Oxidative stress plays a prominent role in the development of endothelial dysfunction. L-arginine and L-citrulline have been demonstrated to have beneficial effects in vascular disease and this thesis aimed to determine whether this protective effect is mediated via an antioxidant mechanism. With evidence to suggest L-citrulline may have antioxidant properties in plants, and the current contradictions that exist concerning the antioxidant properties of L-arginine in cells and in humans, a study aimed at fully characterising the antioxidant properties of both molecules was needed. The ability of L-arginine and L-citrulline to scavenge both superoxide and hydroxyl radicals were investigated with an antioxidant effect only demonstrated against hydroxyl radicals. The mechanism of antioxidant action was shown to be two fold: 1) through inhibition of hydroxyl radical production and 2) through direct scavenging of the hydroxyl radical. Three model systems were developed to test the effect of endogenous and exogenous ROS on biological function. This allowed investigation into whether the antioxidant properties demonstrated in vitro were transferable to physiological and pathophysiological systems. Both amino acids were able to regulate endogenous ROS in platelets but this was shown to have little effect on overall platelet function. In terms of exogenous radicals, L-citrulline but not L-arginine, was able to protect against superoxide mediated endothelial dysfunction in vessels however this was deemed to be through a ROS independent effect Both amino acids inhibited hydroxyl radical mediated lipid peroxidation at concentrations of amino acid found in the plasma. Taken together, these studies suggest that the improvement in vascular function seen upon administration of L-arginine and L-citrulline is in part mediated through an ability to scavenge hydroxyl radicals. However, their inability to directly affect superoxide levels draws into question whether significant antioxidant effects occurs in vivo and would suggest that a certain proportion of their protective effect on the vascular system is mediated via an antioxidant independent mechanism.

Myosin light chain phosphorylation plays a central role in the regulation of paracellular permeability. The main objective of this study was to design and synthesise membrane-permeant peptide inhibitors of myosin light chain kinase and phosphatase that could potentially decrease and increase paracellular permeability, respectively. Elevated myosin light chain kinase activity, as observed in a variety of inflammatory disease, phosphorylates myosin light chain, to increase paracellular permeability. Initial studies showed a peptide inhibitor of myosin light chain kinase termed PIK could rectify increased paracellular permeability in two different cell-based models of inflammation. PIK was also, however, shown to be too labile for use in vivo. From a series of PIK analogues, two candidates prepared using D-amino acids were identified that showed sufficient stability, membrane-permeant properties and retention of specific function of PIK for future in vivo studies. Since the ratio of myosin light chain kinase to phosphatase activity regulates the degree of myosin light chain phosphorylation, it was hypothesised that inhibition of myosin light chain phosphatase would increase paracellular permeability. A membrane-permeant peptide designed to inhibit myosin light chain phosphatase activity termed PIP was identified in a screen of potential candidates through its capability to significantly decrease transepithelial electrical resistance in a polarized human intestinal epithelial cell system in vitro without any apparent cytotoxicity. Further studies will be required to determine the extent to which PIP increases paracellular permeability. Using these novel membrane-permeant peptide inhibitors, studies were performed using polarized monolayers of human intestinal epithelial cells to assess the action of previously identified regulators of paracellular permeability in vitro. Previous studies demonstrated that the non-specific myosin light chain kinase inhibitor ML-7 prevented the absorption enhancing properties of sodium caprate. Treatment with PIK and sodium caprate simultaneously resulted in significant increases in the permeability of inert fluorescent probes while pre- and post-incubation with PIK inhibited sodium caprate effects. These surprising findings suggest a potential application for combination treatment with sodium caprate and PIK to increase paracellular permeability of poorly absorbed drugs.

