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My opening argument is that Romantic aesthetics is grounded in a metaphysics for which epic is a fitting form. This metaphysics presents an ontological picture that endorses a substance monism that either claims mind to be this substance or includes mind as part of some neutral substance. I then argue for what I call 'metaphysical Hegelianism about epic', which is the view that epic's 'inner form', to borrow Georg Lukács's term, is characterised by concern with the limitation of the freedom of individuals by objective reality. Epic is characteristically concerned with two types of limitation particularly: limitation of the will, and limitation of freedom by the presence of other minds. The first of these leads to artistic consideration of the kinds of experience associated with apprehension of questions surrounding free will and determinism. The second leads to artistic consideration of the place of the individual agent in relation to the community. To show the epic's preoccupation with mind-emphasising ontologies and normative models of agency, examples are given from the four main epics I discuss: The Prelude (1798-9, 1805, and 1850 texts), Sordello (first published 1840), Aurora Leigh (first published 1856), and Leaves of Grass (first published 1855). I then ask why epic consideration of these problems can be valuable. With reference to Romantic epistemology, I argue that art can provide knowledge that goes beyond what is possible for propositionally-based knowledge. Consequently, artworks must be experienced for the kinds of unparaphrasable knowledge they contain to be accessed. I suggest that a central function of the scholarship and criticism of art is to assist the experiencer in this experiencing. Finally, I discuss two different ways in which the word 'epic' is sometimes used: to describe the epic form; and to describe the experience of individual works in the epic form that satisfy certain conditions.

End stage liver disease represents a common end point for a number of disease processes, the only current treatment for which is liver transplantation, but the demand for donor organs exceeds the supply. This has led to the use of more marginal donors with an increase in the rates of complications. Mesenchymal stem/stromal cells (MSC) are a multipotent cell capable of modulating the immune system through a number of different processes and represent a potential therapy in post transplantation liver injury. In this study I describe the prospective isolation and culture expansion of murine MSC. In in vitro assays MSC suppressed T lymphocytes and following stimulus with inflammatory cytokines MSC secreted a number of cytokines including Il-10. In the MDR2-/- model intravenous MSC therapy led to a reduction in liver injury with an increase in restorative macrophages. Subcutaneous administration of MSC showed no beneficial effect. MSC were also tested in a hepatic ischaemia reperfusion injury model where no effect was seen. In summary MSC were able to suppress lymphocyte proliferation and secrete anti-inflammatory cytokines in vitro, and in vivo they were able to reduce liver injury in the MDR2-/- model but not the hepatic ischaemia reperfusion injury mode.

Epilepsy is the most common serious neurological disorder. At least 80% of patients with epilepsy can be successfully treated. The other 20% are said to be treatment-refractory and continue to have seizures despite multidrug therapy. One possible explanation for this is an inadequate permeation across the blood brain barrier (BBB). Thirteen specimens of temporal lobe were obtained and sections prepared prior to immunohistochemical examination of P-gp expression. In these patients, all sections showed expression of P-gp in the endothelial cells of blood capillaries/vessels. In addition, 12 out of the 13 sections also showed staining in astrocyte foot processes in which previous studies had failed to localise P-gp. The ability of P-gp to transport carbamazepine (CBZ) was then assessed. Control and mdrla/1b (-/-) mice were given i.p. doses of CBZ in order to establish any differences in the brain concentrations of this drug. No significant difference were seen (192.67 ± 149.85ng g⁻¹ and 389.33 ± 333.16ng g⁻¹ respectively) indicating lack of efflux by murine P-gps. Furthermore, movement across a Caco-2 cell monolayer indicated that CBZ was not actively transported in this model (ratio of Papp_BtoA to Papp_AtoB was 0.78 ± 0.18) and the drug failed to compete for efflux with the fluorescent P-gp substrate rhodamine 123 (median