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It has been proposed that the use of cross-over or dose escalation designs for dose ranging studies in combination with more informative analysis could lead to a better characterisation of the dose response relationship. In example presented, the dose response relationship for the 3-hydroxy-3-methylglutaryl Coenzyme A inhibitors (HMG COA) inhibitor, simvastatin, was estimated from a cross-over study which covered the current recommended dose range (10 to 40 mg). Analysis using nonlinear mixed effects modelling approach demonstrated that the selected doses only covered 20% (70% to 90%) of the upper part of the estimated dose response relationship. It was concluded that a lower dose strength would be required to allow adjustment within the log-linear portion of the dose response relationship. The clinical implications of potential relationships between the pre-treatment cholesterol level and the model parameters were explored through prediction and simulation. On simulating the relationship between dose and the percentage of patients who would achieve reductions to below a recognised target concentration, it was found that a different set of dosages may better optimise clinical response. Where strict experimental design is invalidated by study design or restricted recruitment, the resulting data can be unbalanced and not easily analysed by standard statistical methods. In the example presented, the number and size of doses of dofetilide used to test for PK/PD differences between patients with ischaemic heart disease (ISH) and healthy volunteers were different. A population PK/PD modelling approach was implemented, and no difference between the two groups could be detected. The Cmax and peak QTc ranged were predicted to be narrower following a fixed dose regimen in comparison to a dose per kilogram regimen. However, after incorporating the PK/PD variability, this was not predicted to manifest into an overall increase in the risk of Torsades de Pointes.

Background: The transition away from secondary school is an important time for adolescents, when identity is shaped and autonomy is increased. It is likely that this period can be particularly worrisome for people with intellectual disabilities (IDs) and it is possible that these worries will have an impact on mental well-being. This study sought to shed light on the content and implications of worries during the approach to transition. Methods: Twenty-five participants with mild to moderate IDs and 27 participants without IDs, all aged 15 to 18, were recruited from schools in the West of Scotland. Participants were interviewed using a Worry Interview that had been adapted from previous research carried out with young adults with IDs. They also completed a measure of rumination and distress that related to their most salient worries. Anxiety was measured using the Glasgow Anxiety Scale for People with an Intellectual Disability (GAS-ID). Results: Content analysis of the interviews identified differences between the worries of the two groups of participants that may represent differences in life experiences. The distress that was linked to the worries was positively correlated with anxiety in both groups. The ID group were significantly more anxious than the non-ID group. Conclusions: Consideration should be given to the specific worries of adolescents in the approach to transition. Doing so may allow solutions for their concerns to be identified, thus easing distress and leading to an overall impact on mental well-being. Limitations of the study and ideas for future research are discussed.

The lipid-modifying qualities of nicotinic acid, a B3 vitamin, have been exploited for many years to prevent cardiovascular disease and its associated mortality. Despite its widespread availability and clinical use, the clinical target and precise molecular mechanism by which nicotinic acid acts remains elusive. In order to maximise nicotinic acid’s full potential as a lipid-modulating drug, overcome its related side effects and further develop novel and more potent drugs it is vital to understand its molecular mechanism of action. Fifty years on from its arrival on the market, receptors which this drug acts upon have recently been identified as the G protein-coupled ‘nicotinic acid receptors’. A family of three highly homologous receptors, HM74, HM74A and GPR81 are characterised by their differing affinities for nicotinic acid. Comparison of these homologues revealed a high degree of similarity between them, with the greatest similarity between HM74 and HM74A. The main structural difference between these receptors is the longer C-terminal tail of HM74. As the C-terminal tail of GPCRs is often implicated in receptor regulation it was hypothesised that the differences in this region may result in differential regulation of HM74 and HM74A. This hypothesis was tested by examining the regulation and desensitisation characteristics of each receptor in a heterologous expression system. Both HM74 and HM74A failed to interact with β-arrestin 2 or internalise in response to nicotinic acid. However, both receptors were phosphorylated in an agonist-dependent manner. HM74 but not HM74A was demonstrated to desensitise as result of prolonged nicotinic acid exposure. The importance of the C-terminal region was further analysed by the use of chimeric nicotinic acid receptors, in which the C-terminal tail of HM74 and HM74A were exchanged. It was shown that the C-terminal tail of HM74 may be implicated in the desensitisation characteristics of this receptor. Furthermore, it was shown that HM74 and HM74A display differential characteristics with respect to ERK1/2 phosphorylation.

