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Warfarin is one of the most commonly used oral anticoagulants worldwide and is highly efficacious for the treatment and prevention of thromboembolic disorders. However, due to its narrow therapeutic index and large interindividual variability, it remains a challenging drug to prescribe. Genetic factors (CYP2C9 and VKORCI), together with clinical factors (age and body weight), account for up to 60% of warfarin dose variance but the remaining ~40% variability remains unexplained. A polymorphism rs2108622 in CYP4F2, a vitamin K oxidase, has previously been associated with increased warfarin stable dose requirements, accounting for 1-7% dose variability. In our cohort of prospectively recruited patients (n = 311), we were unable to confirm these results. Interestingly, after fine mapping of the CYP4F2 gene region, we found a SNP rs2189784, which is in LD with rs2108622, to be associated with time to therapeutic INR (Pc = 0.03). Further fine mapping of the CYP4F gene cluster together with the utilisation a bank of well characterized Caucasian surgical liver samples (n = 149) and data from a genome-wide association study (n = 714), showed that CYP4F2 rs2108622 and rs2189784 SNPs were found to be associated with increasing CYP4F2 and decreasing CYP4FII or CYP4Fl2 mRNA expression, respectively. Interestingly, a CYP4Fll variant rsl060467 (in LD with rs2108622) was associated with reduced CYP4F2 rnRNA expression. Furthermore, rsl060467 contributes to 2.5% of warfarin dose variability and was associated with reduced warfarin dose requirement (~1 mg/day, Pc = 0.003), an effect in the opposite direction previously reported with CYP4F2 rs2108622 by Caldwell et al. (2008) and other studies. Warfarin-resistant patients have been reported to harbour VKORCI missense mutations. Extended regions of VKORCI were sequenced in our patients (n = 65) with resistance to warfarin, defined by clinical and pharmacodynamic criteria. Seven novel heterozygous mutations were identified and in silica analyses predicted the promoter c.-160G>C mutation creates a putative Spl transcription factor binding site and that the missense mutation c.79C>G to be deleterious. To confirm these predictions, in vitro functional studies were carried out using EMSA, transient transfection assays, and DNA methylation. c.-160G>C was found to create a weak binding site for Spl transcription factor, and caused an increase in promoter activity by ~20% (P = 0.003). The c.79C>G mutation reduced levels of PIVKA-II by ~10%. Associations of VKORCI genotypes with DNA methylation did not remain significant after correction for multiple testing. The effect of warfarin on the rate of decline of vitamin K-dependent clotting factors, and the role of SNPs in the clotting factor genes, is not known. Using a large prospective cohort of patients (n = 619), SNPs in F7 and F 10 genes showed association with variability in factor VII levels. The rate at which the plasma levels of factors II, X and protein C decline affect how patients respond to warfarin, in particular the achievement of warfarin stable dose and time to therapeutic INR. Furthermore, the change in clotting factor X level accounted for 1.4% of warfarin dose variability. In conclusion, the results presented in this thesis demonstrate that multiple genetic, clinical and biochemical factors account for the variability in warfarin response. Further understanding of such complex interactions, along with the advent of genomics technologies and development of new computational and conceptual tools, will yield insights to the accurate prediction of drug efficacy and toxicity, which will hopefully translate into improved outcomes for patients.

Warfarin is the most widely used oral anticoagulant for the prevention and treatment of various cardiovascular diseases. However, its complications represent an important clinical problem. The drug has a very low therapeutic index and there is significant interpatient and inter-iscmer variability in both pharmacokinetics and pharmacodynamics; these are affected by a large number of genetic and non-genetic factors. Genetic variation in CYP2C9 and VKORCl has been most extensively studied in relation to warfarin dose requirements.

Thromboxane A2 (TXA2) and ADP are both major positive feedback mediators of platelet activation and act through G-protein coupled receptors (GPCRs) on the platelet surface. Aspirin and clopidogrel, two of the main drugs used in the prevention of acute coronary syndromes, function by blocking the actions of these two agonists at their receptors. Since aspirin and clopidogrel have been shown to be more effective when administered together rather than alone, the study of potential cross-talk between TXA2 and ADP receptors (TP and P2Y) is important in improving understanding of the combined mechanism of action of these two drugs.