fluorescence following efflux period were 7 ± 1.8 for CBZ and 10.2 ± 5.5 for controls). The ability of CBZ to mediate induction of P-gp was then examined. Flow cytometry, Western blot analysis and immunohistochemistry all revealed an increase in P-gp expression in ex-vivo lymphocytes exposed to therapeutic concentrations of CBZ (Median fluorescence of antibody 2.0 ± 0.7 for control and 14.5 ± 5.0 for CBZ after 72h; 2P < 0.01). This increase was preceded by an increase in ABCB1 mRNA (6082.8 ± 3230.1 copies per reaction for control and 17743.1 ± 6737.2 for CBZ after 6h; 2P < 0.05). The mechanism of this induction was then assessed by electrophoretic mobility shift assays that indicated the increases in P-gp were associated with increased binding of the pregnane X receptor (PXR) to a consensus DR-4/RORE sequence in the regulatory sequence of the ABCB1 gene (optical density of 260990 ± 61456 for control and 433461 ± 185633 for CBZ after 1h; 2P < 0.05). Correlations between phenotype and genotype were made in human liver. P-gp expression was assessed by Western blot analysis and real time RT-PCR. Samples were genotyped for the two SNPs C3435T and G2677T(A). No correlation was observed between the genotypes and P-gp expression (P-gp/actin ratios: TT at 3435 0.28 ± 0.17, CC at 3435 = 0.2 ± 0.05; GG at 2677 = 0.22 ± 0.12; TT at 2677 = 0.22 ± 0.13) indicating that these SNPs must not be important for expression of this transporter in liver. Finally, C3435T, was examined in a cohort of epileptic patients. The CC genotype was significantly increased in patients that had undergone surgery for epilepsy (x² = 6.8; D.F. = 2; P < 0.05). However, there was no significant correlation between surgery and seizure control, or an increased frequency in those patients on multidrug therapy and so this SNP is unlikely to be a valuable prognostic indicator for drug resistance in epilepsy. In summary, there is an abnormal expression of P-gp in the BBB of refractory epileptics, which is unlikely to impact on the brain concentration of CBZ and it is likely that this increase is, at least in part, a function of exposure to the commonly used antiepileptic drugs. C3435T had no impact on the level of expression of P-gp in human liver, providing further evidence for the tissue specific nature of the correlation between genotype and expression. Finally, it is unlikely that the frequency of the C3435T SNP will be a valuable prognostic marker in these patients.

Antiretroviral therapy is challenging due to the drug-drug interactions (DDIs) arising from the co-medications administered to treat co-morbidities and opportunistic infections. Drugs causing induction/inhibition of enzymes and transporters, or displacement from plasma proteins can alter drug pharmacokinetics (PK) and result in treatment failure due to elevated toxicities or suboptimal efficacy. Furthermore, inter-individual differences conferred by genetic variability responsible for modulating antiretroviral PK complicates treatment. Favourable PK is of paramount importance for successful treatment of any disease. Excipients are frequently used in drug formulations. These excipients not only transform the PK of drugs, but also exert biological effects in the body. These properties may be exploitable to modify PK for optimal outcomes. Evidently, investigations into the pharmacological properties of drugs and excipients are imperative for designing rational formulations and therapies to treat HIV. Knowledge about the effect of genetic polymorphisms on drugs will aid in vigilance of disparate outcomes and help personalising treatment. The NEAT 001 / ANRS 143 was conducted to analyse the efficacy of a novel NRTI- sparing dual combination of darunavir/ritonavir and raltegravir against a standard-of- care triple therapy of darunavir/ritonavir and tenofovir/emtricitabine in HIV-infected antiretroviral naïve subjects. The single nucleotide polymorphisms (SNPs) that have known to have a clinical effect in previous literature were chosen and analysed for their influence on the drugs administered in this study, described in Chapter 2 and 3. In Chapter 4, the plasma protein binding and displacement of protease inhibitors - darunavir, atazanavir, lopinavir and ritonavir - were studied using Rapid Equilibrium Dialysis for their putative implications for DDIs. In Chapter 5, the effects of 25 commonly used excipients were analysed for their effects on P-glycoprotein (P-gp) by assessing the change in the cellular accumulation of a P-gp substrate digoxin in cells over-expressing P-gp. There is little data on the transport of the