Macrovascular contraction and relaxation effects of metformin were measured using a Mulvany Halpern myograph. Mouse aortic ring sections were treated for 1 and 4 hours in vitro with metformin at 10-5M, and for 2, 4 and 8 weeks in vivo with metformin at 250mg/kg/day. The rings were contacted with increasing concentrations of noradrenaline (10-9M, 10-8M, 10-7M, 10-6M) in the absence and presence of metformin. Maximally contracted tissue was then relaxed using increasing acetylcholine concentrations (10-9M, 10-8M, 10-7M, 10-6M). Meformin increased the sensitivity of the aorta to noradrenaline-induced contraction. The maximal effect in vitro was seen after 4 hours giving a 221% increase in contraction after 4 hours at noradrenaline 10-6M. Acetylcholine-stimulated relaxation via endothelium also increased with metformin after 4 hours by 36.85%. The maximal effect of metformin treatment in vivo was seen on aortic contraction after 8 weeks: the effect of melformin treatment on relaxation was less marked at this time. Metformin also increased passive tension generated by the aortic vessel wall after 4 hours, which was reversed by administration of papaverine, which acts directly on vascular smooth muscle. Metformin was shown not to alter nitric oxide production by the mouse aortic wall after 1 and 4 hours in vitro. Metformin lowered basal calcium concentrations, as measured by FURA/2AM, generating a slow sustained increase in calcium release induced by noradrenaline during contraction. This research programme has shown that metformin can increase both the contraction and relaxation capabilities of aortic sections treated both in vitro and in vivo with therapeutic concentrations of metformin at 10-5M. Metformin has been shown to act directly in the vascular wall to alter vascular contractility via effects on both vascular smooth muscle and endothelium, and to influence calcium movements independently of nitric oxide.

The aim of this thesis is to investigate the physicochemical parameters which can influence drug loading within liposomes and to characterise the effect such formulations have on drug uptake and transport across in vitro epithelial barrier models. Liposomes composed of phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC) and cholesterol (0, 4, 8, 16 µM) were prepared and optimised in terms of drug loading using the hand-shaking method (Bangham et al., 1965). Subsequently, liposomes composed of 16 µM PC or DSPC and cholesterol (4 µM) were used to monitor hydroxybenzoate release and transport from Iiposomes. The MIT (3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and crystal violet assays were employed to determine toxicity of the Iiposome. formulations towards the Caco-2 cell line, employed to model the epithelial barrier in vitro. Uptake and transport of mannitol, propranolol, glutamine and digoxin was measured in the presence and absence of Iiposome formulations to establish changes in absorption resulting from the presence of lipid formulations. Incorporation of the four hydroxybenzoates was shown to be influenced by a number of factors, including liposome composition and drug conformation. Methyl hydroxybenzo.ate (MP) was incorporated into the bilayer most effectively with percentage incorporation of 68% compared to 45% for butyl hydroxybenzoate (BP), despite its increased Iipophilicity. This was attributed to the decreased packing ability of BP within the hydrocarbon core of the lipid bilayer compared to MP. Release studies also suggested that the smaller MP was more strongly incorporated within the lipid bilayer with only 8% of the incorporated solute being released after 48-hours compared to 17% in the case of BP. Model transport studies were seen to reflect drug release profiles from the liposome bilayers with significantly (p < 0.01) higher amounts of BP partitioning from the liposome compared to MP, Caco-2 cell viability was maintained above 86% in the presence of all Iiposome formulations tested indicating the liposome formulations are non-toxic towards Caco-2 cells. Paracellular (apical-to-basolateral) transport of mannitol was significantly increased in the presence of DSPC, PC / DSPC:Cholesterol (16:4 µM; 1000 µg). Glutamine uptake and transport via the carrier-mediated route was Significantly (p < 0.01) increased in the presence of PC I DSPC:Cholesterol (16:0; 16:4 µM). Digoxin apical-to-basolateral transport was significantly increased (p < 0,01) in the presence of PC / DSPC:Cholesterol (16:0; 16:4 µM); thus reducing digoxin efflux via P-glycoprotein. In contrast, PC:ChoJesterol (16:0; 16:4 µM) significantly (p < 0.01) decreased propranolol uptake via the passive transcellular route. Bi-directional transport of propranolol was significantly (p < 0,01) decreased in the presence of PC/DSPC:Cholesterol (16:0; 16:4 µM). The structure of a solute is an important determinant for the incorporation and release of a solute from liposome formulations. PC, DSPC and cholesterol liposome formulations are nontoxic towards Caco-2 cell monolayers and improved uptake and transport of mannitol, glutamine. and digoxin across Caco-2 cell monolayers; thus providing a potential alternative delivery vehicle.

