Therapeutic proteins are required for the treatment of a variety of chronic and life threatening diseases and as s such there is a high demand for their rapid production. Chinese hamster ovary (CHO) cells are commonly used for therapeutic protein production because they perform the post-translational modifications required for the correct biological activity in patients. However, when compared to other expression systems, recombinant CHO cell lines produce a relatively low concentration of recombinant protein. There are many studies that have improved recombinant protein production in CHO cell lines by enhancing specific productivity and viable cell density and methods have been developed to isolate high-producing cell lines at an early stage of cell line development.

The goal of most population pharmacokinetic and pharmacodynamic analyses is to develop a model that adequately describes the available data and that can be used for predictive purposes. The assessment of covariates is of utmost importance to enhance predictive performance and ultimately to design rational dosing regimens. The increasing use of pharmacogenetics has opened up a whole new array of covariates that can potentially explain the observed between subject variability. In addition, the lack of information regarding drug therapy in special populations such as paediatrics needs to be addressed. The aims of this thesis were therefore to assess the impact of pharmacogenetics on the pharmacokinetics and/or pharmacodynamics of test compounds and to assess ways of including this covariate information into models. Furthermore, to develop models that can explain the differences between adults and paediatrics in order to develop rational dosing guidelines in the paediatric population.

Physiologically-based pharmacokinetic (PBPK) modelling is a computational technique which includes the anatomy and physiology of the study species (species-specific information), as well as the physicochemical properties of the study compound (compound-specific informafion). This data-rich situation confers many advantages on drug development and discovery, especially in the areas of extrapolation between species, route IS of administration and doses. However, it remains under-used in drug research. This thesis examines the applications of PBPK modelling in three different scenarios, in the light of recent developments in this field, in order to support its wider use in drug research in both preclinical and clinical areas.

HIV infection remains a worldwide concern and new drug treatments are required to tackle drug resistance and to reduce drug-associated host toxicity. Raltegravir, a new anti-HIV drug which targets the HIV integrase enzyme, is now being used in anti-HIV treatment and has shown impressive efficacy and low toxicity. However, the drug shows high pharmacokinetic (PK) variability between subjects and it has been difficult to associate PK parameters with treatment outcome. The aim of this thesis was to improve the current understanding of the factors influencing raltegravir PK and cellular exposure using in vitro and in silico techniques.

Peptides from venom of marine cone snails are regarded as promising novel ligands in drug discovery race. They are highly specific, up to subtype level, leading to new therapeutic insights for clinical and neuropharmacological research and applications. In this study, a novel peptide AMIXA, belongs to the P-superfamily, has been characterised pharmacologically and is classed as a π-conopeptide according to its pharmacological function.

Isolated hepatocytes are one of the most relevant and practical in vitro models for the study of xenobiotic transport and metabolism. Several modifications of the original two-stage collagenase perfusion technique for the preparation of isolated hepatocytes exist. However, there are limited data about the effect of such modifications on xenobiotic transport and metabolism in isolated hepatocytes. The aim of this project is to investigate the effect of the liver digestion enzyme on hepatocyte uptake, metabolism and efflux of xenobiotics. Three different liver digestion enzyme systems were investigated; collagenase type II (CII), collagenase type A with trypsm inhibitor (CA/TI) and a preparation containing collagenase and dispase (C/D).

Cytochrome P450 enzymes, especially those of the CYP3A family, play a major role in the metabolism of many drugs.' Patient response to drugs metabolized by CYP3A4 and CYP3A5 varies considerably and part of this variability is due to genetic polymorphism ot the CYP3A5 enzyme. In this study, human liver microsomes (HLM) were prepared from a panel of 26 liver samples and total soluble protein was evaluated. The CYP3A5 and CYP3A4 protein contents were determined by Western blotting and metabolic studies of individual HLM were performed with three selected substrates, ALP, MDZ and TST using HPLC. Results from the 26 HLM preparations revealed a high variability in CYP3A4 (366.7-1.06 pmol/mg protein, 346 fold) and CYP3A5 (4.26 -0.14 pmol/mig protein, 30 fold) content. As might be expected, the two samples with the CYP3A5*l/*3 genotype expressed higher CYP3A5 protein level than the other 24 samples (CYP3A5*3/*3) indicating the consequence of the CYP3A5*1 allele on CYP3A5 protein expression.