Background Statins are inhibitors of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, in cholesterol biosynthesis. As such, they have been widely used in clinical practice as cholesterol lowering agents to reduce morbidity and mortality from coronary artery disease. There is evidence from clinical studies and in vitro experiments that statins have additional anti-inflammatory properties in atherosclerotic disease, which are unrelated to their lipid lowering activity. Clinical studies have previously suggested that statins might show a beneficial clinical effect in inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Furthermore, preliminary data obtained in models of pulmonary inflammation suggest that the effects manifest in rheumatoid patients can be achieved also in asthma. A proof of concept study was designed to test the hypothesis that atorvastatin improves asthma control and airway inflammation in adults with asthma. Methods Fifty four adults with allergic asthma were recruited to a 22-week crossover randomised controlled trial comparing the effect on asthma control and airway inflammation of oral atorvastatin 40 mg daily with that of a matched placebo. Each treatment was administered for 8 weeks separated by a 6-week washout period. The primary outcome was morning peak expiratory flow. Secondary outcomes included spirometry, asthma control questionnaire (ACQ) score, asthma quality of life questionnaire (AQLQ), provocation concentration to methacholine (PC20) and inflammatory markers: exhaled nitric oxide, sputum differential cell count, sputum supernatant and serum inflammatory markers such as interleukin-6 (IL-6), IL-5, IL-8, sICAM-1, TNF-α, leukotriene B4 (LTB4) and high sensitivity C-reactive protein (hsCRP), and blood lymphocyte proliferation. Results At 8 weeks, the change in mean morning PEF, as compared with baseline, did not differ between the atorvastatin and placebo treatment periods [mean difference -0.5 L/min, 95% CI -10.6 to 9.6, p=0.921]. No statistically significant effect of atorvastatin was seen in evening PEF, or methacholine responsiveness (PC20). Out of all spirometry results, only post-salbutamol FVC showed a statistically significant result, which was slightly lower in the atorvastatin group [treatment difference -0.1L, 95% CI -0.2 to 0.0, p=0.037]. There was also no change in ACQ or AQLQ. No change was seen in exhaled nitric oxide. The total cell counts recovered from sputum were similar after atorvastatin compared to after placebo treatment. After 8 weeks, the mean absolute and relative sputum macrophage count was significantly reduced after atorvastatin compared to placebo [mean absolute difference -44.9x104 cells, 95% CI -80.1 to -9.7, p=0.029]. There was a reciprocal increase in the relative proportion of sputum neutrophils [mean proportion difference 13.1%, 95% CI 1.8 to 24.4, p=0.025], but there were no significant changes in the absolute count of these cells or the counts and proportions of the other sputum cell phenotypes under atorvastatin treatment. The sputum concentrations of inflammatory cytokines and mediators were similar after atorvastatin compared to after placebo treatment other than LTB4 which was significantly reduced [mean difference -88.1 pg/mL, 95% CI -156.4 to -19.9, p=0.014]. No significant difference was seen in the concentration of any serum marker of inflammation between atorvastatin and placebo treatment periods. The change in hsCRP was of borderline significance [mean difference -0.65 mg/L, 95% CI -1.38 to 0.09, p=0.082], but there were no changes in sICAM-1, TNF-α, IL-5, IL-6 and IL-8. There was no significant difference in lymphocyte proliferation. The biochemical effects of atorvastatin therapy were reflected in significant reduction in concentration of serum lipids; cholesterol (mean difference -1.71 mmol/l, 95% CI -1.94 to -1.48 p<0.0001), and HDL-cholesterol (mean difference -0.14 mmol/l, 95% CI -0.26 to -0.02 p=0.026), but not triglycerides. There were significant, albeit modest, increases in mean bilirubin, AST and ALT. There was no difference in compliance, assessed by number of tablets returned and by biochemical results. There was no correlation between changes in LTB4 or IL-8 and sputum macrophage count, sputum neutrophil count, or PEF. The only correlation observed between the variables that were compared was between sputum macrophages and neutrophils. Adverse event rates were similar in patients taking atorvastatin compared with placebo. Equal numbers of patients were lost to follow-up in both arms of the study. One patient died of unrelated causes while taking the placebo medication. Conclusions There were no clinically important improvements in a range of clinical indices of asthma control after eight weeks of treatment with atorvastatin despite expected changes in