Antiretroviral pharmacokinetics is one of the most important predictors of therapy efficacy and correlations between virological suppression and plasma concentrations have been described for all antiretroviral classes. Plasma concentrations are the result of absorption, distribution, metabolism and elimination (AD ME) processes which are mediated by numerous proteins in different tissues. Single nucleotide polymorphisms (SNPs) in genes encoding for individual proteins may be an important determinants drug exposure. The aim of this thesis was to characterise the role of some key SNPs determining antiretroviral plasma concentrations in order to improve the current understanding of dose-dependent efficacy and toxicity. A number of different strategies to investigate the role of genetic variability in antiretroviral exposure were developed in this thesis. The polymorphism 63396 C>T in the PXR gene, a nuclear factor regulating the expression of CYPs and transporter in hepatocytes, was proven to influence unboosted atazanavir clearance using a pharmacogenetic based population phannacokinetic model and patients with 63396TT genotype had a increment in ATV clearance of 17% (Chapter 2). In Chapter 3, the predictors of intracellular phannacokinetic of unboosted and boosted atazanavir were investigated and expression of SLC03Al, an uptake transporter expressed on PBMC was correlated with cellular accumulation of boosted and unboosted atazanavir (rho=0.706, p=0.010; rho = 0.830, P = 0.005, respectively). An in vitro in vivo extrapolation model for efavirenz was developed in Chapter 4 and the effect of CYP2B6 516 G>T on standard regimens or dose reduction was quantified. Efavirenz pharmacokinetics was predicted with good accuracy compared to available data; simulated Ctrough was 2119 ng/ml vs 1764 ng/ml, simulated Cmax was 3725 ng/ml vs 4063 ng/ml. The effect of 516G>T on simulated EFV clearance (GT = - 24% and TT = -58%) was comparable to previously published population phannacokinetic data (GT =-36%, TT =-66%). Maraviroc, a CCR5 inhibitor was identified as a substrate for SLC01B1 with a Km of 33.85 IlM and Vmax of 187.9 finol/oocyte/min and the inhibitory potential of concomitant protease inhibitors on this transporter was investigated using Xenopus laevis oocytes heterologous protein expression system (Chapter 5). Moreover the SNP SLCOIBl 521 T>C was correlated with maraviroc Ctrough in a cohort of HIV infected patients; MVC Ctrough in SLC01B1 521 heterozygote patients (TC) (n=8) was higher than in wild type homozygotes (TT) (n=22), 103 ng/ml (69 - 124) vs 46 ng/ml (28 - 66), p=0.003 (Chapter 6). The convergence of different factors under study permitted partial definition of the complex interplay in which drug-drug interactions and physical characteristics interact with genetic variability to define the phannacokinetic phenotype. These findings will help identify patients with a higher risk of achieving sub-therapeutic or potentially toxic plasma concentrations and will serve as a knowledge-base from which to grow and validate strategies for optimisation of therapy.

Anti-retroviral therapy comprises a combination of three or more drugs from at least two classes. Following long term therapy some patients experience metabolic side- effects including abnormal fat distribution, dyslipidaemia , lipoatrophy, hyperlipidaemia, impaired lipid and glucose metabolism. Metabolic nuclear receptors may be implicated in some of these deleterious side effects. RTV is widely implicated in the deleterious metabolic side effects of ARVs, whilst ATV has been shown to have a better lipid profile than R TV. Previous studies had not determined whether this was due to differences in intra-cellular accumulation of the two drugs. Cellular accumulation of ATV was determined to be 2 fold greater than that of RTV in the hepatic cell line, Huh7. This confirmed that the metabolic disruption and the deleterious side effects associated with RTV were not due to its higher intracellular accumulation. LXRα was significantly up-regulated by RTV, and it was determined to be a regulator of key downstream target gene expression, including SREBP-lc, PPARα, ApoA- I, VLDLR, ApoC-II and ApoC-III. Inhibition of LXRa by 22(S)-hydroxycholesterol and by siRNA knockdown ofLXRa was used to confirm these findings. Biological knockdown of LXRα by siRNA resulted in down-regulation of LXRα gene expression in the presence of RTV, which could provide a potential future therapeutic mechanism for inhibition of LXRa in vivo. LXRα was involved in the regulation of gene expression of bile acid and cholesterol transporters including: ABCAl, ABCB2, ABCGl, ABCG4, ABCG5, ABCG8 and the glucose transporter GLUT4, which were up-regulated by RTV. LXRa was involved in the regulation of ABCB 11 and ABCC2 but data suggested that LXRα was not the master regulator. Gene expression of these metabolic transporters differs in response to R TV and ATV. The actions of R TV can be blocked through modulation of LXRa gene expression. LXRα was also involved with the regulation of ABCB 1 and ABCC 1 gene expression. A polymorphism in LXRα, rs2279238C/T, could influence the risk of HADL development in Caucasian HIV+ populations. The results indicate further investigation would be required in a larger HIV cohort to substantiate this association. Elevated LDL-c levels were observed in HIV+ Caucasians homozygous GG genotype for the ApoB rs7575840GT SNP and with at least one copy of the A allele for the ABCAl rs2230806G/A SNP. The C allele in the LDLR rs1529729C/T SNP was associated with elevated total cholesterol, LDL-c and TG levels. Elevated total cholesterol and increased LDL-c levels were observed when this SNP was combined with the GG genotype for ApoB rs7575840GT and the C allele for the LDLR SNP rs2228671C/T. Decreased HDL-c was observed in HIV+ patients with the T allele of the SCARB 1 SNP rs2278986C/T when associated with a combination of demographic factors and the following SNPs: ABCAl rs2230806G/A, A allele; LDLR rs1529729C/T, C allele; ABCGl rs1893590AlC, C allele; ABCG8 rs6544713C/T, T allele; and ApoB rs693A/G, G allele. In summary the data in this thesis indicates LXRa is a regulator of metabolic target genes involved in cholesterol homeostasis, bile acid metabolism and the response to RTV in a hepatic cell line. The role of RTV as an agonist of LXRα was confirmed. The contrasting lipid profiles ofRTV and ATV are not due to higher intracellular concentration of the former. This thesis has also identified a potential therapeutic use for LXRα modulation. SNPs in LXRa and key cholesterol transporters have correlated with metabolic complications in HIV+ cohorts which merit further investigation in larger patient cohorts to validate the associations.

