Background Cardiovascular disease (CVD)-related events such as myocardial infarction and stroke remain the world’s leading cause of death. The incidence of CVD-related events is expected to rise in the future due to the increase in the global prevalence of obesity and diabetes, in addition to less economically developed countries adopting a western style diet. Atherosclerosis is a chronic inflammatory disease which is the underlying cause of CVD and characterised by the build-up of fatty deposits within the walls of medium and large arteries. Macrophages play critical roles during the pathogenesis of atherosclerosis, including the uptake of modified low-density lipoproteins to form foam cells. CardioWise is a dietary supplement developed by Cultech Limited which contains the anti-inflammatory compounds ω-3 PUFAs, flavanols and phytosterols. The aim of this project was to assess the cardiovascular protective effects of CardioWise and its individual components in isolation using in vitro and in vivo model systems. Results Foam cell formation was attenuated in human THP-1 macrophages treated with CardioWise. In addition, CardioWise reduced pro-inflammatory gene expression, monocyte recruitment and M1 macrophage phenotype polarisation. CardioWise was also found to increase HDL cholesterol levels and attenuate circulating levels of pro-inflammatory cytokines in wild type mice. Further investigation identified (+)-catechin within CardioWise as a key beneficial molecule to explore in greater detail. In vitro experiments demonstrated that catechin reduced monocyte migration and reactive oxygen species generation. Wild type mice treated with catechin were also found to receive anti-atherogenic benefits such as increased HDL cholesterol levels and reduced pro-inflammatory cytokine levels. Conclusion The findings of this study show that CardioWise and catechin are capable of exerting strong anti-inflammatory effects on several stages of atherosclerosis disease development in vitro. Furthermore initial in vivo studies using wild type mice revealed that both treatments are also capable of exerting several cardiovascular protective effects. Reasons for these beneficial effects have been proposed in this thesis and future studies outlined.

The work described in this thesis was aimed at distinguishing between two possible mechanisms for the antiviral mode of action of pyrophosphate analogues. Either, (a) they are converted into analogues of nucleoside triphosphates which inhibit the viral polymerase, or (b) they interact directly with the polymerase possibly by coordinating with an essential metal ion. Several pyrophosphate analogues have been synthesised and screened for activity against herpesvirus DNA polymerase, influenza RNA polymerase and calf thymus DNA polymerase a. Characterisation of a number of these compounds was accomplished by chemical ionisation mass spectrometry. A number of reagent gases were tested and ammonia was found to be the most satisfactory. The work carried out in this thesis suggests that the pyrophosphate analogues do not inhibit the viral polymerases by first being incorporated into the 6-y positions of nucleoside triphosphates. The proposed nucleoside triphosphate analogues have been synthesised and these are neither substrates for, nor inhibitors of the enzymes. Furthermore, when [2-^H] -phosphonoacetate was incubated with the standard polymerase assay mixtures, all the radioactivity recovered from the assays was in the form of starting material, none could be detected in the form of nucleoside triphosphate analogues. However, it appears that the pyrophosphate analogues complex with an essential metal ion of influenza RNA polymerase. Dissociation constants (Kd i) for complexes formed between zinc ions and pyrophosphate analogues at pH 8.0 have been determined by gel filtration and there is a correlation between the Kd i of an analogue and its effectiveness as an inhibitor of influenza RNA polymerase.