Changes in the pattern of activity of neurones within the basal ganglia are relevant in the pathophysiology and symptoms of Parkinson’s disease. The globus pallidus (GP) – subthalamic nucleus (STN) network has been proposed to form a pacemaker driving regenerative synchronous bursting activity. In order to test whether this activity can be sustained in vitro a 20o parasagittal slice of mouse midbrain was developed which preserved functional connectivity between the STN and GP. Using single-unit extracellular recording, dopamine (30 µM) produced an excitation of STN cells. This excitation was independent of synaptic transmission and was mimicked by both the D1-like receptor agonist SKF38393 (10 µM) and the D2-like receptor agonist quinpirole (10 µM) but not by the D2-like agonists sulpiride (10 µM) and eticlopride (10 µM). Using whole-recordings, dopamine was shown to induce membrane depolarisation. This depolarisation was caused either by a D1-like receptor mediated increase in a conductance which reversed at -34 mV, consistent with a non-specific cation conductance, or a D2-like receptor mediated decrease in conductance which reversed around -100 mV, consistent with a potassium conductance. Bath application of dopamine altered the pattern of the burst-firing produced by NMDA an apamin towards a more regular pattern. This effect was associated with a decrease in amplitude and increase in frequency of TTX-resistance plateau potentials which underlie the burst activity.

Two drugs, troglitazone and atovaquone, were selected based upon them being poorly soluble in aqueous media and being relatively lipophilic. These drugs were incorporated into solid dispersions, of various drug to polymer ratios, to improve their solubility. Three polymers were chosen to be the carrier in the solid dispersions; two gastric soluble polymers hydroxypropyl methylcellulose, polyvinylpyrrolidone and one enteric polymer, hydroxypropyl methylcellulose phthalate. Dissolution runs, in Fasted State Simulated Intestinal Fluid (FaSSIF), were performed upon the drugs, physical mixtures and the solid dispersions. The results showed that incorporation of the drug into the solid dispersion enhanced the dissolution of both drugs, the enhancement was more pronounced with troglitazone. Increasing the polymer:drug ratio and the polymer used had an effect upon the dissolution of the drugs. Physical characterisation of the dispersions showed that troglitazone was more compatible with the polymers than atovaquone. Examination of the dissolution of troglitazone in various gastric media yielded the information that the selection of dissolution medium is important. Exposure of the troglitazone dispersions to gastric medium prior to dissolution in FaSSIF showed that there are advantages to using an enteric polymer. A computer model was derived in an attempt to predict the in vivo performance of the dispersions. The influence of the carrier upon the dissolution of the drugs from a solid dispersion was investigated. It was shown that the polymers improved the dissolution rate of troglitazone when pre-dissolved. Investigation into the ability of each polymer at preventing the recrystallisation of the drug from a supersaturated solution found that polymers helped slow down the rate of recrystallisation. Two methods were investigated, ultra-violet spectroscopy and micro-viscometry, to analyse the dissolution of the polymers. This showed that the troglitazone dispersions were, to some extent, controlled by the dissolution rate of the polymer, whereas the atovaquone dispersions were less controlled by the polymer dissolution.

The preparation and characterisation of collagen: PCL, gelatin: PCL and gelatin/collagen:PCL biocomposites for manufacture of tissue engineered skin substitutes are reported. Films of collagen: PLC, gelatin: PCL (1:4, 1:8 and 1:20 w/w) and gelatin/collagen:PCL (1:8 and 1:20 w/w) biocomposites were prepared by impregnation of lyophilised collagen and/or gelatin mats by PCL solutions followed by solvent evaporation. In vitro assays of total protein release of collagen:PCL and gelatin: PCL biocomposite films revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the biocomposite samples that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. Good compatibility of all biocomposite groups was proven by interaction with 3T3 fibroblasts, normal human epidermal keratinocytes (NHEK), and primary human epidermal keratinocytes (PHEK) and dermal fibroblasts (PHDF) in vitro respectively. The 1:20 collagen: PCL materials exhibiting good cell growth curves and mechanical characteristics were selected for engineering of skin substitutes in this work. The tissue-engineered skin model based on single-donor PHEK and PHDF with differentiated confluent epidermal layer and fibrous porous dermal layer was then developed successfully in vitro proven by SEM and immunohistochemistry assay. The following in vivo animal study on athymic mice revealed early complete wound healing in 10 days and good integration of co-cultured skin substitutes with adjacent mice skin structures. Thus the co-cultured skin substitutes based on 1:20 collagen: PCL biocomposite membranes was proven in principle. The approach to skin modelling reported here may find application in wound treatment, gene therapy and screening of new pharmaceuticals.

