Autotaxin is an extracellular phospholipase D that catalyses the hydrolysis of lysophosphatidyl choline (LPC) to bioactive lipid lysophosphatidic acid (LPA). LPA has been implicated in many pathological processes relevant to cancer, including cell migration and invasion, proliferation, and survival. Patients with ovarian cancer often present with an accumulation of ascites fluid in the intraperitoneal cavity which contains LPA at concentrations up to 80 micromolar. Autotaxin is also found at increased levels in the ascites fluid of patients with ovarian cancer and is over-expressed in ovarian cancer tumours that are resistant to chemotherapy. Maintaining a high local concentration of an autotaxin inhibitor within the peritoneal cavity is likely to be important to ensure sufficient and prolonged inhibition of autotaxin. The residence time of small molecular weight drugs in the peritoneal cavity may not be adequate because they are quickly absorbed through the peritoneal capillaries into systemic circulation. Polymers are becoming an increasingly useful tool in the delivery of drugs and have the potential to improve the properties of small molecules, including intraperitoneal residence time. In this thesis, the synthesis of polymer-autotaxin inhibitor conjugates is described, followed by the biological evaluation of the conjugates. Firstly, the synthesis of a dendrimer-S32826 conjugate and its biological evaluation is reported. S32826 is a LPA analogue with a high potency against autotaxin. This dendrimer-autotaxin inhibitor conjugate was found to inhibit autotaxin activity using two different substrates and two different sources of autotaxin, and to decrease the migration of an ovarian cancer cell line modified to overexpress autotaxin. Furthermore, the conjugate potentiated activation of caspase 3/7 induced by carboplatin. However, conjugation of the drug to the dendrimer significantly reduced its potency. Subsequently the synthesis of an icodextrin-autotaxin inhibitor conjugate and its biological evaluation was undertaken. Structure-based drug design was used to identify an appropriate locus to cross link icodextrin to an autotaxin inhibitor described by Albers et al. with a thiazolidinedione core. The icodextrin-autotaxin inhibitor conjugate was also found to inhibit autotaxin activity using two different substrates and two different sources of autotaxin. Furthermore, the conjugate was found to reduce migration of an ovarian cell line modified to over express autotaxin. Conjugation to icodextrin led to an increase in solubility and a decrease in permeability compared to the free drug. Finally, the icodextrin-autotaxin inhibitor conjugate was administered to the peritoneal cavity of mice. After 24 hours, 30% of the drug was still detected in the peritoneal cavity. These observations suggest that the drug conjugate may be useful in the treatment of ovarian cancer.

The sphingosine 1-­‐phosphate receptor modulator fingolimod / Gilenya / FTY720 has become an effective and commercially available therapeutic agent for the treatment of relapsing-­‐remitting multiple sclerosis. Fingolimod is phosphorylated by sphingosine kinase in vivo to the pharmacologically active S-­‐fingolimod phosphate. The original aim of the work was to synthesise novel phosphate delivery prodrug analogues of fingolimod and determine whether or not these novel analogues could provide an improved therapeutic profile. The principal phosphate delivery prodrug method to be investigated was phosphoramidate “ProTide” chemistry. ProTide fingolimod analogues were found to have a poor level of stability and readily degrade to unwanted cyclised structures at room temperature and when exposed to very mildly basic conditions. In order to mitigate the poor stability issues it was considered possible that forming ProTide analogues of mono-­‐alcohol S1P receptor modulators, as opposed to diol fingolimod, would lead to greater stability. The synthesis of mono-­‐alcohol S1P receptor modulator benzyl ether derivative analogues published by Tsuji et al was attempted and successfully achieved. Previously reported ProTide synthesis and in vitro testing methods were employed. Carboxypeptidase, human serum, base stability, acid stability and cell lysate processing experiments were conducted in the School of Pharmacy. Homology modelling was employed to determine S1P1 selectivity of benzyl ether derivative analogues and novel structures. ProTide benzyl ether derivative analogues were found to have a far greater level of stability than ProTide fingolimod analogues and in vitro processing experiments showed that they are processed to the desired pharmacologically active monophosphate. The research signifies the development of an entirely new family of potential therapeutic agents.