Inorganic arsenic and elevated glucose concentrations increase endothelial production of superoxide, which impairs endothelium-derived nitric oxide bioavailability and associated nitric oxide-dependent arterial relaxations. However, there is now evidence in animal models of diabetes mellitus that relaxations attributed to endothelium-dependent smooth muscle hyperpolarization or endothelium-derived hyperpolarizing factor (EDHF) may be augmented and serve to compensate for the loss of nitric oxide, thereby maintaining arterial responsiveness. The effects of arsenite and elevated glucose concentrations on "EDHF-type" relaxations were thus investigated in isolated rabbit iliac artery rings using the G-protein-coupled agonist acetylcholine and the sarcoendoplasmic reticulum Ca2+ ATPase inhibitor cyclopiazonic acid. Arsenite and elevated glucose both potentiated EDHF-type relaxations evoked by cyclopiazonic acid. Differential effects of arsenite and glucose against EDHF-type responses to acetylcholine were identified in that arsenite attenuated relaxation, whereas glucose potentiated relaxation. Further experiments showed that the arsenite- and glucose-augmented components of relaxation were reversed to control levels by the hydrogen peroxide scavenger catalase and the NADPH oxidase inhibitor apocynin. Arsenite-augmented responses were also reversed by the cell-permeable superoxide dismutase/catalase mimetic manganese porphyrin. It follows that hydrogen peroxide derived from NADPH oxidase may augment EDHF-type relaxations in diabetes mellitus and arsenic toxicity, thus maintaining endothelial control of arterial tone when nitric oxide bioavailability is impaired by oxidative stress. These results are consistent with the demonstrations that hydrogen peroxide augments EDHF-type relaxations in the rabbit iliac artery by promoting endothelial Ca2+ mobilization and enhancing the opening of endothelial Ca2+-activated K + channels, and that the increased activity of these channels underpins augmented EDHF-type arterial relaxations in animal models of diabetes.

There is currently an unsustainable rate of exploitation of certain species of plants and animals for use in traditional medicines. Bear bile and rhino horn are derived from endangered species and are traditionally combined with medicinal herbs as anti-bacterial and anti-inflammatory agents. This study was designed to assess herbs for use as possible alternatives for these two products. Herbs were selected based on their traditional use as antiinflammatory and anti-bacterial agents. Chemical methods were used to confirm the plant species of the herbs and to measure concentrations of metals and pesticide residues as a means for assessing their quality. Antibacterial activities were determined using a direct bioautography technique. Anti-inflammatory activities of the herb extracts and isolated compounds were ascertained using an in vitro nuclear factor kappaB (NF-KB) activity, as assessed by IL-6 luciferase gene reporter assay. A novel assay was developed to estimate drug-herb interactions by measuring the effects of selected herbs and drugs on the production of eicosatrienoic acids from hepatic cytochrome P450 (CYP450) metabolism of arachidonic acid. In addition, CYP3A4 enzyme assays were conducted. The pesticide residue and heavy metal concentrations of the tested herbs was found not to exceed the existing legally permitted concentrations in foodstuffs, but the rhino horn sample contained elevated levels of Cd, Pb, Hg and Zn. Rhino horn was not found to be an effective anti-bacterial or anti-inflammatory agent In the assays used in this study. Seventeen herbs demonstrated anti-bacterial activity. Also, nine herbs demonstrated inhibitory NF-KB activity. Preliminary results indicate that co-administration of Scutel/aria baicalensis, Salvia miltiorrhiza, Rehmann/a glutinosa or Coptidis Rhizoma with drugs metabolised by CYP3A4, could lead to possible drug-herb interactions. Based on the Information obtained in these studies nine herbs are proposed as alternatives to rhino horn; eight herbs and two Kampo medicines are proposed as alternatives to bear bile.

NMDA receptors are ionotropic, glutamate receptors which mediate fast excitatory transmissions within the central nervous system. They form tetrameric or pentameric heterologous complexes from seven NMDA receptor subunits NRl, NR2A-D and NR3A-B which each convey distinct expression patterns, functional and pharmacological properties to the receptor complex. Due to its involvement in excitatory transmission, over-excitation of the NMDA receptor, particularly the NR2B subunit, has been the focus of pharmaceutical therapeutic targeting for neurodegenerative conditions and chronic pain. This thesis discusses the potential importance and clinical effectiveness of targeting NMDA receptors and the difficulties in drag development arising from the receptor's heteromeric nature. The work herein focuses on the pharmacological characterisation of two novel NR2B-selective antagonists Compound A and Compound B， the physiological and pharmacological effects of NR3 subunit inclusion in the NMDA receptor complex, and the modifications of NMDA receptor physiology and subunit expression during chronic pain states. This research provides novel evidence to suggest that Compound A and Compound В bind with a high selectivity and affinity towards NR1/NR2B containing receptors. It provides novel evidence for a differential cytoprotective effect of the NR3 subunits showing significant cytoprotection in NR1/NR2B, but not NR1/NR2A, receptors and shows that NR3B inclusion in the receptor can differentially modulate the binding affinities ofNR2B-selective antagonists. This study also shows evidence for increased activity of spinal and supra-spinal NR2B-containmg receptors indicating NMDA receptor modulation and involvement in a chronic pain model.