Chronic myeloid leukaemia (CML) is a clonal stem cell disorder characterised by the Philadelphia chromosome. The treatment and outcomes of CML patients have improved with the introduction of tyrosine kinase inhibitors (TKI). Imatinib is associated with complete cytogenetic response (CCR) rate of 71% at 12 months, as documented by large phase 3 clinical trials. I carried out a large population study in the Merseyside, Cheshire and North Wales area, which showed a maximal CCR rate of 65% over 5 years of observation. This suggests there is a higher rate of imatinib failure in a general unselected CML population compared to large clinical studies which have strict exclusion criteria. My population study also confirmed that second generation TKIs can produce high CCR rates in imatinib intolerant/resistant patients. There are a number of mechanisms of imatinib resistance. The hOCT1 transporter has been shown to be an important predictor of response to imatinib treatment. However, it has been suggested there are other drug transporters involved in imatinib transport which may have prognostic significance. This thesis examined the role of SLCO1A2, OCTN1 and OCTN2 in the transport of TKIs. Transfected cell lines expressing high levels of the respective drug transporter were made using the AMAXA nucleofection process. The cell lines with the highest gene expression, as quantified by TaqMan PCR, were then selected and used in radioactive uptake experiments. Imatinib was confirmed to be a substrate for SLCO1A2. However, the mRNA expressions levels of SLCO1A2 did not have any prognostic correlation to outcome. A review of patient co-medication also showed inhibitors of SLCO1A2 had no effect on CCR and major molecular response (MMR) rates in imatinib treated patients. OCTN1 and OCTN2 did not transport imatinib. Nilotinib and dasatinib are not substrates for SLCO1A2, OCTN1 or OCTN2. Drug drug interactions have also been implicated in drug resistance. Imatinib and metformin are actively transported by hOCT1. It was postulated that varying concentrations of metformin could potentially affect the uptake of imatinib by competitive inhibition. A metformin concentration of 768µM was required reduce imatinib uptake by 50%. However, this concentration is much higher than therapeutic metformin levels, therefore these drugs do not interact at therapeutic concentrations. This thesis shows imatinib is an effective treatment in CML but the CCR rate is lower than in published phase 3 trials. SLCO1A2 transports imatinib but RNA levels have no prognostic significance. Imatinib and metformin do not interact at normal therapeutic concentrations.

Drug induced liver injury (DILI) is a major cause of patient morbidity and mortality inferring considerable burdens onto healthcare and pharmaceutical sectors. As a consequence, substantial resources are directed towards triaging potentially dangerous new compounds at all stages of drug development. However, despite these efforts, hepatotoxic compounds remain the greatest cause of post-marketing drug withdrawal. One of the major factors preventing efficacious screening of new compounds is the lack of a truly representative in vitro model of hepatotoxicity. This thesis describes our efforts to utilise innovative and emerging techniques to further understand and develop in vitro models of hepatotoxicity. One such technique is the generation of hepatocyte-like cells from induced pluripotent stem cells (iPSCs). iPSC-derived hepatocyte-like cells offer a reproducible, physiologically-relevant, genotypically normal and population-representative model of hepatotoxicity; however, current differentiation protocols are not capable of producing hepatocyte-like cells beyond a relatively immature phenotype, limiting their use for toxicological studies. As part of the cellular reprogramming process the epigenome of the somatic cell undergoes dramatic changes; however, the studies have shown that this ‘resetting’ of the epigenome to a pluripotent state is an imperfect process, resulting in an altered differentiation propensity skewed towards the lineage of origin. We evaluated if using human hepatocytes as the starting cell type and utilising the inherent ‘epigenetic memory’ associated with iPSCs could enhance the maturity of hepatocyte-like cells. Despite a trend towards improvement in phenotype, no significant differences were found between isogenic hepatocyte-derived and fibroblast-derived iPSCs. The further development of hepatocyte-like cells is limited by the inability of current culture systems to adequately support the hepatic phenotype. Once placed into culture, primary human hepatocytes, the gold standard model of hepatotoxicity, quickly lose the metabolic qualities required for modelling drug induced liver injury. Thus, without a culture system which supports the hepatic phenotype, the differentiation of hepatocyte-like cells will remain sub-optimal. Using iTRAQ proteomics we attempted to identify the driving factors responsible for the process of hepatocyte dedifferentiation. Our results identified numerous novel factors, including HSF2, SMARCB1, ZEB1 and FOXO1 which may drive the selective loss of metabolic phenotype. The proteomic assessment of hepatocyte dedifferentiation also highlighted the loss of Nrf2-related proteins during culture. Further investigation of Nrf2 in hepatocytes revealed a potentially negative relationship between Nrf2 induction and the key metabolic enzyme, CYP3A4. Furthermore, Nrf2 gene and protein expression was shown to increase during hepatocyte-like cell differentiation. Taken together, these results suggest that Nrf2 may negatively regulate the hepatic phenotype, potentially preventing the establishment of a mature phenotype during hepatocyte-like cell differentiation. A mechanistic evaluation of Nrf2 during differentiation and dedifferentiation is therefore required to gain a fuller insight into the role it plays in the maintenance and acquisition of the hepatic phenotype. In summary, this thesis presents our contribution to the further understanding, development and enhancement of in vitro hepatotoxicity models, using innovative techniques to assess the impact of epigenetic memory on HLC differentiation, identifying novel influencing factors driving the loss of phenotype in hepatocyte culture systems and evaluating the influence of Nrf2 on the hepatic phenotype during differentiation and dedifferentiation.

