The transporter hypothesis has been postulated to explain pharmacoresistance in epilepsy. Despite over a decade of research surrounding the drug transporter hypothesis, the role that solute carrier (SLC) transporters might play in this theory remains largely unaddressed. Hence, the major focus of this thesis was to investigate and identify SLC transporter systems of interest that are expressed at the blood-brain barrier (BBB) and to determine which, if any, of commonly prescribed antiepileptic drugs (AEDs) are substrates for such transporter systems. Characterisation of AED transport was undertaken using widely reported model systems such as Xenopus laevis oocytes and the human cerebral microvascular endothelial cell line (hCMEC/D3), together with novel stably-transfected MDCK II cell lines. Organic anion transporter 1A2 (OATP1A2), the monocarboxylate transporter (MCT) family and the organic anion transporter (OAT) family were specifically selected for investigation. Valproic acid and gabapentin showed the greatest evidence for SLC-mediated transport by OAT1/OAT3 and MCT1 respectively, while other compounds were largely unremarkable in this respect. Valproic acid transport increased OAT1 overexpressing cells compared to control but decreased in OAT3 overexpressing cells. Gabapentin uptake increased in MCT1 transfected Xenopus laevis oocytes and was shown to decrease in hCMEC/D3 cells in the presence of a panel of MCT inhibitors. The induction/suppression of expression of SLC transporters by AEDs was explored in the hCMEC/D3 cell line, in an attempt to understand how AEDs might influence the functionality of endogenous transport pathways. A number of AEDs were observed to induce/suppress expression of transporter genes involved in transport and detoxification. A further study explored the fundamental physiochemical properties of AEDs, which is relevant to their penetration into the brain. A number of AEDs, including lamotrigine, gabapentin and topiramate, observe adequate uptake in the hCMEC/D3 model of the BBB despite having physiochemical properties, such as a high polar surface area and negative log D value which may limit passive entry into the brain. This would suggest that a carrier mediated system may be involved in the uptake of these drugs into the brain. The work described in this thesis has shown that a number of AEDs may be subject to carrier mediated uptake into the brain. Individual differences in transporter expression at the BBB may be responsible for variability in brain concentrations of AEDs. However, at present, this does not provide us with an adequate explanation for why some people with epilepsy experience pharmacoresistant seizures.

Drug-induced nephrotoxicity is a limiting factor to the efficacy and safety of various therapeutics including the aminoglycoside antibiotics. Aminoglycosides, such as gentamicin, cause proximal tubule injury in a significant proportion of individuals they are given to. The onset of this adverse drug reaction is currently managed by the monitoring of serum peak and trough levels and measurement of the classic renal functional markers serum creatinine and blood urea nitrogen. The limitations of these biomarkers are well established but novel, sensitive proximal tubule-specific biomarkers, such as kidney injury molecule-1, are gradually coming to the fore. Still, management of aminoglycoside nephrotoxicity is lacking a personalised strategy whereby the risk of a patient developing proximal tubule injury can be established at the individual level before exposure. Prior studies of gentamicin nephrotoxicity pin-pointed that HMG-CoA reductase inhibitors, also known as statins, could inhibit the accumulation of gentamicin in vitro and therefore reduce the cytotoxicity of the drug. In order to study HMG-CoA reductase and its relationship to aminoglycoside accumulation further, an LC-MS/MS based assay was developed and validated to measure the product of the enzyme, mevalonic acid, in the urine of rats and humans. Urinary mevalonic acid was converted to mevalonolactone at pH 2, extracted alongside a deuterated internal standard using ethyl acetate and quantified by reversed-phase LC-MS/MS. The assay had a broad dynamic range of 0.0156–10 μg/mL with precision <15% CV and accuracy 85–115% to suit the natural variation within species and between non-clinical and clinical samples. To demonstrate the utility of the assay and to ascertain the natural diurnal oscillations in HMG-CoA reductase activity, mevalonic acid was quantified in the urine of rats, mice and healthy children. In rats the excretion of mevalonic acid was significantly greater in urine collected during 22:00–10:00 h (mean 9.7 ± 2.3 μg/mg UCr) compared with 10:00–22:00 