Loss of upper-limb function is a problem following stroke. Recent research has led to the emergence of new treatments but progress is hampered by lack of reliable objective measures of impairment, and understanding of the underlying impairment mechanisms associated with loss and recovery of functional activity. The aim of this research was to identify, using neuromechanical measurement methods, inter-relationships between motor impairments, and correlates of motor impairments with functional activity limitation in the upper limb of acute and chronic stroke survivors. An instrumented rig has been developed to measure impairments: muscle weakness, active range of movement, motor control accuracy in rhythmic and discrete tracking tasks, spasticity, coactivation, contracture and non-neural stiffness. In pilot studies, signal processing and data analysis techniques have been used to generate novel, clinically and physiologically relevant indices to quantify impairments. In a Main Study, 13 older impaired participants in the acute phase post-stroke, 13 in the chronic phase 14 age-matched unimpaired participants underwent rig assessments and performed a test of upper limb activity. A sub-group of impaired participants were tested on two days for test-retest reliability evaluation. Statistical tests have confirmed the validity of the impairments to distinguish between acute and chronic patients and unimpaired individuals, except coactivation during discrete movements and non-neural stiffness. Repeatability coefficients for the active test indices have been presented as benchmark values for use in future trials. The muscle activation indices showed lower repeatability which highlights the challenge of using these to measure change over time. The impairments that contributed to lower motor control accuracy were reduced extensor weakness, delayed extensor onset timing, coactivation and smaller extension AROM and PROM; coactivation was more strongly associated with motor control accuracy than with spasticity or stiffness. The most important contributors to functional activity in the acute group was extensor weakness, and in the chronic group was motor control accuracy and coactivation (rhythmic task). Contracture was important contributor in both groups, and was associated with weakness and loss of active range of movement rather than spasticity. The findings support the notion that rehabilitation strategies should focus on increasing muscle strength and prevention of contracture. However, assessment of more complex impairments like motor control accuracy and coactivation may be crucial to better target therapy, especially in the later phases post-stroke.

No description available.

No description available.