Efavirenz displays many desirable pharmacokinetic properties such as a long half-life allowing once daily dosing and potency against HIV. Despite these favourable properties efavirenz-containing therapy is associated with the development of central nervous system (CNS) toxicities. Current investigations indicate that high plasma concentrations of efavirenz play a putative role in the development of CNS side effects, but there is a current paucity of data relating to the underlying mechanisms of toxicity. Various nanotechnologies have been explored in attempts to mitigate some of the limitations with efavirenz. While there has been progress in increasing the bioavailability of efavirenz there has been no attempt to assess the impact of increased exposure to efavirenz on CNS toxicity. The body of work presented in this thesis aimed firstly to investigate the underlying mechanism of efavirenz CNS toxicity and secondly to assess uptake and CNS toxicity of efavirenz and a novel solid drug nanoformulation (SDN) of efavirenz. The work presented in this thesis utilised a variety of in vitro, in vivo and in silico methodologies. Chapter 2 utilised allelic discrimination polymerase chain reaction in order to investigate the association of single nucleotide polymorphism (SNPs) in the gamma aminobutyric acid receptor with early treatment discontinuation of efavirenz. In order to assess the effects of SDN efavirenz on the occurrence of CNS toxicities, an in vivo model of anxiety (elevated plus maze) was employed (chapter 3). Chapter 4 detailed the development of a robust and sensitive liquid chromatography tandem mass spectrometer assay for the detection of efavirenz in multiple matrices. The uptake of efavirenz and SDN efavirenz in the CNS was investigated utilising cellular uptake and inhibition studies (chapter 5). Physiologically based pharmacokinetic (PBPK) simulations were used to investigate the distribution of efavirenz in plasma, cerebrospinal fluid (CSF) and brain tissue (chapter 6). Despite an initial trend with Rs211014 and Rs6556547 (univariate analysis) of the training cohort, these SNPs were not found to be significant in the multivariate analysis or in either analysis of the test cohort. Following multiple doses rats treated with efavirenz, but not SDN efavirenz, exhibited anxiety-like behaviour in the EPM. The profile of changes indicated some clear behavioural effects that are likely to be linked to drug-related CNS effects. In particular, a tendency of efavirenz to increase time spent on the central platform may be indicative of anxiogenesis. Cellular accumulation of efavirenz was reduced significantly by montelukast and amantadine, with the reduction in accumulation by prazosin bordering on significance (indicating efavirenz may be a substrate for OCT1 and an SLCO transporter). Additionally, cellular accumulation of SDN efavirenz particles was reduced by dynasore, indicating dynamin-mediated uptake. PBPK simulations predicted efavirenz accumulation in brain tissue, with a tissue to plasma ratio 15.8. The natural occurrence of conditions such as depression involves a complex interplay of factors influencing neurotransmission. This makes identifying single predictors of efavirenz CNS toxicity more difficult. The data presented in this thesis may be built upon to understand the mechanisms governing efavirenz disposition in the CNS and factors influencing the occurrence of CNS toxicity.