serum lipids. There were however changes in airway inflammation and in particular, a reduction in the absolute sputum macrophage count after atorvastatin compared to placebo and an associated reduction in sputum LTB4 and a trend towards lower CRP. The lack of any evidence of clinical benefit of atorvastatin in allergic asthma confirms and extends the findings of a smaller randomised placebo controlled crossover trial of simvastatin in 16 subjects with asthma, which showed no change in clinical outcomes or inflammatory markers. It is unlikely that altering duration of treatment, washout period or type of statin used would have changed the outcome of the study. However, as all patients were receiving inhaled corticosteroid as part of their asthma therapy, it is possible that this may have masked any modest anti-inflammatory effects of the statin. Baseline asthma inflammation may also have been too low to show any significant improvement. Despite the postulated anti-inflammatory actions of statins, it seems that they may not be appropriate for the inflammatory phenotype associated with atopic asthma. The reduction in alveolar macrophage count found in patients with allergic asthma may however have relevance to the treatment of chronic lung diseases such as COPD in which alveolar macrophage function has been implicated in the pathogenesis.

In the last few decades, evidence has been accumulating for a role for xanthine oxidoreductase (XOR)-generated toxic reactive oxygen species (ROS) in a variety of pathological conditions that affect different organ systems. This enzyme in mammals exists in two inter-convertible forms: xanthine dehydrogenase (XDH) (the predominant intracellular form under physiological conditions) and xanthine oxidase (XO). A combination of XO and its oxidizable substrate xanthine (X) (or hypoxanthine (HX)) is widely used as a model to produce ROS and to study their effects in a variety of cell culture studies. However, the effect of the combination of XOR and the reduced nicotinamide adenine dinucleotide (NADH) in cell cultures is much less studied. NADH is another oxidizable substrate for XOR that binds to a different site on the enzyme from that of X binding. The aim of this project was to investigate some aspects of the in vitro toxicity of XOR, which might provide more insights into its in vivo toxicity. The main investigation was a comparison between the well studied X / XO and the much less studied NADH / XO toxicity models. Also, secondary studies were undertaken to investigate those aspects of X / XO toxicity where there are uncertainties about them. These studies were performed using primary cell cultures. Cell cultures are now widely used to study different diseases, and although they have their drawbacks, they have their advantages over the in vivo studies. For this project, primary cultures of cerebellar granule neurons (CGNs) were used. In the beginning, some problems were encountered with CGNs. The main problem was the immediate damage induced to the neurons (including those in the control groups) at the intervention/experiments day (i.e. day 8 or 9 after plating) by manipulating the cultures (i.e. aspirating the culture medium, adding treatment and control vehicles, and adding the restoration medium). After several months of investigation, it was serendipitously discovered that the immediate damage seen in the neurons (including those in the control groups) when they are manipulated at the experiments/intervention day was due to glutamate excitotoxicity (through activating its N-methyl-D-aspartate (NMDA) receptors). The source of glutamate was the fresh serum which is present at 10% V/V in the fresh culture medium that is added to the cultures at that day. After solving this problem, it was possible to conduct reliable experiments to investigate XO toxicity models. Regarding investigating XO toxicity, it was found that both of the X / XO and NADH / XO combinations were toxic to cultures of CGNs. However, the concentration of NADH needed to cause the toxicity was much higher than that of the other substrate, X, which is in agreement with previous cell-free experiments that showed that NADH is a much weaker substrate than X for the bovine milk XO used here. Blocking the site of X binding on XO prevented X / XO toxicity, but did not prevent NADH / XO toxicity. On the other hand, blocking the site of NADH binding prevented both X / XO and NADH /XO toxicities. Another difference between the two systems was that deactivating either superoxide or hydrogen peroxide (both are ROS) generated by XO prevented NADH / XO toxicity, whereas although deactivating hydrogen peroxide prevented X / XO toxicity, deactivating superoxide generated from this combination did not. In the NADH / XO system, an extracellular metal contaminant (likely contaminating XO powder/preparation) seemed to be involved in the toxicity. The two toxicity models were similar in the mediation of toxicity by intracellular iron ion. In X / XO toxicity, although superoxide generated extracellularly from the combination has no role in the toxicity, intracellularly produced superoxide seemed to play a role. Conclusions: 1. Culturing/experimental conditions have been optimised for viability studies in CGNs cultures. 