Disturbances in the acid-base balance, such as acidosis and alkalosis, alone or in the presence of postprandial or pathological hyperlipidaemia can alter the pharmacological and toxicological outcomes of statin therapy. Both acid-base imbalance and hyperlipidaemia are quite common among statin users. Statins are commonly prescribed for elderly patients who have multiple co-morbidities such as diabetes mellitus, cardiovascular and renal diseases. These conditions are risk factors for the development of metabolic acidosis. In addition, patients with abnormal plasma lipoproteins levels are usually treated with statins. There is also a general consensus by clinicians to recommend such patients to use unsaturated fat and fatty acids such as olive oil for prevention of cardiovascular and atherosclerotic diseases. The use of such oils is associated with transient but significant elevation in plasma triglyceride-rich lipoproteins (TRL), mainly chylomicrons. The effect of disturbances in acid-base balance on the inter-conversion of simvastatin and pravastatin between lactone and hydroxy acid forms have been investigated in physiological buffers, human plasma, and cell culture medium over pH ranging from 6.8–7.8. The effects of such inter-conversion on cellular uptake and myotoxicity of statins were assessed in vitro using C2C12 skeletal muscle cells under conditions relevant to acidosis, alkalosis, and physiological pH. Results indicate that the conversion of the lactone forms of simvastatin and pravastatin to the corresponding hydroxy acid is strongly pH-dependent. At physiological and alkaline pH, substantial proportions of simvastatin lactone (~87% and 99%, respectively) and pravastatin lactone (~98% and 99%, respectively) were converted to the active hydroxy acid forms following 24 hours of incubation at 37°C. At acidic pH, conversion occurs to a lower extent, resulting in greater proportion of statin remaining in the more lipophilic lactone form. However, pH alteration did not influence the conversion of the hydroxy acid forms of simvastatin and pravastatin to the corresponding lactones. Furthermore, acidosis has been shown to hinder the metabolism of the lactone form of statins by inhibiting hepatic microsomal enzyme activities. Lipophilic simvastatin lactone was found to be more cytotoxic to undifferentiated and differentiated skeletal muscle cells compared to the more hydrophilic simvastatin hydroxy acid, pravastatin lactone, and pravastatin hydroxy acid. Enhanced cytotoxicity of statins was observed under acidic conditions and is attributed to increased cellular uptake of the more lipophilic lactone or unionised hydroxy acid form. Statins association with plasma lipoproteins was examined using an in silico model, artificial chylomicrons-like lipid particles, rat and human lipoprotein fractions under conditions of physiological and altered pH levels. The effect of statins association with plasma lipoproteins on cellular uptake and myotoxicity of these drugs was also assessed at different pH levels using C2C12 cells that overexpress lipoprotein lipase (LPL). Lipophilic simvastatin displayed considerable association with plasma lipoproteins. The association was more significant with the non-polar lipoprotein fractions (TRL and Low-density lipoprotein [LDL]). This association contributed to increased cellular uptake of statins by C2C12 cells through LPL-mediated process, resulting in a higher intracellular concentration of statins in hyperlipidaemic conditions. These high intracellular concentrations of statins induced significantly higher cytotoxicity in hyperlipidaemic environment comparing to normolipidaemic conditions. Furthermore, a combination of low pH environment (representing acidosis) with hyperlipidaemia enhanced the association of lipophilic statins with plasma lipoproteins and increased cellular uptake and myotoxicity of these drugs. These studies suggest that comorbidities such as hyperlipidaemia, especially when coincident with acidosis, can enhance the statin-associated muscle toxicity, and therefore require extra caution and close monitoring by prescribing clinicians. Hydrophilic rather than lipophilic statins could be a preferable choice in this patient population.

Ovarian cancer is the second leading cause of death among women in the gynaecological category of cancers. The current post surgery treatment which involves the use of platinum-based therapy with its attendant adverse drug-resistance often results in the return of the cancer. This research work, explored the role of natural products as the major source of new drugs by the evaluation of the anti-ovarian cancer activities of three selected medicinal plants and the semi-synthesis of analogues of an anti-cancer agent; thymoquinone from Nigella sativa. Using a bioassay-guided approach, an investigation of the cell growth inhibition of the extracts/fractions of these plants on four human ovarian cancer cell lines, A2780, OVCAR 4, OVCAR 8, and CIS-A2780 showed that Acalypha wilkesiana, Margaritaria discoidea and Rutidea parviflora had promising anti-ovarian cancer activities. This is the first report of the anti-cancer activities of M. discoidea and R. parviflora. The bioactive compounds of the plants were isolated by HPLC, identified by mass spectrometry/NMR spectroscopy and investigated for cytotoxicity. Gallic acid, (IC50 range of 6.2±0.3 – 26.9±4.1 μM) was the active compound in A. wilkesiana. The significantly bioactive compounds of M. discoidea were securinine, (IC50 range of 2.7±0.7 – 8.7±0.1 μM), betulinic acid, (IC50 of 16.0±1.9 μM) and gallic acid. While palmatine, (IC50 range of 5.5±0.9 – 7.9±0.5 μM) was the major active compound in R. parviflora. Twenty-one thymoquinone analogues including eleven semi-synthetic ones were evaluated for cytotoxicity. A synthetic 2-dimethylamino-5-methyl-1,4-benzoquinone demonstrated optimum activity (IC50 range of 4.7±0.5 – 11.2±1.9 μM) and superior aqueous solubility. Palmatine, securinine and 2-dimethylamino-5-methyl-1,4-benzoquinone showed induction of apoptosis via increased caspase 3/7 activity. Palmatine demonstrated PAPR cleavage by western blot analysis. 2-dimethylamino-5-methyl-1,4-benzoquinone showed synergy with carboplatin and paclitaxel. These studies have provided scientific evidences for the potential treatment of ovarian cancer with these traditional medicinal plants and hit compounds for future optimization towards clinical trials.

