The findings of this project have added to the pool of information reported in the literature regarding the application of the nasal route for the delivery of insulin and other peptide drugs. The preliminary studies reported in this project were apparently the first studies performed to investigate the potential use of chitosan in nasal delivery systems. Nasal delivery systems were investigated in rat and sheep models. The efficacy of chitosan as a nasal absorption enhancer for insulin was compared to that of several other compounds which had been reported in the literature to enhance nasasl [i.e. nasal] drug absorption. Erythrocyte haemolysis studies were also performed to evaluate the membrane damaging effects of the various compounds tested. The grade of chitosan predominantly used was a medium viscosity glutamate salt (MVCSN) which was 82% deacetylated and had a molecular weight of about 162,000. Other grades of chitosan of similar degree of deacetylation were also investigated for comparison with MVCSN (low viscosity grades of chitosan glutamate (LVCSN) and lactate (CSN lactate), medium viscosity chitosan hydrochloride (CSN HC1) and high viscosity chitosan base (HVCSN)). The efficacy of chitosan in enhancing the nasal absorption of both insulin and salmon calcitonin, used as an alternative peptide, was demonstrated in rat and sheep models. Nasal insulin delivery systems were extensively investigated in rat and sheep models. In the rat model, insulin / LVCSN formulations at pH -4 were more effective than formulations at pH -7 in enhancing intranasal insulin absorption which was assessed indirectly from the degree of hypoglycaemia following dose administration. The reduced absorption in the latter formulation which was in the form of a suspension was attributed to complex formation between insulin and LVCSN. In the rat model, the absorption enhancing efficacy of MVCSN was second only to that of LPC. This was encouraging in view of the severe membrane damaging effects that LPC solutions have been shown to cause. In contrast, chitosan solutions have been shown to be relatively non-toxic to biomembranes. In the sheep model, a formulation incorporating MVCSN was much more effective than a formulation containing LPC in promoting nasal insulin absorption. These differences were attributed to the animal models used to investigate nasal absorption. The degree of nasal absorption enhancement was improved by increasing the solution concentration of MVCSN until an optimal concentration was attained (approximately 0.5% and 0.35% in rat and sheep models, respectively). Further evaluation of nasal insulin / chitosan formulations in sheep, suggested that the formulation concentration of chitosan was important for its absorption enhancing efficacy and at optimal chitosan concentration nasal insulin absorption was limited by the dose concentration of insulin. In both rat and sheep models, the nasal administration of hypotonic or isotonic formulations of insulin with chitosan did not influence the degree of nasal absorption enhancement attained. However, in rats, a hypertonic formulation was shown to further improve nasal insulin absorption which was attributed to the combined effects of the chitosan and the increased tonicity of the formulation on the nasal membrane. The grade of chitosan used in the nasal absorption studies appeared to influence the degree of absorption enhancement obtained. In the rat model there was no difference in the absorption enhancing efficacy of CSN lactate and MVCSN although the performance of HVCSN was marginally reduced. In contrast, in the sheep model, MVCSN was more effective than LVCSN and CSN lacate in enhancing nasal insulin absorption although there was no difference in the performance of MVCSN and CSN HCL. In studies in the rat, MVCSN was shown to have a transient effect on the permeability of the nasal mucosa to insulin which lasted about 30 minutes. This supports the claims that chitosan is non-damaging to the nasal mucosa. Erythrocyte haemolysis studies showed that MVCSN was non-damaging to rat erythrocyte membranes at concentrations which were higher than the concentrations used in nasal absorption studies. This was encouraging since the other compounds investigated for comparison with chitosan in this project were shown to be potent haemolytic agents at concentrations which were much lower than the concentrations which were effective for nasal absorption enhancement. MVCSN was less damaging to erythrocyte membranes than the other grades of chitosan tested. This project demonstrated that chitosan enhanced the nasal absorption of insulin in rat and sheep models. In the sheep model the bioavailability of nasal insulin, relative to the subcutaneous route, was generally less than 5%. However, the hypoglycaemia which followed nasal insulin / chitosan dose administration was encouraging and a similar degree of efficacy in humans could be feasible for the therapeutic application of nasal insulin.