There is interest in developing a vaccine to prevent heterosexual transmission of HIV. Currently research focuses on delivering candidate vaccine in simple aqueous gels vaginally. In this thesis a range of polymeric gels were developed and formulated into freeze-dried tablets (FDTs) to improve both retention and stability of gel formulations. The characterisation techniques used included differential scanning calorimetry, thermogravimetric analysis, rheological and textural profile analysis, drug release and efficacy in animal models. A universal placebo gel was manufactured for the vaginal administration of a candidate HIV vaccine to animals. The results showed induction of HIV specific antibodies, but the effect was not sustained. Then, single polymeric FOTs made from Noveon, hydroxypropylmethylcellulose and hydroxyethylcellulose (HEC) were formulated to obtain sustained drug release. Noveon FDTs showed the most sustained drug release. A second animal study was conducted using another candidate HIV vaccine in gelatin and mannitol FOTs designed to achieve immediate and intermediate drug release via the sublingual route. The results showed inability of the antigen to induce protective antibodies against the HIV-l virus. Next a range of FOTs using combinations of three polymers including each of the mucoadhesive polymers (Noveon, chitosan, sodium carboxymethylcellulose-NaCMC), plus HEC and polyvinylpyrrolidone-PVP were developed to improve sustained drug delivery. Results revealed that the RSV formulations showed more sustained drug release profile than the universal placebo gel. The rheological and textural properties of the formulations were determined. Noveon and chitosan formulations were selected as best formulation for vaginal drug delivery. A pilot stability study of these two formulations revealed BSA instability over the 9-week study period. Although a stable vaccine was not achieved, this work shows the potential for gel-based FD tablets to become widely used in the sustained delivery of HI V microbicides and vaccines vaginally in place of aqueous gels.

Human immunodeficiency virus (HIV) is a worldwide problem, with more than 34 million people infected with HIV/AIDS in 2011. At the end of 2011, in Nigeria, an estimated 3.7% of the adult population were living with HIV/AIDS. HIV services in Nigeria are secondary-care led, involving multidisciplinary teams and access to free antiretroviral. However, evaluations of service provision from both patient and healthcare professional perspectives, especially, pharmacists in Nigeria have never been conducted, and are the aims of this research. This study involved grounded theory methodology, using In-depth semi-structured interviews with adults infected with HIV, pharmacists, and administrators involved in the management and care of those patients at Maitama District Hospital in Abuja. HIV pharmacists working for the NHS in the UK were interviewed for comparative purposes. Thirty-five patients were interviewed. Five concepts were identified that influenced how they accessed hospital services after diagnosis. These include faith in God and antiretroviral, social issues with emphasis on HIV stigma and discrimination, patient journeys at the hospital with delays and repeat visits, obstacles such as ARV unavailability and their expectations. Also, five concepts were identified from the pharmacists’ interviews which include clinical service, impressions of service provided, social issues the patients encountered, the obstacles faced with clinical service provided and expectations for improvement. Ten patients were shadowed on their clinic days to observe the patient journey articulated. Furthermore, the administrators interviewed re-affirmed the opinions of the patients and pharmacists about many patients attending HIV clinic, few staff attending to patients, medicines unavailability, especially ARV drugs, and lack of working space for staff. Delays, few pharmacists/many patients and shortage of ART as barriers to service provision ii emerged as dominant themes across the three groups of interviewees in Nigeria. Also, it has been found that there is a wide gap between HIV patients’ hospital management in the UK and Nigeria as regards availability of antiretroviral, staff strength, number of patients in attendance on clinic days and weekly clinic days. Pharmacy was found to have a substantial role in the management of HIV/AID patients but it appears from this study that service improvements, both human and material resources are needed. Twenty three recommendations, which are further synthesised into six potential areas, are made, which, if implemented, would dramatically improve the service provision for HIV/AIDS patients at Maitama District Hospital.