Chapter 1 is an introduction of this thesis highlighting the importance of natural products in drug discovery utilising chemical modification of the original motif, especially together with an application of various bioisosteric approaches. Then, it gives an overview of a natural product, plantazolicin A which contains multiazoles with peptide bioisosteric properties. Other azole containing natural products including Thiazole/Oxazole Modified Microcins (TOMMs) are also introduced here. The current practical synthetic methods of the azoles such as thiazole and oxazole are discussed, especially with newly developed rhodium(II)-catalysed oxazole formation reaction via rhodium carbenoids derived from -diazocarbonyl compounds. Chapter 2 describes the total synthesis of plantazolicin A with the retrosynthetic plan by using the carbene chemistry, mainly starting from two precursors to prepare the key intermediate I and II. Each synthetic method is detailed including the choice of the optimum protecting groups and their development. The multi-oxazoles are formed via rhodium(II)-catalysed oxazole formation reactions with -diazocarbonyl compounds and the detailed procedures are explained. The two key intermediates I and II are combined together to give the main plantazolicin A scaffold and the detailed investigation to remove the protecting groups are also discussed here. A conformational study was carried out with extensive NMR nOe study together with molecular modelling to find the most stable conformational energy. A hairpin-like 3D structure of plantazolicin A is revealed here. In Chapter 3, the design of analogues of plantazolicin A is discussed and the synthesis is detailed using rhodium(II) catalysed oxazole formation reaction, following the success of the total synthesis of plantazolicin A. The analogues are tested against the growth of bacteria, especially methicillin-resistant Straphylococcus aureus (MRSA). The detailed structure-activity relationship (SAR) is also discussed here. Chapter 4 summarises the results of chapter 2 and 3, and chapter 5 contains full experimental details for all the work carried out.

Occupational therapy (OT) lecturers are at the interface between student education and training and professional practice and therefore have unique insights into the contemporary factors shaping their professional identity and that of the profession. Two main contemporary and interrelated factors have been identified. Firstly, neoliberalist government policies which encourage the marketisation of health and social care provision, which has implications for how the profession is defined and for professional autonomy and values, ways of working, education and training. Secondly, how knowledge is constructed and used within evidence based practice and whether this is compatible with an emerging occupational therapy body of knowledge. These factors are being played out in the fields of representation, regulation and higher education, shaping OT lecturer professional identity and therefore approaches to teaching and learning. This was understood as a structure and agency relationship based on Bourdieu’s theories on ‘Habitus’, ‘Field’ and ‘Capital’ (Bourdieu 1998, 1990, 1977). To do this, nine narrative inquiry focused interviews of occupational therapy lecturers from two universities were carried out. These narratives were understood within wider organisational contexts using a document analysis. A thematic analysis was applied to both interviews and documents. Five main themes have been identified: professional identity and artistry; professional and philosophical body of knowledge; doing research, evidencing practice and the neoliberal agenda; the representation and regulation of the College of Occupational Therapists (COT) and the Health Care and Professions Council HCPC); and moving into new ways of working – the selling of OT. These have implications for the development of OT professional habitus, the relationship between COT and the profession and approaches to teaching and learning.