h (mean 3.4 ± 1.3 μg/mg UCr). In a human paired urine study, in 60% of individuals, morning collections had significantly greater concentrations of mevalonic acid than evening collections where the morning excretion was, on average, 105% greater. The diurnal rhythm of HMG-CoA reductase activity was investigated in relation to aminoglycoside nephrotoxicity in a repeat-dose gentamicin rat model. A strong positive relationship between pre-dose mevalonate excretion, gentamicin accumulation and kidney injury in the renal cortex was observed. Animals administered gentamicin at 10:00 h experienced greater gentamicin accumulation and kidney injury, compared to animals on the 22:00 h dosing schedule which corresponded to greater mevalonate excretion in the hours prior to 10:00 h compared to 22:00 h. These data support the idea that there is a contributory relationship between HMG-CoA reductase activity, the uptake of gentamicin and subsequent nephrotoxicity. Investigations with the aminoglycoside tobramycin did not reach the same conclusion as no clear relationship was observed. In contrast to the HMG-CoA reductase focussed research, a non-targeted metabonomic approach to understanding gentamicin nephrotoxicity was undertaken. Multivariate analyses of 1H-NMR spectra from gentamicin-exposed rats revealed multiple major perturbations in the urine and serum metabolome, prior to kidney injury molecule-1 elevation (urine OPLS-DA model 12 h post-dose Q2Y=0.93, p=0.007). Depletion of metabolites related to energy production and elevation of metabolites implicated in oxidative stress suggests gentamicin had a profound effect on the mitochondria of the proximal tubule epithelial cells. Quantification of metabolites such as the TCA cycle intermediates could be a non-invasive alternative to monitoring the toxicity of aminoglycosides prior to overt renal functional changes. Multivariate analyses of 1H-NMR urine spectra were also subjected to a pharmacometabonomic approach whereby the pre-dose or early post-dose metabolomewas integrated with post-dose kidney injury molecule-1 measurements in order to group individuals based on their differential response to gentamicin. Early-intervention metabolite signatures were identified to have an inverse relationship to kim-1 excretion, providing further evidence that the TCA cycle intermediates could be useful prognostic biomarkers of gentamicin nephrotoxicity. Analysis of pre-dose profiles identified gut- microbial metabolite 3-HPPA as correlated to the post-dose toxicity of gentamicin; follow up studies demonstrated that 3-HPPA excretion also had a positive relationship to urinary mevalonic acid. Hence, the pharmacometabonomic analyses implicated gut microbial and host HMG-CoA reductase activity as related to the extent of gentamicin nephrotoxicity, which certainly warrants additional investigations. The adoption of targeted and non-targeted biomarker identification techniques has proven successful in this research. Mevalonic acid and HMG-CoA reductase are promising mechanistic factors which may affect susceptibility to aminoglycoside nephrotoxicity in man and research will be facilitated by the development of the LC-MS/MS assay described herein. Certainly, the use of statins as a prophylactic measure against aminoglycoside nephrotoxicity will be explored. Comprehensive analysis of the metabolome has identified the importance of the perturbation of energy metabolism and oxidative stress in the onset and development of gentamicin nephrotoxicity and in addition, integration of these vast data sets with the novel biomarker kidney injury molecule-1 has revealed that the gut microbiome could also influence an individual’s susceptibility to this adverse drug reaction.

In 2006, Baker reported the isolation of palmerolide A, a polyketide derived marine macrolide, from the Antarctic tunicate Synoicum adareanum collected in the shallow waters surrounding Anvers Island. This unique macrolide displayed potent levels of cytotoxic activity in the human melanoma cell lines (UACC-62, LC₅₀ = 18 nM and M14 LC₅₀ = 76 nM), while also inhibiting V ATPase with an IC₅₀ of 2 nM. There have been eight further palmerolides isolated from Synoicum adareanum, which have been shown to possess cytotoxicity activity. The synthetic route devised for palmerolide A, utilised a convergent approach relying on the initial synthesis of three subunits. These subunits were to be coupled via a Horner-Wadsworth Emmons reaction, a Julia-Kocienski olefination, then followed by formation of the macrolcycle. This approach offers both flexibility and convergence, as alterations to the subunits would give access to other members of the palmerolide family and their analogues such as palmerolide E.

