEM. The model has been successfully reformulated into a KPD-model that describes the relationship between warfarin dose and INR response, and that is applicable to both adults and children. From a clinical perspective, this is a very important change since it allows the use of information on dose and INR that is available routinely. The model incorporates both patient and clinical characteristics, such as age, weight, CYP2C9 and VKORC1 genotype, and baseline and target INR, for the prediction of an individualised starting dose. It also enables the use of information from previous doses and INR observations to further individualise the dose a posteriori using a Bayesian forecasting method. The NONMEM model has been transferred to a user-friendly, platform independent tool to aid use in clinical practice. The tool can be used for a priori and a posteriori individualisation of warfarin therapy in both adults and children. The tool should ensure consistent dose adjustment practices, and provide more efficient individualisation of warfarin dosing in all patients, irrespective of age, body weight, CYP2C9 or VKORC1 genotype, baseline or target INR. The expected outcome is improved warfarin therapy compared with empirical dosing, with patients achieving a therapeutic and stable INR faster and avoiding high INRs that increase the risk of bleeding.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 75-83).

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmaceutics. Title from title-page of electronic thesis. Bibliography: leaves 289-311.

Highly active antiretroviral therapy (HAART) has improved the life expectancy of HIV–1–infected patients dramatically since it was launched in 1996, but there are still many challenges in the provision of HAART, especially to children in resource limited countries. Efavirenz (EFV), a non–nucleoside reverse transcriptase inhibitor (NNRTI) forms part of the recommended national first line antiretroviral treatment regimen for children older than 3 years and weighing more than 10 kg in South Africa. Limited pharmacokinetic information on EFV plasma concentrations in sub–Saharan HIV–1– infected children is available. EFV is primarily metabolised by hepatic CYP2B6 isoenzymes. The CYP2B6 gene is characterised by extensive inter–individual variability in hepatic expression and activity. The single nucleotide change, 516G>T, on the CYP2B6 gene has consistently been associated with elevated EFV plasma levels in different ethnic populations and these are more frequently observed in populations of African descent. The recommended therapeutic range of EFV plasma levels is 1–4 ug/ml and the Cmin should be above 1 ug/ml. In this prospective study (PK/PD.EFV.07) cohort, 60 black children, both genders, with no prior exposure to antiretroviral therapy and eligible for antiretroviral therapy (ART) were enrolled and followed up at 1, 3, 6, 12, 18 and 24 months post HAART initiation. They all attended the outpatient clinic at Harriet Shezi Children’s Clinic, Chris Hani Baragwanath Hospital, Soweto, South Africa. The required ethics approval was obtained to conduct this study. The objectives of this investigation were to: develop and validate a suitable LCMS/ MS method to accurately determine plasma EFV levels from this study population, determine the prevalence and effect of CYP2B6 516G>T polymorphism on EFV plasma levels, determine the population pharmacokinetic clearance (CL/F) value of EFV, identify covariates that influence the clearance of EFV in HIV–1– infected children, and investigate specific pharmacodynamic effects and therapeutic outcomes of this EFV–based regimen within this paediatric population over the 24 months post–HAART initiation. The main findings of the measured mid–dose EFV plasma concentrations showed that sub–therapeutic concentrations (<1 ug/ml) accounted for 18% (116/649), within therapeutic range (1–4 ug/ml) represented 52.5% (341/649), and concentrations above the therapeutic range (>4 ug/ml) represented 29.5% (192/649). A significant number of the samples (47.5%) were outside the accepted therapeutic range during this 24 month follow–up period. Possible reasons contributing to this include genetic variation in drug metabolism and non–adherence. Genotype results on all 60 study participants were: 23% 516 T/T homozygotes, 42% 516 G/G homozygotes and 35% 516 G/T heterozygotes. The 516 T–allelic variant frequency was relatively high at 41%. This also supports and explains why such a large number (29.5%) of the mid–dose interval plasma samples were above (>4 ug/ml) the accepted therapeutic range. Repeated measures ANOVA confirmed that CYP2B6 516 G/G, G/T and T/T genotypes were consistently predictive of the log EFV concentrations at all times (P = 0.0001). The total median (IQR) EFV plasma concentrations over the 24 months post–HAART when pooled, were 6.36 (3.47 - 7.28) for T/T, 2.55 (1.62 - 3.59) for G/T, and 1.41 (1.02 - 1.74) ug/ml for G/G groups respectively (P<0.00001). Multiple comparisons by groups revealed that the EFV plasma concentrations between the T/T and G/G (P=0.000002) and between G/T and G/G (P=0.009) were statistically significant. However, the differences between the EFV plasma concentrations of the T/T and G/T groups were not significantly different (P=0.074). This supports previous results that the presence of the 516 T–allelic variant is responsible for the higher EFV plasma concentrations within individuals presenting with this single nucleotide mutation on the CYP2B6 gene. This EFV–based treatment was well tolerated even at plasma concentrations above the therapeutic range (>4 ug/ml) and most side effects subsided spontaneously. 89% of the participants were virally suppressed at 24 months post–HAART. The efficacy of this EFV–based treatment did not affect the three genotype groups differently and they showed similar improvement in their immunological (CD4–cell count and CD4%) markers and reduction in viral load over the 24 months post– HAART initiation. We found no association of the CYP2B6 516G>T polymorphism and side effects reported after 1 month of treatment within this study population. The final population pharmacokinetic (PK) estimates for EFV clearance (CL/F) were, 2.46, 4.60, and 7.33 l/h for the T/T, G/T, and G/G respective genotype groups. The volume of distribution (V/F) estimate was 89.52 l. The importance of interoccasion variability (IOV) in a PK model for a longitudinal study was again highlighted by this investigation. To our knowledge, this is the first study in black South African HIV–1–infected children with measured sequential EFV plasma concentrations which also investigated the influence of the CYP2B6 516G>T polymorphism on EFV plasma concentrations and the population clearance (CL/F) value of EFV in a longitudinal study over a period of 24 months post–HAART initiation. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.