Δ9-tetrahydrocannabinol (THC) is the major lipophilic cannabinoid in the cannabis plant and is responsible for many of its pharmacological effects. Cannabis and cannabinoids are often administered orally in foods or lipidic formulations. Marinol® is an oral lipidic formulation containing THC dissolved in sesame oil, and is approved to treat nausea associated with cancer chemotherapy. THC exerts its pharmacological effects by binding to cannabinoid receptors 1 and 2, both of which are involved in a large variety of downstream signalling pathways. Some of these pathways regulate cell death. In agreement with this involvement, THC has been shown to be immunosuppressive in vitro and in vivo. Previous work on synthetic cannabinoids has shown that they can be transported in the lymphatic system in significant amounts when administered orally. The lymphatic system is a passive circulatory network of vessels and lymphocyte-rich nodes that has three major functions. The first function is to maintain body fluid balance. The lymphatic system is open-ended, unidirectional, and collects excess fluid at blood capillaries and tissues. Large molecules that cannot re-enter the blood also enter the lymphatic system. The second function of the lymphatic system is to mount immune responses: after lymphocytes are produced in bone marrow and thymus, they migrate to the lymphatic system and are organised into lymph nodes, which are immunological checkpoints that screen passing fluid for antigens, where innate or adaptive immune responses can be mounted onto them. The third function of the lymphatic system is the absorption of dietary lipids. Lipids are a heterogeneous group of molecules that include lipids, triglycerides and vitamins which are found in food. Lipophilic lipids are emulsified in the gastrointestinal tract by bile and phospholipids, forming mixed micelles. The micelles provide an interface for pancreatic lipase to digest triglycerides into fatty acids and glycerol. These micelles dissociate when they approach the unstirred water layer of the intestinal wall, and the contents are absorbed into specialised enterocytes. Absorption of long-chain fatty acids triggers the production of chylomicrons (CM), a large triglyceride-rich lipoprotein which transports digested fats through the lymphatic system. Triglycerides can then be released from CM by the action of lipoprotein lipase, which is expressed in a variety of cells and tissues. Lipophilic molecules and drugs can associate with CM, and share their fate in the body. Transport through the lymphatic system associated with CM bypasses the liver, which could reduce the first-pass metabolism of a drug. Lipophilic drugs with a high association with CM are prime candidates for lymphatic transport if administered orally. The use of Marinol® by cancer patients receiving chemotherapy could target THC to the lymphatic system, further suppress the immune system of those taking it and possibly making them more vulnerable to secondary infection. Therefore, it is important to confirm whether THC is transported by the intestinal lymphatic system, and whether it is selectively targeted there at immunosuppressive concentrations. To address this research question, analytical assays needed to be developed and optimised to detect THC in a variety of different biological matrices. HPLC-UV methods were successfully developed and validated to determine concentrations of THC in plasma, CM emulsions, and simulated intestinal fluid. The first step to determine the lymphatic transport of THC is to assess the fraction available for absorption in the small intestine. As mentioned previously, Marinol® is a lipidic formulation of THC, and the emulsification and digestion of lipids are not taken into account with traditional drug dissolution techniques. However, an in vitro lipolysis model is a technique that mimics lipid digestion and can reliably predict the absorption of a drug dissolved in a lipidic formulation. After establishment and optimisation of the model, up to 45% of THC dissolved in a lipidic formulation (sesame oil) was solubilised and made readily available for absorption in simulated intestinal fluid. As the concentration of THC in formulation increased, the fraction of drug solubilised decreased. The lymphatic transport potential of THC was then assessed using three techniques – association with CM, pharmacokinetic and biodistribution studies. THC had a high association with CM (72.5%), and its absolute oral bioavailability was improved three-fold when administered in a lipidic formulation (18.5%) compared to a lipid-free formulation (5.73%). At times of peak plasma concentration, the level of THC in mesenteric lymph node homogenate was 30 times higher than plasma, and was at immunosuppressive levels shown in previous in vitro studies (8.6 µg/mL). The mesenteric lymph nodes drain the small intestine, and would be the first nodes that dietary fats pass after being absorbed from the small intestine. Finally, the antiproliferative effect of THC was assessed by exposure to peripheral blood mononuclear cells isolated from healthy individuals and cancer patients recovering from chemotherapy. THC produced a dose-dependent reduction in cell proliferation, with significant differences from vehicle above 10 µg/mL. THC had a reduced effect on cells isolated from cancer patients compared to cells isolated from healthy individuals. THC associated with CM emulsion also had a similarly reduced effect to cells isolated from healthy individuals compared to the effect of THC dissolved in organic solvent. Collectively, this work confirms that THC is transported via the intestinal lymphatic system, and can be concentrated there at extremely high levels, shown to be immunosuppressive in previous in vitro studies.