The biosynthetic pathway which leads, in Cephalosporium acremonium, to the production of the commercially important β-lactam antibiotic Cephalosporin C (CPC) has been the subject of extensive biochemical studies and is now well characterized. In contrast, genetic analysis in this organism was limited until the application of protoplast fusion techniques facilitated parasexual analysis and allowed a genetic map to be established. (Hamlyn 1982; Hamlyn et al 1985). Subsequently, work leading to our understanding of the genetic basis of the CPC biosynthetic pathway in C. acremonium began. (Perez-Martinez 1984; Perez-Martinez and Peberdy in preparation). The studies described here were aimed at extending this understanding to a point at which individual genes implicated in the pathway could be identified and positioned on the linkage map. A programme of mutagenesis resulted in the production of a number of 'blocked' mutant strains of C. acremonium which were phenotypically particular steps of the CPC biosynthetic pathway. The segregation of several of these mutations relative to other genetic markers was examined. Crosses designed to detect complementation between mutations resulting in a 'blocked' phenotype were carried out and involved strains produced in other laboratories in addition to those characterized during this work. Complementation was shown between two mutations which apparently affected the same step in CPL biosynthesis (the conversion of penicillin N into deacetoxycephalosporin C) and evidence for the linkage of one of the mutations (cnp-6) to a mutation resulting in a requirement for inositol was obtained. During the course of the complementation studies, it was noted that the haploid and heterozygous products obtained following C. acremonium protoplast fusion crosses did not always behave in the typical manner described previously. (Hamlyn 1984). The persistent heterogeneity of these fusion products and the possible implications of this are discussed.

The herbal extract of Artemisia has been regarded to be anti-hyperglycaemic since olden times and is commonly used by diabetics in Libya. The present work was designed to evaluate, test and determine which fraction or component of the herb had the hypoglycaemic effects in normal and streptozotocin-induced diabetic rats. The plant extract was administered to the animals in their drinking water and body weight, food and fluid intake and urine volume were all monitored daily. Food and fluid intake and body weight gain in normal rats were not altered by treatment with the plant extract but there was a rise in the urine glucose in the first six rats but rats 7, 8 and 9 were not affected by treatment with plant. Urine volume was increased in all rats suggesting Artemisia judaica is a mild diuretic. The streptozotocin-induced diabetic rat model, used in this study, was associated with the characteristic diabetic symptoms of hyperphagia, hyperglycaemia, polydipsia, weight loss and urinary glucose excretion. When a crude aqueous extract of Artemisia was given in their drinking water, it had little effect on these symptoms after 10 days of treatment. Urine glucose was reduced in the last two days and ketones in the urine were abolished by this treatment. Diabetes mellitus is known to affect many and varied parameters in rat liver. Insulin, biguanides and sulphonylureas are known antidiabetic diabetic treatments. Artemisia judaica extract was tested for its effect on hepatic steroid metabolism and glycogen phosphorylase a activity in comparison with the above drugs. Clearly Artemisia does act as an insulin-mimetic in these assays by reversing all the effects produced by the administration of streptozotocin. In particular the changes in the enzyme activities of cytochrome P-450 (2E1, 2B and 2C) on androst-4-ene-3, 17-dione metabolism are all reversed by the administration of Artemisia extract to diabetic rats.