Background: Aminoglycoside antibiotics are commonly used in paediatric clinical practice, especially for the treatment of neonatal sepsis and pulmonary exacerbations in cystic fibrosis (CF). However, megalin-mediated endocytosis of the aminoglycosides by renal proximal tubule epithelial cells leads to toxicity, and may result in acute kidney injury and chronic kidney disease. Current approaches to identify and prevent toxicity are limited. Several novel biomarkers have shown utility in preclinical studies for the identification of aminoglycoside-induced nephrotoxicity, but clinical data and an understanding of their clinical utility is lacking. The potential of statins to prevent aminoglycoside-induced nephrotoxicity by inhibition of megalin-mediated endocytosis has been previously demonstrated in vitro and in a rat model, but its potential in man is unclear. Aims: Firstly, to investigate the utility of novel urinary biomarkers for the early identification of aminoglycoside-induced nephrotoxicity in children. Secondly, to develop a novel intervention using statins to prevent aminoglycoside-induced nephrotoxicity in children with CF. Methods and Results: Urine samples were collected from 41 premature neonates at least once per week, and daily during courses of gentamicin. Three urinary biomarkers were measured using Luminex-based (Kidney Injury Molecule-1 (KIM-1) and Neutrophil Gelatinase-associated Lipocalin (NGAL)) and colorimetric assays (N-acetyl-β-D-glucosaminidase (NAG)). All three biomarkers were elevated during treatment with gentamicin, but when adjusted for potential confounders, only the elevation in KIM-1 remained significant (mean difference from not treated, 1.35ng/mg urinary creatinine; 95% CI 0.05-2.65). Electrochemiluminescent assays for both KIM-1 and NGAL were validated, and were compared to Luminex-based assays by analysing samples from healthy children in the UK (n=120) and the US (n=171). 95% reference intervals for both biomarkers were derived using quantile regression. Urine samples were collected from a cohort of children with cystic fibrosis (n=158) at outpatient clinic appointments and during exposure to tobramycin. Biomarkers were measured using the validated electrochemiluminescent assays. Elevations in both KIM-1 and NGAL (median peak fold-change was 2.28 (IQR 2.69) and 4.02 (IQR 7.29) respectively) were observed during exposure to tobramycin. In a multiple regression model, baseline KIM-1 was associated with the number of previous courses of IV aminoglycoside (p < 0.0001; R2=0.11). An in vitro model of aminoglycoside-induced nephrotoxicity was developed using a conditionally immortalized proximal tubule epithelial cell line (ciPTECs). Dose and time-dependent toxicity was demonstrated with neomycin, gentamicin, and tobramycin (from most to least potent). In rats, the addition of rosuvastatin significantly reduced nephrotoxicity compared to gentamicin alone (p < 0.01). In guinea pigs, dose-dependent inhibition of gentamicin-induced nephrotoxicity was seen with rosuvastatin (at a minimum concentration of 0.94mg/kg/day, p < 0.0001), but not with simvastatin. In vitro models demonstrated that neither rosuvastatin nor atorvastatin had any effect on the minimum inhibitory concentration of tobramycin for Pseudomonas aeruginosa. Conclusion: Urinary KIM-1 has shown potential as a biomarker of both acute and chronic proximal tubular injury associated with exposure to aminoglycosides in children. Inhibition of aminoglycoside-induced nephrotoxicity by statins was demonstrated in further animal models, allowing the selection of a statin and dose (rosuvastatin 10mg) which have been taken forward into a clinical trial which will test this hypothesis in children with CF, utilising urinary KIM-1 as the primary outcome measure.