anti-tuberculosis agent linezolid which is frequently co-administered with antiretroviral drugs. Linezolid was assessed for transport by P-gp, MRP and the BCRP in Chapter 6. Darunavir and ritonavir plasma levels were significantly lower in patients receiving raltegravir compared to those receiving tenofovir/emtricitabine, suggesting DDIs. SLCO1B1 521T > C (rs4149056) and SLCO3A1 G > A (rs4294800) were associated with higher darunavir and ritonavir plasma concentrations, respectively. It was seen that the protease inhibitors bind to both AAG and albumin, suggesting a compensatory mechanism between the plasma proteins. P-gp inhibition by excipients was demonstrated by the following order of effects: Cremophor EL > Vit-E-PEG > Brij 58 > Tween 80 > NaCMC > Tween 20 > CTAB > Solutol HS 15 > AOT. Linezolid was found to be a substrate for P-gp, MRP and the BCRP and the results suggested involvement of additional transporters in linezolid disposition. These findings not only corroborate previously published findings, but also presents novel findings that throw light on the mechanism of genetic variability, plasma protein displacement and induction/inhibition of transporters giving rise to DDIs.

The WHO currently recommends tenofovir (TFV) disoproxil fumarate (TDF) or TFV alafenamide (TAF) as a preferred first-line antiretroviral therapy (ART) for the treatment and prevention of HIV. However, mild to moderate TFV-related renal toxicity is an uncommon but significant complication for TDF therapy. Efavirenz (EFV) is also approved for first line treatment, with the approval of a lower dose treatment, 600mg to 400mg, offering an opportunity for a greater number of patients to receive ART for the same amount of current public health expenditure, whilst enabling the maintenance of viral load suppression and partial mitigation of adverse events. However, EFV displays broad inter-patient and inter-population variability in its pharmacokinetics and displays a range of dose dependent adverse effects. In order to gain further understanding of the impact patient genetics has on response to ART, studies investigating the effect of single nucleotide polymorphisms (SNPs) within multi ethnic populations receiving either TFV or EFV were completed. Additional work was conducted to investigate potential drug-substrate interactions at the transporter level within proximal tubule cells (PTCs), and the ramifications this may have for diagnosis of renal impairment. This work was conducted through assessing the relationship between SNPs found within genes encoding transporters on the apical membrane of PTCs and a diagnosis of renal dysfunction, defined as kidney tubular dysfunction or chronic kidney disease within a Ghanaian population (Chapter 2). Additionally the association between pharmacogenetic variants linked to TDF metabolism or TFV excretion and TFV plasma and urine concentrations, in a majority Caucasian patient population, receiving TFV as part of their ART was investigated (Chapter 3). This thesis sought to assess whether SNPs in genes involved in EFV and nevirapine metabolism were linked to previously observed changes in the contraceptive subdermal implant levonorgestrels (LNG) pharmacokinetics, when prescribed alongside EFV, 19 within a Ugandan population (Chapter 6). In order to study the effect of transporter interactions at the site of TFV toxicity, the proximal tubule, a transiently transfected Human Embryonic Kidney 293 cell line overexpressing either multidrug and toxin extrusion protein (MATE) 1 or MATE2K was developed and assessed for correct functionality (Chapter 4). This cell line was then utilised for drug-substrate interaction studies between TAF and TFV and either the endogenous biomarker creatinine or the type 2 diabetes drug metformin (Chapter 5). These studies provided a greater understanding of the contribution pharmacogenetics provides to the interaction observed between EFV and LNG, and outlined a genetic association between TFV transporter SNPs and TFV plasma and urine concentrations. Utilisation of these findings in future pharmacogenetics studies would aid in the understanding of the impact of genetic variants in different populations, and the consequences this has for achieving sustained virological response to ART. The study of the effect of TAF and TFV on MATE1 and MATE2K transport of metformin and creatinine produced novel data on these interactions. Overall, the studies included within this thesis have clinical impact through further elucidating the potential mechanisms of toxicity or treatment failure within patients receiving ART.