Previous analysis of the i.r. and U.V. spectra of dicoumarol (a powerful anticoagulant and uncoupler of oxidative phosphorylation) has failed to establish the detailed structure of the molecule. In this thesis the i.r. and u.v. spectra of dicoumarol and its derivatives have been re-examined, and with the additional data available from n.m.r. spectroscopy the structure of the molecule has been found to be the hydrogen bonded structure below. [illustration omitted]. The pKa values of dicoumarols have been shown to be consistent with this formulation. Other compounds (such as dimethones and 4-hydroxy-6-methyl-x-pyrones) have been shown to have a similar structure. The tautomerism of dimedone has been investigated by n.m.r spectroscopy, and interpreted in terms of an equilibrium between keto, enol and enol dimers, the last mentioned involving hydrogen bonding between the hydroxyl protons and carbonyl groups of two enol tautomers. A number of coumarin derivatives have been added to respiring beef heart mitochondria and variations in the rate of respiration of the mitochondria with changing concentration of coumarin noted. On the basis of the mode of interaction of the compound and the mitochondria, the compounds tested may be divided into three classes, a) those that uncouple and inhibit b) those that only uncouple, and c) those that are inactive. All compounds in the class a) are capable of intramolecular hydrogen bonding in which the hydrogen bonded hydroxyl proton and the carbonyl group to which it is bonded form part of an 8-membered ring. Those of the class b) are able to form only intermolecular hydrogen bonds. The mechanism of action of these compounds is consistent with their action at two different sites in the energy-linked oxidation reactions of respiring mitochondria. A comparison of the activity of the compounds as uncouplers and inhibitors of oxidative phosphorylation with their activity as anticoagulants suggests there may well be a connection between the two processes, though the present state of knowledge does not permit any common mechanism to be put forward.

A total of 6 steroidal ligands have been synthesised from simple starting materials using Sonogashira coupling reactions. The ligands are based on testosterone substituted in the l7a-position with ethnynyl-pyridine, ethnynyl-quinoline or ethnynyl-isoquinoline. A range of platinum(II) complexes have been synthesised using these ligands of the type trans- and cis- [Pt(NH3)2(L)(Cl][N03], where L is the steroidal ligand, alongside control complexes containing simple aromatic amines for comparison. The addition of the steroid imparted a substantional ability of the steroidal platinum(II) complexes to alter the structure of B-DNA. Furthermore, the steroid imparted cytotoxicity onto the complexes, which were very much more active that the steroidal ligand alone or platinum(II) complexes containing only an aromatic amine. Moreover, forcing the steroidal skeleton closer to DNA using 3-substituted pyridine rings and cis-geometries caused greater DNA unwinding, which is likely to relate to direct interactions between the DNA helix and the steroidal skeleton.

This study aimed to explore the construct of Masters level clinical practice. A mixed methods approach converging quantitative and qualitative data was undertaken. Consensus of behaviours indicative of the construct was explored through a quantitative Delphi study. Participants represented a total population sample of Masters course tutors in healthcare (n = 48). Round 1 requested behaviours indicative of the construct. Quantitative content analysis informed the behaviours explored in round 2, where participants rated their relative importance. Round 3 asked participants to rank the behaviours in order of importance. Descriptive and inferential analysis enabled interpretation of consensus. The construct was also explored through an in-depth qualitative case study, using semi-structured interviews and participant observation. Purposive sampling selected the `case' of a manipulative physiotherapy course and the participants for the study. Analytic categories were derived from the data using a constant comparative process until saturation of the data were achieved. Theoretical propositions to identify the components of the construct were developed. The response rate for the Delphi study was very good (79.1%, 77.1% and 70.8% for rounds 1-3 respectively). Rounds 1 and 2 achieved good consensus enabling 21 agreed 'important' behaviours to be taken into round 3. The ranking process in round 3 afforded consensus overall, but also highlighted some differences between professions regarding the prioritisation of components of the construct. There was good convergence of the data with the case study, with clinical reasoning and knowledge identified as the most important components of the construct. The study has identified generic components of the construct of Masters level clinical practice. In addition specific components and their prioritisation for the speciality of manipulative physiotherapy are identified. Development of this work by exploring several case studies to enable further consideration of professions and specialities through analytic generalisation would be beneficial.