This study consisted of two stages. Stage 1 investigated the reproducibility and practicality of two observational sedation assessment scales for use in critically ill children. The two scales were different in design, the first being simple in design requiring a single assessment of the patient. The second was more complex in design requiring assessment of five patient parameters to obtain an overall sedation score. It was established that nursing staff preferred the second, more complex sedation scale mainly because it was perceived to give a more accurate assessment of level of sedation and anxiety rather than merely level of sedation. Stage 2 investigated the pharmacokinetics and pharmacodynamics of midazolam in critically ill children. 52 children, aged between 0 and 18 years were recruited to the study and 303 blood samples taken to analyse midazolam and its metabolites, 1-hydroxymidazolam (1-OH) and 4-hydroxymidazolam (4-OH). A significant correlation was found between midazolam plasma concentration and sedative effect (r=0.598, p=0.01). It was found that a midazolam plasma concentration of 223ng/ml (±31.9) achieved a satisfactory level of sedation. Only a poor correlation was found between dose of midazolam and plasma concentration of midazolam. Similarly only a poor correlation was found between sedative effect and dose of midazolam. Clearance of midazolam was found to be 6.3ml/kg/min (±0.36), which is lower than that reported in healthy children (9.11-13.3ml/kg/min). neonates produced the lowest clearance values (1.63ml/kg/min), compared to children aged 1 to 12 months (8.52ml/kg/min) who achieved the highest clearance values. Clearance was found to decrease after the age of 12 months to values of 5.34ml/kg/min in children aged 7 yeas and above. Patients with renal (n=5) and liver impairment (n=4) were found to have reduced midazolam clearance (1.37 and 0.74ml/kg/min respectively). Disease state was found to affect production of 1-OH. Patients with renal impairment (n=5) produced the lowest 1-OH midazolam plasma ratio (0.059) compared to patients with head injury (0.858).

Cachexia inducing tumours are known to produce a glycoprotein called proteolysis inducing factor (PIF), which induces skeletal muscle atrophy via increased protein degradation and decreased protein synthesis. The objective of this study was to investigate the signalling pathway by which PIF reduces protein synthesis in skeletal muscle and to determine the link, if any, to the ability to induce protein degradation. In murine myotubes PIF induced an increase in expression of the active form of the dsNRA dependent protein kinase (PKR), as well as the phosphorylated form of the translation initiator elF2a, possibly through the release of calcium, at the same concentration as that inhibiting protein synthesis. Inhibition of PKR reversed the inhibition of protein synthesis by PIF and also the induction of protein degradation through the ubiquitin-proteasome pathway by a reduction in the nuclear migration of NK-?B. The expression of phosphorylated forms of PKR and elF2a was also increased in the muscle of cancer patients experiencing weight loss, and in gastrocnemius muscle of mice bearing the cachexia inducing MAC16 tumour, as well as in the tumour itself. Treatment of mice bearing the MAC16 tumour with a PKR inhibitor attenuated muscle atrophy and inhibited tumour growth, through the inactivation of PKR and the consequent reduction of nuclear accumulation of NF-?B. A decreased translational efficiency of the elF-4F complex of initiation factors through dephosphorylation of 4E-BP1 and an increase eEF2 phosphorylation was seen in response to PIF in vitro. The same pattern of events also occurred in gastrocnemius muscle of mice bearing the MAC16 tumour demonstrating weight loss, where a depression of mTOR and p70S6K activation was also observed as weight loss increased.