The work is devoted to the application and further development of modern statistical methods to study pharmacokinetics of drugs. Specifically, it deals with applications and development of repeated measures analysis, so called 'population approach' methods, in the field of pharmacokinetics. hi the first part of the thesis, a new, model-free approach is developed and tested. It introduces a model-free measure of patient's exposure to drugs, and then investigates the relationships between the exposure level and covariates using various statistical techniques. Classification tree models (CART) and regression analysis are used to study various subpopulations of interest. It is shown, via simulations, that the model-free method is capable to identify predictors of exposure in a wide range of variability in the data. The non-linear mixed effect modelling is used to confirm the results of the model-free investigation. Model-free approach is successfully applied to several drugs. Non-linear Mixed Effects population models developed for the same data agree with its results. Limits of the new method are also identified. Specifically, it does not allow the estimation of the variability: either the within-subject (intra-individual) variability in response, or between-subject (inter-individual) variability of the pharmacokinetic parameters in the population. The second part of the thesis is devoted to applications of the Non-linear Mixed Effect methodology to population pharmacokinetics and dose-response analysis. Population pharmacokinetic and dose-response models of several drugs are developed. Pharmacokinetic models allow for complete characterisation of the drug's pharmacokinetics and its relationships to safety and efficacy. The developed models are used to explore the relationships between the exposure (individual Bayes estimates) and demographic predictors of exposure, and safety and efficacy of the drug. Finally, the developed models are used in simulations to guide the design of new studies.

The production of biotherapeutics including antibodies and antibody fragments is a rapidly expanding market with an increasing number of products being approved for use. One of the major platforms used for production of such therapeutics is Escherichia coli, which offers a rapid production at low production costs. The favoured location for targeting these biotherapeutic is the periplasm of E. coli as this environment supports the formation of disulphide bonds and simplifies the purification process. There are a number of periplasmic release procedures currently practised in industry including osmotic shock. However their limitations call for the development of an improved generic periplasmic release method. This project demonstrates how the polymer poly(styrene-co-maleic acid) (SMA) can be applied as a novel and alternative periplasmic release agent. The amphipathic polymer self-assembles into discs encapsulating membrane proteins and thereby destabilises the outer membrane consequently releasing the periplasm. Data presented here show that SMA releases the model target proteins with a higher yield at equal or higher target purity than the conventional methods. Furthermore the developed methods was analysed and refined to be compatible with existing downstream and first steps for its adaptation on industrial scale were made.

Antiepileptic drugs (AEDs) are prescription only medications which were firstly introduced in the 1880s to treat epilepsy. However, the rapid growth in the drug discovery market led to a new generation of AEDs with multiple mechanisms of action. These new drugs represent a promising treatment for many diseases in addition to epilepsy such as neurological disorders, psychological disorders and substance and alcohol abuse treatment as substitutes for benzodiazepines and methadone. However, their multiple roles triggered their misuse potential and concern on their abuse potential was raised in the literature, the media, and by many addiction organizations. Hence, this research highlights some of the AEDs which have raised concern and discusses their therapeutic effects, mechanism of action as well as their overdose and abuse probability from a forensic toxicology point of view. Some AEDs have a narrow therapeutic index and require therapeutic drug monitoring in order to attain the optimum response. The majority of published analytical methods focuses on their analysis in serum and plasma within therapeutic ranges and includes a maximum of 11 AEDs in one analytical step. Therefore, a robust and accurate method was developed for the simultaneous analysis of 15 common AEDs and two of their major metabolites in whole blood using LC/MS/MS. The method was validated according to the standard practices for method validation in forensic toxicology (SWGTOX, May 2013) over a wide concentration range to include AED therapeutic and toxic concentrations which make it suitable for both clinical and forensic analysis. Stability studies are of great importance in forensic cases where it takes up to a few weeks for autopsy, sampling, drug screening and finally confirmation analysis. However, reports specifically addressing the stability of antiepileptic drugs in whole blood are relatively scarce compared with those for drugs of abuse. Thus, using the previous method, the stability of AEDs in whole blood was investigated under different storage conditions. The LC/MS/MS method developed for AEDs analysis in whole blood was successfully transferred to another laboratory and extended to include 18 AEDs and 4 metabolites. It was revalidated for AEDs analysis in serum and plasma in addition to whole blood. Before any new method can be adapted to routine forensic analysis, it has to be validated using authentic samples. A total of 467 previously processed samples were reanalysed using the transferred method. The results were compared to the reference laboratory's values and these showed a very good correlation. The prevalence of AED abuse, namely gabapentin and pregabalin, was investigated among prisoners. 904 urine samples were collected from 8 prisons in Scotland over a one month period. Firstly, a simple and accurate method was developed and qualitatively validated for 21 AEDs in urine to screen the urine samples. Secondly, the method was quantitatively validated for the positive AEDs. Drug analysis in hair has multiple applications in clinical laboratories and forensic toxicology. However, only a few papers have considered conventional AEDs analysis in hair for therapeutic drug monitoring purposes. As part of this research, AED extraction from hair samples was investigated. Six different digestion methods and 4 clean-up procedures were compared for 16 AEDs. An LC/MS/MS method was qualitatively validated using the extraction procedure that attained the highest recovery with all AEDs. Subsequently, two authentic hair samples were tested and the method was quantitatively validated for the positive AEDs in these samples.