The first-order degradation rate constant (kdeg) of drug metabolising enzymes (DMEs) is a known source of uncertainty in the prediction of time-dependent drug-drug interactions (DDIs) in physiologically-based pharmacokinetic (PBPK) modelling. There is a large disparity or paucity of published kdeg and related half-life (t1/2) values for DMEs. Physiologically-relevant kdeg values should ideally be derived in vivo to facilitate accurate DDI predictions. However, direct measurement of DME degradation in humans is not routinely possible and indirect measurements utilising changes in levels of specific endogenous substrates have only been described for a few DMEs. This thesis aims to develop an in vitro method of measuring DME protein degradation rates to improve the prediction accuracy of time-dependent DDIs. One in vitro approach of measuring protein degradation rates involves inhibiting de novo protein synthesis, followed by tracking residual protein or activity decline over time. Pharmacological protein synthesis inhibitor agents are commonly used for this purpose but may cause cytotoxicity. Four commonly used inhibitor agents were assessed for their capacity to inhibit protein synthesis without overt cytotoxicity. However, all selected compounds were too cytotoxic for subsequent use in kdeg studies. Small-interfering ribose nucleic acid (siRNA) can be added to in vitro systems to initiate gene-specific silencing by inhibiting messenger RNA (mRNA) translation. It was hypothesised that siRNA would inhibit de novo protein synthesis with less cytotoxicity owing to its specificity. CYP3A4 is the most widely studied cytochrome P450 (CYP) enzyme in terms of DDIs because of its well-recognised role in xenobiotic metabolism. Primary human hepatocytes were treated with CYP3A4-specific siRNA to suppress mRNA translation, followed by the tracking of enzyme activity and protein loss over time to derive kdeg. CYP3A4 kdeg was calculated at 0.019 (± 0.044) and 0.020 (± 0.0003) h-1 from protein and activity loss, respectively. These values were in good agreement with existing published values. The siRNA approach was subsequently applied to determine CYP2B6 kdeg. The CYP2B6 kdeg values derived from siRNA-treated hepatocytes were 0.081 (± 0.009) h-1 from protein loss and 0.058 (± 0.010) and 0.062 (± 0.006) h-1 from activity loss, which were assessed by different methods. The CYP2B6 kdeg values derived from untreated hepatocytes were similar to values in literature. This novel approach can now be used for other less well-characterised DMEs that are associated with time-dependent DDIs. Cellular protein abundance is a balance between synthesis and degradation. Dysregulation of the lysosomal or proteasomal protein degradation mechanisms affects steady-state protein levels and impacts on overall cellular functions. It was hypothesised that single nucleotide polymorphisms (SNPs) in the CYP3A4 protein degradation machinery could affect CYP3A4 protein abundance and downstream activity. Five SNPs were investigated for associations with plasma atazanair (ATV) concentrations, which was a surrogate measure for CYP3A4 activity. No associations were found and this was likely due to the lack of clear understanding of the specific mechanisms that commits CYP proteins for degradation. Further work in this field will identify targets that may be exploited in the future for more accurate measurements of DME kdeg. The data presented in this thesis enhances the understanding of methods used to study protein degradation and this can be applied to multiple fields of cellular research. Importantly, work herein has generated a novel approach to measuring kdeg of proteins that can be applied to other less-well characterised enzymes for better prediction of time-dependent DDIs.