As the prevalence of Chronic Myeloid Leukaemia (CML) grows, due to the therapeutic success of tyrosine kinase inhibitors (TKI), we are witnessing increased incidences of drug resistance. Some of these patients have failed all currently licensed TKIs and have no mutational changes in the kinase domain that may explain the cause of TKI resistance. This poses a major clinical challenge as there are currently no other drug treatment options available for these patients. Therefore, our aim was to identify and target alternative survival pathways against BCR-ABL in order to eradicate TKI-resistant cells. To investigate alternative survival mechanisms in TKI-resistant CML cells, ponatinib-resistant cell line models were generated, which show resistance to all current TKIs, despite complete inhibition of BCR-ABL activity. Additionally, DNA sequencing revealed no mutational changes within the BCR-ABL kinase domain, which may explain TKI resistance and RNA-sequencing showed an impaired transcriptional response following ponatinib treatment when compared with parental TKI-sensitive cells. Using these models, we demonstrated that the TKI-resistant cells acquired alternative activation of mTOR. Using clinically relevant dual PI3K and mTOR inhibitors; NVP-Bez235, VS-5584, apitolisib and gedatolisib, we validated the PI3K-AKT-mTOR pathway as a therapeutic target in vitro in TKI-resistant CML cell lines and more importantly in bone marrow derived mononuclear cells from CML patients resistant to TKIs and with no known kinase domain mutational changes. We demonstrated in vitro that TKI-resistant cell lines are highly sensitive to PI3K and mTOR inhibitors, with EC50 values less than 30 nM compared to ponatinib, 647.3 nM. These inhibitors reduced cell viability by causing a significant induction of apoptosis and significant decrease in the clonogenic growth of primary TKI-resistant CML patient samples. Furthermore, we showed that NVP-Bez235 induced autophagy as a protective mechanism following PI3K/mTOR inhibition. The combination of NVP-Bez235 with pharmacological (Hydroxychloroquine (HCQ)) or specific autophagy inhibition, via ATG7 knockdown, the efficacy of NVP-Bez235 was enhanced shown by the dramatic reduction in clonogenic growth of TKI-resistant CML patient cells. In addition, we validated this in vivo using a murine model by transplanting luciferase tagged TKI-resistant cells, treated with NVP-Bez235 in combination with HCQ, which significantly reduced tumour burden and increased survival rates compared to controls. These data suggested that the PI3K-AKT-mTOR pathway may be a key player responsible for TKI-resistance and that pharmacological inhibition of this pathway, with the additional inhibition of autophagy, may represent a potential new treatment option for TKI-resistant CML patients, when resistance is driven by a BCR-ABL-independent mechanism.

Hyperactivated microglia plays a key role in regulating neuroinflammatory responses which propagate damage to neurons. In recent years, substantial attention has been paid in identifying new strategies to abrogate neuroinflammation. Tiliroside, a dietary glycosidic flavonoid found in several medicinal and dietary plants is known to possess anti-inflammatory and antioxidant activities. This study is aimed at investigating the molecular mechanisms involved in the inhibition of neuroinflammation by the tiliroside. Neuroinflammation inhibitory effects of tiliroside (2-6 μM) were investigated in BV2 microglia stimulated with a combination of LPS (100 ng/ml) and IFN (5 ng/ml). Results show that tiliroside significantly reduced the production of pro-inflammatory cytokines IL-6, TNFα, IL-1β while increasing the production of anti-inflammatory cytokine IL-10 in LPS/IFN-stimulated BV2 microglia. The compound reduced NO production in LPS/IFN-stimulated BV2 cells through inhibition of iNOS protein expression. Tiliroside also suppressed COX-2 protein expression and inhibited PGE2 production in activated microglia. Western blotting and functional experiments revealed that inhibition of neuroinflammation by tiliroside was shown to be mediated through inhibition of NF-B and p38 MAPK signalling pathways. Also, the compound activated SIRT1 and inhibited the expression of acetylated-NF-B-p65 in LPS/IFN-activated BV2 microglia. Further experiments revealed that inhibition of neuroinflammation by tiliroside is not dependent on SIRT1. Tiliroside increased the levels of Nrf2, HO-1 and NQO1 antioxidant proteins, indicating an activation of the Nrf2 protective mechanisms in the microglia. Furthermore, transfection of BV2 cells with Nrf2 siRNA resulted in the loss of anti-inflammatory activities of tiliroside. Results of neurotoxicity experiments showed that neuroinflammation-induced neurodegeneration, DNA fragmentation, ROS generation and calcium accumulation were significantly reduced in HT22 neurons when exposed to conditioned medium from BV2 microglia that were pre-treated with tiliroside prior to stimulation with LPS/IFN. Results from this study suggest that tiliroside inhibits neuroinflammation in LPS/IFN-activated BV2 microglia by targeting NF-B and p38 MAPK signalling pathways. Furthermore, the compound activated Nrf2 antioxidant mechanisms in the microglia, which appears to contribute to its anti-inflammatory activity. The study also established that tiliroside protects HT22 neurons from neuroinflammation-induced toxicity.

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