2. The combination of NADH and XO induces damage to CGNs, where although blocking the NADH binding site prevents this damage, blocking the X binding site does not. It is feasible that the oxidation of NADH by some forms of XOR (other than the one used here) that are known to be very efficient in oxidizing NADH might produce in vivo toxicity. 3. A possibility raised by this study is that a metal (like the metal contaminant proposed to play a role in NADH / XO toxicity in this study) might contribute to XOR toxicity in vivo. 4. Intracellular superoxide often mediates XOR toxicity. 5. The results add support to many previous studies which suggested that intracellular hydroxyl radical (or a similar species) is involved in XOR toxicity.

The prolyl oligopeptidase family enzymes are a group of medically significant proteins distributed in all kingdoms of life that have several unifying structural and functional features. Members of the family hydrolyse short peptides, normally no more than 30 amino acids long. This strict substrate specificity is regulated by β-propeller domains that restrict access to the active site. Conformational changes allow access to the active site by small peptides but block access to larger, structured peptides. Mechanistic details such as these were revealed when the structure of prolyl oligopeptidase was solved by X-ray crystallography in 1998. Since then, numerous crystal structures of family members have been solved, increasing understanding of the structure-function relationships of these important enzymes. Many members of the family are either proven or potential drug targets. As such, structural characterization of these enzymes and their interactions facilitates the design of pharmaceutical compounds. With this in mind, this research was undertaken, using X-ray crystallography to further understand the interactions between particular members of the prolyl oligopeptidase family and inhibitor compounds. Four specific, potent inhibitors were visualized in complex with the active site of prolyl oligopeptidase. All four inhibitors follow a similar template which was shown to be successful when one of them advanced to phase 3 clinical trials as a treatment for Alzheimer‟s disease. The structure of oligopeptidase B from Trypanosoma brucei, which is involved in cell invasion leading to Trypanosomiasis, was solved to 2.5Å, revealing a number of interesting features and enabling various avenues of study. Finally, a plant derived glutamyl endopeptidase known to be regulated by endogenous inhibitors was overexpressed, purified to a high degree and shown to be correctly folded and active. Establishing this procedure allows for the commencement of crystal trials, as well as study by other biophysical methods.

The thesis describes a number of examples of the use of recently developed mass spectrometry experimental approaches to characterise biologically important mixtures. The recently introduced field of ambient ionisation mass spectrometry has been utilised in the rapid, sensitive, information rich characterisation of pharmaceutical formulations. Little, or no, sample treatment was required and the experiments were shown to provide detailed information on active ingredients in the presence of a number of other components. A number of ambient ionisation approaches including DART, DESI and DAPCI were compared and advantages and disadvantages of each approach outlined and discussed. The exciting technology of ion mobility has recently been commercially interfaced with mass spectrometry (IMMS). This has been utilised in a series of fundamental experiments that probe the interaction of varied cations with isomeric oligomers of carbohydrates. The approach enables conformational changes to be rapidly measured over a wide (500-6000 Da) mass range. Changes in conformations were observed for multiply cationised species which agree with previously measured solution phase measurements. The IMMS approach has also been used successfully to characterise a number of Nlinked glycans released from glycoproteins. The experiments enable isomeric structures to be differentiated and present an opportunity to develop a rapid, high information content screen. Estimated cross sectional measurements have been calculated and found to be in good agreement with those obtained from conventional drift cell approaches.