Statins inhibit 3-hydroxy-3-methylglutaryl coeczyme A reductase (HMGCR), the rate- limiting enzyme in cholesterol biosynthesis, and are widely used to treat hypercholesterolaemia, a risk factor for cardiovascular disease. Myopathy (muscle toxicity) is an adverse effect that is thought to occur due to the secondary effects of inhibiting HMGCR; however the mechanism of this myopathy is not fully understood. Therefore, the aim of this study was to use two human cell lines as an in vitro model for comparing the effects of a variety of statins on both cholesterol synthesis and protein prenylation between the liver, the intended target tissue for therapeutic benefit, and skeletal muscle, the major site of toxicity. Initially, the differentiation of RD rhabdomyosarcoma muscle cells was characterised in terms of morphological and molecular changes that occur, using microscopy and TaqMan analysis respectively, and a 5-day cell differentiation period was chosen for use in later experiments. The initial hypothesis to then be tested was that the process of protein prenylation exhibits circadian variation, as has already been shown for cholesterol synthesis, and this might enable temporal separation between the cholesterol-lowering efficacy and the toxicity of statins. However, the ability of the RD cell line to establish and maintain circadian rhythmicity in vitro could not be verified and so the circadian contributions to cholesterol metabolism and protein prenylation in liver and muscle in this model could not be investigated. Using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay, significant, dose-dependent reductions in cell viability were observed in both liver and muscle cells on treatment with each of the statins (P = 0.0041, atorvastatin; P < 0.0001, all other statins, two-way analysis of variance (ANOVA)). However, greater cell toxicity was observed with the more lipophilic statins, simvastatin (acid), lovastatin (acid) and cerivastatin, and also in RD muscle cells compared to Huh7 liver cells. Taken together, these data represent the first comprehensive comparative analysis of statin toxicity in human liver and muscle cell lines. Cholesterol metabolism was then examined in these cells: Although cholesterol was present in all cell types, the levels in RD cells were somewhat lower than in Huh7 cells, probably due to much lower levels iii of HMGCR as measured at both the transcript and protein level; and 48 hour treatment with 10j.JM simvastatin and pravastatin, a~d 1 j.JM cerivastatin, seemed to lead to a decrease in total cellular cholesterol. \ Subsequently, protein was extracted from Huh7 and RD cells treated with simvastatin, cerivastatin or pravastatin, and then subjected to analysis by western blot to detect any changes in the prenylation status of selected small GTPases. Inhibition of prenylation was found by directly analysing changes in the unprenylated form of Rap1A, using a specific antibody, and by examining the reduction in membrane association of H-Ras, M-Ras and Rab1A. Treatment with simvastatin and cerivastatin inhibited the prenylation of Rap1A, and seemed to also affect membrane association of the small GTPases H-Ras, M-Ras and Rab1A. In both muscle and liver cells the inhibition of prenylation of Rap1A, a Ras-related GTPase, was 10-fold more sensitive to treatment with cerivastatin than simvastatin. Inhibition of Rap1A prenylation in RD muscle cells was also 10-fold more sensitive to simvastatin and 3-fold more sensitive to cerivastatin treatment than in liver cells. Hydrophilic pravastatin, which showed the lowest toxicity in MTT assays, did not affect the prenylation of any of the small GTPases examined, as far as was detectable using western blot analysis. In conclusion, this work has shown that RD muscle cells show both greater sensitivity to the toxic effects of statins and preferential disruption of protein prenylation in response to statin treatment when compared to Huh7 liver cells. This further supports a mechanism whereby disrupting production of intermediates in the cholesterol synthesis pathway, including the isoprenoids used in protein prenylation, is a significant cause of statin-induced muscle toxicity. This study also highlights the' importance of examining the effects of drugs at both the target site of action and at the site of toxicity, rather than only studying one site in isolation, in order to gain insight into the mechanism of adverse drug effects. iv

Anticoagulation response to warfarin is unpredictable because of the wide interindividual variability in dose requirement and the drug's narrow therapeutic index. The aim of my PhD project was to ftirther investigate and identify factors that contribute to the variability in warfarin dose requirements. Identification of the factors contributing to the variability could help toward improving the safety of warfarin through personalized therapy.