This thesis describes investigations of the usage of unconventional therapeutic methods to treat diabetes mellitus, with particular reference to Asian patients. Findings suggest that usage of unconventional therapeutic methods may persist in diabetic patients regardless of their language, religious belief, ethnic and cultural background or psychological states and adherence. The thesis is presented in two parts. Study 1, a preliminary study, was conducted in Thailand. Groups of adults with diabetes mellitus, aged 17 - 70+ years, were studied to assess the extent to which unconventional therapeutic methods were used, and to examine the possibility that such usage is associated with their psychological states and unsatisfactorily feelings toward orthodox medicine. Data collection was achieved through a combination of well-established and well-evaluated questionnaires and a structured interview. A scale to assess attitudes to diabetes was found to be reliable in this sample, but scales to measure diabetes knowledge and treatment satisfaction were not. Study 2 was a study of British Indo-Asians in Foleshill, Coventry, England. The extent to which medicinal plants were used was explored and compared between two different cultural and religious backgrounds of adults with diabetes: (1) born in an Asian country and (2) born in England. The majority of participants were old with low educational background and income. A number of modifications were made to the structured interview used in study 1 to make it more appropriate for this sample. The two studies suggest that usage of medicinal plants is common among diabetics in Thailand and among British Indo-Asian diabetics born in an Asian country. Only a minority of users of medicinal plants in both countries were willing to discuss their usage of medicinal plants with their physicians. This could be because users believed that their physicians might not approve the usage of non-orthodox treatment. In Thailand, usage of medicinal plants was significantly associated with one factor - a lack of basic diabetes knowledge. In the study in England, a typical user was characterised as an Asian female born in an Asian country, who had a low income, used betel-nut, had a preferencef or a doctor's ethnicity, and had low treatment satisfaction and adherence scores.

The aim of the investigation was to develop a less empirical way of selecting an appropriate polymeric stabilising agent that would effectively maintain good dispersibility of a given drug substance with known physico-chemical properties. This was achieved by quantifying the adsorption of different polymers onto a range of drug substances from different solvent environments and to establish which physico-chemical properties of the polymers control their adsorption or non-adsorption onto a particular drug. The pharmaceutical actives, two proprietary compounds SB-223412, SB-204269 and loperamide HCl were investigated. The drugs are particulate, with very poor water solubility. The drug particles were examined using SEM. The mean particle size was 1.1, 1.4 and 1.0µm respectively. The polymeric excipients chosen for this investigation were the water-soluble polymers hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), carboxymethylcellulose (CMC), gum arabic (GA), guar gum (GG) and lambda-carrageenan (l-carrageenan). The physico-chemical methods developed in this work help improve the current empirical selection of an appropriate polymer in pre-formulation studies. The methods include an initial bottle test screen. The aim of the screen was to establish which polymers were the most effective in producing stable dispersions of the chosen drug substances. The bottle test screen results showed l-carrageenan and HPMC to be effective stabilisers on SB-223412 and loperamide HCl particles respectively. GA, HPMC and 0.15M NaCl l-carrageenan caused partial dispersion on SB-223412, SB-204269 and loperamide HCl respectively. Stable dispersions were monitored by photon correlation spectroscopy (PCS) for changes in particle size after their preparation and also for up to three months thereafter. Samples remained dispersed at three months. The adsorption characteristics of the stabilising and partially stabilising polymers were quantified by the construction of adsorption isotherms under various conditions of pH and ionic strength. All isotherms were low affinity with a pronounced shoulder region followed by a plateau. Generally all stabilising polymers adsorb to approximately the same amount. Clear differences were seen in the amount adsorbed of partial dispersions. The isotherms provided information regarding how much polymer was required to saturate the surface of a given mass of drug and also some insight into the stabilising mechanism. The combined techniques provided very useful qualitative and quantitative information about the physico-chemical, intermolecular and structural properties of pharmaceutical dispersions in a pre-formulation stage.