Background: Proton pump inhibitors (PPIs) have become the cornerstone of medical treatment for acid-related gastrointestinal disorders. To date, there is a distinct lack of understanding about the recent UK trends in PPI use and evidence about the association between the increased risk of these drugs and the potential adverse effects, in particular the risk of infection, remains questionable. The publication of contradictory findings in several research studies further compounds this situation. Aim and Objectives: This thesis aimed to examine the epidemiology of PPI use in general practices in the UK, and the side effects of PPI, mainly their proposed infective complications. The specific objectives were: • To determine the prevalence and pattern of PPI prescription, and to identify the practices employed to reduce PPI use in the UK general population. • To examine the risk of community-acquired pneumonia before and after the administration of PPI and to assess whether unmeasured confounding explains this association. • To determine whether the mechanism by which PPIs induce an increased risk of infection is supported by the same mechanism acting in another cause of achlorhydria, pernicious anaemia. Methods: This thesis describes work conducted using the UK’s Clinical Practice Research Database (CPRD) and, for some studies in this project, a subset of the CPRD linked to the hospital records from the Hospital Episodes Statistics (HES) database. Firstly, the CPRD was used to estimate the annual prevalence of PPI use during the period 1990-2013. In this study, new users of PPI therapy who had five years of follow-up data were identified to describe patterns of cessation and duration of PPI use. Secondly, cohort (analysed using Cox regression and prior event rate ratio) and self-controlled case series studies were conducted to examine the risk of community-acquired pneumonia and PPI exposure. Thirdly, a cohort of pernicious anaemia patients was used to estimate the risk of infections (community-acquired pneumonia and Clostridium difficile infection) compared to controls to examine whether a reduction in gastric acidity might be the underlying mechanism of the increased risk of these infections. Findings: 1) There was a considerable increase in the administration of PPI prescriptions in UK general practice such that both the period and point prevalence of PPI use increased between 1990 and 2012 (period prevalence increased from 0.2% to 14.8% and point prevalence from 0.03 % to 7.7%). Of new users of PPI therapy, 27% used PPI therapy over a long-term basis (≥1 year continuously), while 4% remained on PPI therapy for five years. Clear attempts to step down the dosage were identified in 41% of long-term users. 2) Among 320, 000 patients, including 160 ,000 new PPI users, the risk of community-acquired pneumonia was 1.67 (95% confidence interval (95%CI) 1.55 to 1.79) times higher for patients exposed to PPIs than it was for the controls. Among the 48,451 PPI-exposed patients with a record of community-acquired pneumonia, the relative incidence rate ratio was 1.19 (95%CI 1.14 to 1.25) in the 30 days after a PPI prescription but was higher in the 30 days before a PPI prescription (1.92, 95%CI 1.84 to 2.00). This reduction in the increased risk in PPI users after prescription was also reflected in the prior event rate of 0.91 (95%CI 0.83 to 0.99). 3) A total of 45,467 pernicious anaemia patients were identified and matched to 449,635 controls. Patients with a pernicious anaemia diagnosis had a higher risk of developing community-acquired pneumonia than the control group (adjusted hazard ratio(HR)1.24, 95%CI 1.21 to 1.26); however, this risk decreased when a stricter definition of pernicious anaemia was applied, and the data was further restricted to incident diagnosis. The findings also suggest that pernicious anaemia patients have a 57% increased risk of Clostridium difficile infection (adjusted HR 1.57, 95% CI 1.40 -1.76) and this association persisted when we limited the analysis to a subgroup with a more restrictive definition of pernicious anaemia diagnosis, or to incident cases. Conclusions: This research revealed that there was a high prevalence of PPI prescribing in the primary care setting and that there are considerable opportunities available to reduce the cost and side effects of PPI use through improving adherence to recommended withdrawal strategies. In addition, the studies investigating the proposed infective complications of PPI use on which we focussed in this thesis add important data to the development of a safety profile of PPI use.