The work in my thesis focuses on developing highly sensitive tests, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the measurement of antiretroviral drugs within plasma, cells, and cerebral spinal fluid from clinical studies evaluating compartmentalised antiretroviral therapy (ART) pharmacokinetics and forgiveness for missed or late dosing. Firstly I developed and validated a LC-MS/MS assay to quantify the antiretroviral (ARV) drugs, lopinavir, darunavir, ritonavir and raltegravir in peripheral blood mononuclear cells (PBMCs). This intracellular assay included optimising methodology for cell separation to minimise loss of drug (Chapter 4). All drugs eluted within an 8 minute run time. Matrix effects were minimal (-2.9%). Calibration curves were validated over a concentration range of 0.4-150ng/mL. Intra and inter assay variation ranged between 0.01-2.29% for precision and 96.76-102.32% for accuracy. Secondly I developed and validated a LC-MS/MS assay to simultaneously detect and quantify 10 ARVs; maraviroc (MVC), nevirapine (NVP), rilpivirine (RPV), raltegravir (RAL), atazanavir (ATV), darunavir (DRV), amprenavir (APV), ritonavir (RTV), lopinavir (LPV) and etravirine (ETV) in cerebral spinal fluid (CSF). All drugs eluted within a 10 minute run time. Calibration curves were validated over the following concentration ranges; LPV, MVC, RTV = 0.78-100 ng/mL, RAL, APV, ATV, RPV, ETV, DRV = 1.95-250 ng/mL and NVP = 19.5-2500 ng/mL (r2 values >0.99; quadratic 1/x). Intra and inter assay variation ranged between 1.59-15% for precision and -10.5-6.4% for accuracy. Carryover was <20% of the lower limit of quantification for all drugs. The recovery was >70% and the CV% at low, medium and high concentrations was less than 20% for all drugs. Thirdly I developed and validated a LC-MS/MS assay to quantify intracellular tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) within PBMCs. This work was very technically challenging and something that was not being done by any other laboratory within the United Kingdom. Analytes eluted within 12 minutes run-time with adequate separation. Calibration curves were validated over the following range TFV-DP=0.35-10.91 ng/mL, FTC-TP=0.38-103.17 ng/mL (r2 values >0.99; linear 1/x). The lower limit of quantification was <20 %, signal to noise was >5% and carryover <0.1%. The precision was; TFV-DP=6.3-11% and FTC-TP=6-18.6%, and accuracy was TFV-DP=97.5-100.8%, FTC-TP=98-100.3%. Finally, all assays developed and validated were successfully applied to collaborative clinical trials (see Communications; Published Research Papers). Benefits are expected to accrue from this work in the design of more forgiving therapy regimens for HIV patients, and better drug selection to specifically target HIV replicating within sanctuary sites.

Initial predictions of drug response and drug-drug interactions (DDIs) are made following high-throughput in vitro screening. Such assays are indispensible in the pharmaceutical industry to determine the metabolism, transport and pharmacokinetics of new chemical entities. However, they often fail when extrapolated to in vivo response due to unsuitable pharmacokinetic or pharmacodynamic prediction. The primary aim of this thesis was to investigate and understand the differences in the expression profiles of drug disposition genes, between transformed hepatic cell lines and primary human hepatocytes. Primary human hepatocytes were also analysed to determine uptake contribution, induction and genotype of key drug disposition-relevant genes. The loss of hepatic phenotype in HepG2 and Huh7 cells is partly due to the altered expression of transcriptional regulators including; chaperones, co-chaperones, co-activators and co-repressors. Indeed, Chapter 2 of this thesis shows lower levels of the Gadd45β and PGC1α gene expression in HepG2 cells corresponds to a deficient expression and activity of cytochrome P450 3A4 (CYP3A4), with the levels reducing further as cell passage increases, in comparison to primary human hepatocytes. HepG2 cells were transfected with a novel complex transfection of Gadd45β and PGC1α with the aim to improve CYP3A4 activity in Chapter 3. CYP3A4 activity was improved by 54% and induction response was enhanced in comparison to control cells with no off-target effects. Over the last decade it has become apparent that transporters can play a significant role in the disposition of many drugs. Organic anion transporting polypeptide (OATP) transporters have received considerable recent attention since they mediate sodium-independent uptake of a broad array of xenobiotics. A method to determine the specific contribution