Chapter 1 gives an overview of the relevance of studying therapy adherence in Parkinson’s disease. Chapter 2 examines drug induced neurological syndromes and considers the validity of patients’ concerns about taking prescribed medications. Chapter 3 compares different methods of assessing therapy adherence. Chapter 4 studies factors associated with sub-optimal medicine usage in 54 patients. Chapter 5 reports a study of patient perceived involvement with management decisions and an assessment of satisfaction with the movement disorder service in 107 patients. Chapter 6 explores patients’ beliefs about antiparkinson medication in 129 patients. Chapter 7 examines the effect on Parkinson’s patients of emerging data about drug side effects, specifically fibrosis due to ergot-based dopamine agonists. Chapter 8 reports on an educational intervention designed to improve Parkinson drug timing compliance. In summary, this thesis provides important new information about medicine taking in Parkinson’s disease. A fifth of PD patients take less than 80% of prescribed antiparkinson medication. Electronic monitoring is the only reliable method of accurately detecting sub-optimal medication usage. Patients who take less than 80% of prescribed medicines are more likely to be younger, have concomitant depression, be prescribed more tablets per day and have poorer quality of life. Patients are more satisfied if they are involved in management decisions and have increased intention to comply with prescribed medication if there is better communication. Poorer quality of life is associated with less intention to comply with prescribed medication. Timing of medication intake is generally irregular but can be improved by informing patients of the continuous dopaminergic theory and providing specific drug timings. Once daily drugs are taken more consistently than drugs with more frequent doses.

A dietary-independent thiamine deficiency has been found in experimental and clinical diabetes. Decreased tissue availability of thiamine due to its increased renal clearance is associated with the development of diabetic nephropathy and possibly other microvascular complications of diabetes. Increased thiamine clearance in diabetes is caused by impaired renal reuptake of thiamine. It is proposed that tissue-specific thiamine deficiency may occur in diabetes at sites of complications development – kidney retina and peripheral nerve. The aim of this project was to investigate the involvement of glycaemic control on renal clearance of thiamine and thiamine metabolism in tissues and characterise the pharmacokinetics of thiamine in experimental diabetes – including fitting to a multi-compartment pharmacokinetic model. Thiamine metabolism and pharmacokinetics was studied in streptozotocin (STZ)-induced diabetic rats and normal healthy controls. The effect of correction of hyperglycaemia by intensive insulin therapy and the effect of high dose thiamine therapy was investigated. A multi-compartment model was developed to describe the pharmacokinetics of thiamine metabolism in diabetic and healthy states. This was facilitated by development and validation of a new method of analysis of thiamine by stable isotopic dilution analysis liquid chromatographytandem mass spectrometry (LC-MS/MS). The results show that there is increased renal clearance of thiamine in STZ diabetic rats after only 12 weeks of diabetes and this is corrected by intensive insulin therapy. Depletion of thiamine was found in retina of STZ-diabetic rats and was corrected by intensive insulin therapy. Pharmacokinetic modelling of thiamine revealed in diabetes thiamine inflow of the kidney was increased 2 fold and urinary excretion increased 4 fold; rate constants for thiamine inflow and outflow of the retina were decreased 5 fold and increased 2 fold, respectively, suggesting the retina has decreased uptake and retention of thiamine; and peak [13C3]thiamine content of sciatic nerve was decreased 90% (P < 0.05). Thiamine inflow and outflow of the heart and skeletal muscle were little changed in diabetes. The LC-MS/MS assay detected thiamine and previously unknown physiological metabolites – O-acetylthiamine and oxythiamine. The latter accumulated in clinical renal failure and may impair thiamine pyrophosphate function. I conclude that hyperglycaemia is a key and likely causative factor linked to tissue thiamine deficiency and thiamine washout of the body in diabetes. The kidney, retina and nerve have disturbed thiamine handling in diabetes – increased excretion, decreased uptake and/or accumulation. This may predispose to increased risk of thiamine deficiency and vascular complications of diabetes. These effects are likely mediated by impaired transcriptional regulation and expression of thiamine transporters in diabetes where functional impairment of transcription factors Sp1 and Nrf2 have recently been implicated.