Corticosteroid medicines have anti-inflammatory and immunosuppressant effects. Corticosteroids are prescribed for a wide range of conditions in children. The duration of treatment ranges from a few days (short course) to long term. They are usually given orally in the different dosage form of tablets, syrup or soluble tablets. There are a wide range of adverse drug reactions (ADRs) associated with corticosteroids. This thesis initially evaluates the incidence of each individual ADR in children using systematic reviews. Prospective studies of palatability and pharmacoepidemiology using different methods are also used to evaluate aspects of using corticosteroids in children in Saudi Arabia and the UK. A systematic review of the toxicity of short-course oral corticosteroids in children was conducted. Six electronic databases were searched for articles that evaluated the toxicity of oral corticosteroids in children for up to and including 14 days of treatment. Thirty eight articles including 22 randomised controlled trials (RCTs). The review found that the three most frequent ADRs were vomiting, behavioural changes and sleep disturbance, with incidence rates of 4.3 – 5.4% of patients. Infection was one of the most serious ADRs. A systematic review of the toxicity of long-course oral corticosteroids in children was conducted. One hundred studies including 33 prospective cohort studies and 21 RCTs met the inclusion criteria. The review found that the three most frequent ADRs were weight gain, growth retardation and Cushingoid features, with incidence rates of 18.1 – 21.1% of patients. Infection was one of the most serious ADRs, with twenty one deaths. Hypothalamic-pituitary-adrenal (HPA) axis suppression was detected in 249 of 429 patients in whom it was measured. Based on the findings that were highlighted from the systematic review, a prospective observational/interview study was performed. This study evaluated the tolerability and palatability of oral prednisolone and dexamethasone in children in Saudi Arabia and the UK. Palatability was evaluated by asking patient/parent’s opinions of the taste and acceptability of the medication. Tolerability in particular nausea, vomiting and abdominal pain was evaluated by direct questioning of the patient/parents after each administration. Dexamethasone sodium phosphate solution was the most palatable preparation. Prednisolone base tablets were rated the least palatable and were also the least well tolerated. Palatability scores seemed to improve with second doses. A prospective pharmacoepidemiological study of corticosteroids use in Saudi Arabian children was conducted. This study aimed to evaluate the prescribing pattern of corticosteroids for children in the Emergency, outpatient clinics and paediatric wards in the Gurayat General Hospital (GGH) in Saudi Arabia. A total of 1000 patients were approached for the study. Most of whom were asthmatic, eczema, bronchiolitis, and croup patients. A total of 1209 prescriptions were prescribed from different departments. The three most frequently prescribed corticosteroids medications were hydrocortisone ampoules (24.4%), prednisolone tablets (16.4%) and mometasone furoate ointment (9%). This research has contributed to the field of corticosteroids in children by providing more information about the most common and serious ADRs and determining their relative risk levels.