of OATP1B1 in the hepatic uptake was successfully optimised and applied for 5 therapeutic drugs in Chapter 4. Future application of this strategy is likely to have broad importance in determining relative contribution that individual transporters play in drug disposition. To prevent accumulation and toxicity of xenobiotics, biotransformation and transport of foreign compounds occurs. However, these processes can be altered by induction or inhibition mechanisms. Rifampicin is a first line drug in tuberculosis (TB) treatment but it is a potent inducer of CYPs and transporters. DDIs during TB treatment are common but the induction potential of different rifamycins has not been comprehensively ranked. Chapter 5 investigated the induction potential of rifampicin, rifapentine and rifabutin. Rifampicin significantly induced CYP3A4, ABCB1, OATP1B1 and ABCC2 in primary human hepatocytes. Induction by rifabutin was observed for CYP3A4, OATP1B3 whilst rifapentine only significantly induced OATP1B1. This work serves as a basis for further study into the extent to which rifamycins induce key metabolism and transporter genes. Nuclear receptors (NR) regulate the expression of CYPs and drug transporters influencing pharmacokinetics. PXR and VDR have been found to synergistically increase CYP3A4 expression and activity in intestinal cell lines. This effect has been observed in vivo with seasonal variations apparent for CYP3A4 substrates. In Chapter 6, novel associations between vitamin D receptor polymorphisms and expression of it and its target genes involved in drug disposition were shown in D2 intestinal biopsies. This thesis reports generation of model systems and their application to enable many questions to be answered relating to pharmacokinetics and DDIs. The thesis forms a solid platform from which to further investigate these issues in future studies.

The failure of highly active antiretroviral therapy may be due to pharmacological factors such as drug transporters and metabolism enzymes. Drug transporters and metabolism enzymes played complementary roles in drug absorption, distribution, metabolism and excretion by biotransformation and counter-transport, particularly in the intestine while nuclear receptors as transcription factors regulate the expression of drug transporters and metabolism enzymes. In this thesis, a positive correlation between nuclear receptors expression and the expression of ABC transporters and OATP transporters in intestine were observed while a negative correlation was found between the gene expression of nuclear receptors and cytochrome P450 enzymes in intestine. Single nucleotide polymorphisms in genes could potentially impact on gene expression of drug transporters and metabolism enzymes. The polymorphisms of nuclear receptors were associated with the expression of ABC transporters. Drug concentrations have a high inter-individual variability in patients receiving the same dose of antiretroviral drugs, which could affect outcome of antiretroviral therapy. There are many factors that may affect plasma concentrations such as age, gender, body weight, ethnicity, genetic factors and so on. In a Ghanaian cohort, a negative correlation was found between the body weight and the EFV plasma concentration. Genetic factors such as the polymorphisms of cytochrome P450 enzymes also influenced efavirenz plasma concentrations. Meanwhile, efavirenz plasma concentrations were associated with the viral load in plasma within a UK cohort. Nanomedicine involves new and promising technologies that may enable and improve the targeted delivery of antiretroviral drugs. The permeability of lopinavir in the Caco-2 cell line was improved by formulation of nanodispersions. However, the permeability of efavirenz was decreased for all nanodispersions in MDCKII and MDCKII-ABCB5 cell lines. Comparing efficiency of efavirenz nanodispersions transcellular permeability in MDCKII and MDCKII-ABCB5 cell lines indicated that ABCB5 is able to transport efavirenz when incubated as dissolved molecule or nanodispersion. It is support by the copy number variation of ABCB5 had no relationship with EFV plasma concentrations. In summary, this thesis has attempted to determine the pharmacological factors influencing pharmacokinetics of HIV drugs, including drug transporters, metabolism enzymes and nuclear receptors. Data illustrating the factors that influence efavirenz plasma concentrations which are important for viral suppression were also generated. Furthermore, the nanodispersion technology is worthy of further study in order to improve drug delivery and drug distribution of antiretroviral drugs.