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The simple anion inorganic nitrite (NO2 ) has previously been considered a relatively inert nitric oxide (NO) metabolite. However, recent evidence shows that NO2" exhibits an enhanced vasodilator effect in hypoxia. It has been suggested that this effect is mediated by intra-vascular reduction of NO2" to NO by deoxygenated-haemoglobin. This thesis investigated the effects of low-dose NO2" supplementation in man, in three different environments where the actions of NO2" may be potentiated. Firstly, the effect of systemic sodium nitrite (NaNO2) administration upon forearm and pulmonary haemodynamics were assessed in healthy volunteers in both hypoxia and normoxia conditions created by a controlled environmental chamber. The study- infusion resulted in an approximate doubling of plasma NO2", with similar pharmacokinetics observed in both hypoxia and normoxia. Forearm vasodilation occurred in hypoxia but not normoxia. In addition a pulmonary vasodilator effect was present in hypoxia only, and was not dependent upon simultaneous elevation of plasma NO2". The same dose of NaNO2 was given to patients with proven inducible myocardial ischaemia in the second study: a double-blind placebo-controlled clinical trial. Objective markers of myocardial ischaemia were measured by tissue velocity imaging performed during dobutamine stress echocardiography. The results showed that low-dose NaNO2 acts as an anti-ischaemic agent despite no vasodilator effect being present in normoxia. A third set of experiments were performed to assess the potential role of NaNO2 as an ischaemic conditioning agent in a forearm model of ischaemia/reperfusion injury. Here NaNO2 was able to act as a pre-conditioning but not a post-conditioning agent. Two key results subtend this thesis. Firstly, tissue levels of NO2" are more important than intra-vascular levels, as its hypoxia-enhanced actions appear to be modulated from this site. Secondly, NaNO2 may find clinically relevance in the future as a targeted vasodilator, providing a therapeutic effect to tissues in need only.

Despite having revolutionized the treatment of psychiatric illnesses, atypical antipsychotic agents raise an increasing medical concern regarding their association with prominent body weight gain and metabolic abnormalities resulting from chronic treatment. As a consequence, the use of atypical antipsychotic medication has been linked to a substantial increase in the development of obesity, type 2 diabetes and cardiovascular diseases in patients undergoing therapy. In this study, the primary aim was to develop a mouse model of atypical antipsychotic-induced body weight gain and adiposity. Moreover, the chosen antipsychotic agents, clozapine and olanzapine, were investigated in a fibroblast-like cell line model (7-F2) and in primary bone marrow cells, in order to test a possible direct contribution by these agents in causing adipogenesis and altered lipid metabolism at the cellular level in peripheral tissues. It was found that the ability to produce a reliable and robust mouse model, capable of mimicking the clinical situation was obstructed by variability and inconsistency of the experimental outcomes. This prompts the suggestion that caution should be exercised in the interpretation of results from previous models and also, to question their predictive validity. Furthermore, although a morphological study on 7-F2 cells showed that clozapine and olanzapine do not enhance the differentiation of fibroblastic cells into adipocytes, mRNA over-expression of genes involved in adipocyte formation and metabolism suggest that these antipsychotics incite de novo formation of fat cells in the bone marrow. Overall, although the results are of a preliminary nature, they emphasize the need for in-depth examination of any possibility that clozapine or olanzapine might directly trigger an increase in adipocyte numbers (hyperplasia) or alter adipocyte size (hypertrophy).