Multimodal analgesia has been proposed as a useful strategy to reduce postoperative pain while decreasing opioid consumption and thus opioid adverse events. Gabapentin is one such agent although previous results have been heterogeneous. This thesis aimed to review randomised controlled trials of gabapentin for reducing pain, opioid adverse effects and the haemodynamic response to intubation while attempted to predict clinical effectiveness from these trials using meta-regression. Extending this principle, we evaluated other multimodal analgesic agents to identify whether heterogeneity could be explained by various clinical and methodological covariates. Our gabapentin review included 133 randomised controlled trials and demonstrated its efficacy in reducing pain scores, opioid consumption and opioid adverse events such as nausea, vomiting and pruritus. However, gabapentin increased the risk of sedation. Gabapentin was effective at reducing the haemodynamic response to intubation in 29 randomised controlled trials although trials failed to report on clinically relevant outcomes. Gabapentin exhibited no pre-emptive analgesic effect in 4 randomised controlled trials. There was evidence of considerable statistical heterogeneity on meta-analysis of gabapentin for pain scores and 24-hour morphine consumption. Meta-regression analysis showed however that baseline risk predicted the majority of the heterogeneity between studies. Extending this approach to other multimodal analgesics from 344 randomised controlled trials; we demonstrated this was true for analgesic agents in general. In addition to baseline risk, methodological limitations, especially inadequate allocation concealment, explained some of the residual heterogeneity. There was evidence of funnel plot asymmetry for most analgesic agents, suggesting publication bias. However, this may be a product of trials with higher baseline risk having larger standard errors, rather than true publication bias. Indeed, when we simulated meta-analyses with no publication bias, with both effect size and standard deviations dependent on baseline risk, funnel plot asymmetry was still evident (p < 0.001). Therefore, conventional funnel plots may be an unsuitable method of detecting publication bias where baseline risk predicts between-study heterogeneity. We present an alternative method using meta-regression residuals that corrects funnel plot asymmetry in the presence of no publication bias. Finally, due to concerns that methodological limitations exaggerated effect estimates, we used trial sequential analysis to determine whether sufficient low risk of bias evidence exists to reject type I and type II errors in the analyses of analgesic adjuncts. We demonstrated there is currently insufficient evidence from low risk of bias trials to be confident of the efficacy of the majority of analgesic adjuncts.

Background. Antipsychotic medications are widely prescribed for schizophrenia and other related psychiatric conditions, but can have serious financial costs and adverse effects. The evidence base concerning the efficacy; effectiveness; and adverse effects of antipsychotic medications is extensive but variable in quality and applicability, and controversy continues to exist as to whether the newer medications are superior to the older antipsychotics. Locally derived data on the use and effectiveness of these medications can inform their future use, and complement the national and international studies. Aims & objectives To review the pertinent literature regarding antipsychotic medication, and to examine the use and clinical effectiveness of antipsychotic medication in a local context. Also to develop a valid but pragmatic scale to monitor the adverse side effects of antipsychotic medications. Methods A pre-existing case register was analysed to describe the contemporary patterns of antipsychotic usage. For the original data containing studies, one prospective and two retrospective attributions of clinical global impression (CGI) scores, as well as continuation and hospitalization rates were examined. The side effect scale (GASS) was devised after literature review and patient consultation, and tested on consenting patients in comparison to a well established existing scale (LUNSERS) and on healthy individuals. Results Data from the Glasgow city case register shows antipsychotics are widely and appropriately prescribed there but polypharmacy is common. In the prospective 6 month study, olanzapine and risperidone produced significant improvements in CGI but lack of power precluded similar conclusions with amisulpride, clozapine, and quetiapine. In the retrospective studies, clozapine was clinically superior to other oral antipsychotics but there was no significant clinical difference between the main 3 depot or long acting antipsychotics studied. The new side-effect scale – the GASS - was found to be easy to use and as discriminating as the LUNSERS. Discussion There can be difficulties generalizing data from short term RCTs to routine clinical practice. However this thesis demonstrates that simple but robust measures such as the CGI or GASS can be used to structure and inform everyday clinical practice. Consistent with the evolving debate on the relative merits of individual medications, this thesis showed there was little difference in clinical effectiveness between various oral antipsychotics, with the exception of clozapine. The lack of a significant difference between the old and newer long acting injectable antipsychotics is a new finding, and this area merits further study. Conclusions Structured routine monitoring of outcomes is possible in the NHS with regard to antipsychotic medication. Oral and LAI (or depot) antipsychotic medications continue to differentiated more by their adverse side effect profile rather than their relative effectiveness, with the exception of clozapine A new short, inclusive, and valid side-effect monitoring scale – the GASS - is introduced.