Background Rupture of the Achilles tendon occurs in over 11,000 people annually in the UK. Traditional management using cast immobilisation is being slowly replaced by immediate weight bearing rehabilitation, but currently there is no consensus regarding the exact protocol to be used. The aim of this thesis was to develop an immediate weight bearing rehabilitation protocol for patients who have sustained an acute rupture of their Achilles tendon to inform a definitive evaluation of its effectiveness. To achieve this aim a framework (by the Medical Research Council) for defining and developing interventions with several components was used to underpin the structure of this thesis. Pre-Clinical Development The first two objectives of this thesis were focussed on the ‘pre-clinical’ development phase. Firstly, a systematic review of the evidence base identified the components that define immediate weight bearing rehabilitation. Two of these were evaluated in controlled gait analysis studies to inform and develop a rationale for the intervention to establish what changes are expected and why. The key findings showed that rigid orthoses designs with a large degree of plantarflexion, increased heel pressures, reduced forefoot pressures and decreased the amount of time spent in the terminal stance and pre-swing phase of the gait cycle. Feasibility and Piloting The first clinical phases (feasibility/piloting) followed, which included testing procedures, establishing likely recruitment and follow up. Alongside this, a further systematic review was undertaken to identify what outcome measures are used in research for this injury to determine effectiveness. The Achilles tendon Total Rupture score was the only disease specific patient reported measure identified with supporting validation research. Further evaluation of its measurement properties found the score to be internally consistent, responsive and with good construct validity. Conclusions This thesis defines the rehabilitation components, proposes a theoretical framework and tests this in practice. The results will ensure that rehabilitation after an acute Achilles tendon rupture is based on a systematically developed protocol rather than ad hoc practice. This will now be used to inform future definitive research in this area.

Most drugs have a considerably low solubility in the environmentally friendly hydrofluoroalkane propellants (HFAs) currently used in pressurized metered dose inhalers (pMDIs). As a consequence, instability can occur from crystal growth and Ostwald ripening of the system altering the therapeutic performance of the pMDI. Understanding and being able to predict the behaviour of such drugs in the propellant will help in selecting the correct co-solvents and/or surfactants to increase the stability of such formulations. When anhydrous beclomethasone dipropionate (BDP) is suspended in tricholoromonofluoromethane (CFC-11) rapid crystal growth occurs, leading to the formation of a clathrate. Since chlorofluorocarbon (CFCs) propellants have been replaced by HFAs, many questions arose concerning the ability of BDP to form clathrates in the HFA and any stability issues that arise from such reformulation. Clathrates are crystalline compounds consisting of a lattice of one type of molecule that hosts a second type of “guest” molecule within its structure. Since the solid state chemistry can significantly alter the physical interactions within a suspension formulation, it is crucial to determine the most stable crystalline form in the presence of the propellant. Successful formation of BDP CFC-11 clathrates were observed in this work as well as positive outcomes in terms of reduction in the surface energy and the force of adhesion within a model pMDI formulation (even after processing i.e. size reduction). Following this, HFA-134a and 227-ae were selected to determine any potential clathrate formation and to monitor their stability within a pMDI formulation. The focus of this project was to determine the stability of BDP and budesonide in HFA propellants, as well as the appropriateness of each formulation for pMDI use. This project considered the potential use of complementary surface and solid-state analytical tools to provide fundamental understanding of clathrate formation and their physicochemical characteristics. A special interest in understanding the fundamentals behind the process of Ostwald ripening, a process that affects drug particle size and their related stability and hence ultimately dose consistency was also considered. Atomic force microscopy (AFM) was used in order to determine its applicability in studying Ostwald ripening and surface activity of the different APIs in model propellant. Furthermore, the effects of a range of parameters that included storage time, co-solvents and surfactants on Ostwald ripening were taken into account. The work presented in this thesis has demonstrated that the formation of a propellant clathrate is favourable for APIs that to improve formulation stability through a reduction in particle surface energy. However, isolation and full characterisation of such HFA clathrates remains challenging due to their decreased stability when removed from the high pressure media of the pMDI device. This thesis shows that a combination of co-solvent and surfactant provides an effective reduction in instabilities due to Ostwald ripening of the pMDI formulation and a better control of particle size of the APIs within the formulation. This work provides a platform for future formulation development for pMDIs.