Improved methods of insulin delivery are required for the treatment of insulin-dependent diabetes mellitus (IDDM) to achieve a more physiological profile of glucose homeostasis. Somatic cell gene therapy offers the prospect that insulin could be delivered by an autologous cell implant, engineered to secrete insulin in response to glucose. This study explores the feasibility of manipulating somatic cells to behave as a surrogate insulin-secreting β-cells. Initial studies were conducted using mouse pituitary AtT20 cells as a model, since these cells possess an endogenous complement of enzymes capable of processing proinsulin to mature insulin. Glucose sensitive insulin secretion was conferred to these cells by transfection with plasmids containing the human preproinsulin gene (hppI-1) and the GLUT2 gene for the glucose transporter isoform 2. Insulin secretion was responsive to changes in the glucose concentration up to about 50μM. Further studies to up-rate this glucose sensitivity into the mM range will require manipulation of the hexokinase and glucokinase enzymes. Intraperitoneal implantation of the manipulated AtT20 cells into athymic nude mice with streptozotocin-induced diabetes resulted in decreased plasma glucose concentrations. The cells formed vascularised tumours in vivo which were shown to contain insulin-secreting cells. To achieve proinsulin processing in non-endocrine cells, co-transfection with a suitable enzyme, or mutagenesis of the proinsulin itself are necessary. The mutation of the human preproinsulin gene to the consensus sequence for cleavage by the subtilisin-like serine protease, furin, was carried out. Co-transfection of human fibroblasts with wild-type proinsulin and furin resulted in 58% conversion to mature insulin by these cells. Intraperitoneal implantation of the mature-insulin secreting human fibroblasts into the diabetic nude mouse animal model gave less encouraging results than the AtT20 cells, apparently due to poor vascularisation. Cell aggregations removed from the mice at autopsy were shown to contain insulin secreting cells only at the periphery. This thesis provides evidence that it is possible to construct, by cellular engineering, a glucose-sensitive insulin-secreting surrogate β-cell. Therefore, somatic cell gene therapy offers a feasible alternative for insulin delivery in IDDM patients.

Pulsed field gel electrophoresis of 82 intestinal spirochaete isolates showed specific differentiation of Serpulina pilosicoli and Serpulina hyodysenteriae although considerable heterogeneity was observed, especially amongst S. pilosicoli isolates. In several cases genotypically similar isolates originated from different animals suggesting that cross-species transmission may have occurred. The Caco-2 and Caco-21HT29 cell models have been proposed as potentially realistic models of intestinal infection. Quantitation of adhesion to the cells showed isolate 3 82/91 (from a bacteraemia) to adhere at significantly greater numbers than any other isolate tested. This isolate produced a PFGE profile which differed from other S. pilosicoli isolates and so would be of interest for further study. Comparison of bacteraemic and other S. pilosicoli isolates suggested that bacteraemic isolates were not more specifically adapted for adhesion to, or invasion of the epithelial cell layer than other S. pilosicoli isolates. Genotypically similar isolates from differing animal origins adhered to the Caco-2 model at similar levels. Generation of a random genomic library of S. pilosicoli and screening with species specific monoclonal antibody has enabled the identification of a gene sequence encoding a protein which showed significant homology with an ancestral form of the enzyme pyruvate oxidoreductase. Immunoscreening with polyclonal serum identified the sequences of two gene clusters and a probable arylsulphatase. One gene cluster represented a ribosomal gene cluster which has a similar molecular arrangement to Borrelia burgdorjeri, Treponema pallidum and Thermatoga maritima. The other gene cluster contained an ABC transporter protein, sorbitol dehydrogenase and phosphomannose isomerase. An ELISA type assay was used to demonstrate that isolates of S. pilosicoli could adhere to components of the extracellular matrix such as collagen (type 1), fibronectin, laminin, and porcine gastric mucin.

Skeletal muscle proteolysis in cancer cachexia is mediated by a sulphated glycoprotein with a relative molecular mass of 24kDa, termed Proteolysis-Inducing Factor (PIF). PIF induced a significant increase in protein degradation, peaking at 4.2nM PIF (p<0.001), 'chymotrypsin-like' activity of the proteasome (p<0.001) and increased expression of components of the ATP-ubiquitin dependent proteolytic pathway. This was attenuated in vitro by pre-incubation with the PKC inhibitor calphostin C (100μM) and NF-κB the inhibitors SN50 (18μM), curcumin (50μM) and resveratrol (30μM), 2 hours prior to the addition of PIF. In vivo studies found the IKK inhibitor resveratrol (1mg/kg) to be successful in attenuating protein degradation (p<0.001) and upregulation of ubiquitin-dependent proteolysis in MAC16 tumour bearing mice. C2C12 myoblasts transfected with mutant I?B? and PKC? inserts did not elicit a PIF-induced response, suggesting the importance of the transcription factor NK-κB and PKC? involvement in PIF signal transduction. 15(S)-HETE acts as an intracellular mediator of PIF and exerts an effect through the activation of PKC and subsequently IKK, which phosphorylates I?B? and allows NF-κB to migrate to the nucleus. This effect was negated with the PKC inhibitor calphostin C (300nM). A commercially produced PIF receptor antibody was raised in rabbits immunised with a peptide containing the partial N-terminal sequence of the PIF receptor. The PIF receptor antibody was successful in attenuating the PIF-induced increase in skeletal muscle catabolism and protein degradation in vitro at 10μg/ml (p<0.001) and 3.47mg/kg in vivo (p<0.001). The data suggest great potential in the development of this antibody as a therapy against cancer cachexia.