No description available.

Neointimal formation is a complex process that occurs due to the over compensatory healing response produced by the vessel following injury. Three key events, SMC proliferation, SMC migration and the inflammatory response, occur in unison to drive the formation of a neointima. Cannabinoids/endocannabinoids have been shown to elicit antiproliferative, anti-migratory and anti-inflammatory effects, highlighting modulation of the endocannabinoid system as possible therapeutic strategy. The aims of this study were to; (i) develop an organ culture model of neointimal formation, and investigate the presence of the endocannabinoid system, (ii) investigate the functional response of anandamide (AEA) in the murine carotid artery, (iii) investigate the effects of cannabinoids on SMC proliferation, and (iv) to establish the effects of cannabinoids on SMC migration. The organ culture model developed in this study demonstrated the presence of both CB receptors on SMCs, LCMS/MS analysis of tissue samples showed that endocannabinoid concentration was significantly (2-arachidonoylglycerol / 2-AG) increased in injured artery sections. Isolated vessel studies demonstrated that AEA produces a small (~20%) relaxation of the murine carotid artery which was not dependant on the production of active metabolites, but involved activation of the CB1 receptor. Studies investigating the effects of cannabinoids on cell proliferation revealed that paradoxically both a CB2 agonist and a CB2 antagonist reduced markers of cell proliferation without any effect on cell viability; high concentrations of AEA (10μM) reduced SMC proliferation, however this was associated with an apparent cytotoxic/cytostatic effect. The preliminary data from cell migration studies suggests that a CB2 agonist may function to reduce stimulated cell migration and that 2-AG can increase migration of unstimulated SMCs. In conclusion, although further research is required, the data within this thesis provides evidence that the endocannabinoid system (in particular the CB2 receptor) may have the potential to be manipulated for therapeutic gains in terms of restenosis.

In order to assess pharmacokinetics and pharmacodynamics (PK/PD) accurately, it is necessary to obtain measurements of the absolute concentrations of compounds in the brain. A major shortcoming of using microdialysis to measure PK/PD is that microdialysis measurements do not give us absolute concentrations of solutes in the brain, since the relationship between dialysate concentrations and true extracellular fluid (ecf) concentrations surrounding the probe is unknown. Several methods have been devised to circumvent this problem. The present study employed a novel method, MetaQuant (MQ) microdialysis, which achieves near 100% recovery, and so enables the measurement of absolute ecf concentrations. I examined the effect of the D4 receptor agonist, PD168077 on extracellular dopamine levels (that is PD) in medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) of freely moving rats, while simultaneously measuring brain concentrations (that is PK) of the drug. Thus we were able to estimate the PK/PD profile of the drug in the two brain regions. Compared with basal extracellular levels, subcutaneous administration of PD168077 caused significant increase in dopamine in mPFC. Activation of dopamine D4 receptors in the mPFC may improve cognitive function, which is highly impaired in individuals with schizophrenia. Moreover, it has been consistently shown that phencyclidine (PCP) produces robust cognitive disruption, in a novel object recognition (NOR) test. I studied the efficacy of PD168077 to attenuate sub-chronic PCP induced deficit in the NOR task. Sub-chronic PCP induced a robust cognitive disruption and PD168077 (10 mg/kg, s.c. dose) reversed this disruption. Further MQ dialysis data showed that PD168077 (10 mg/kg, s.c. dose) increased dopamine levels in mPFC that was depleted due to PCP suggesting a mechanism for the observed alleviation of PCP induced cognitive deficits.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) selectively binds and activates the N/OFQ peptide (NOP) receptor. At the cellular level N/OFQ inhibits cAMP accumulation and Caˆ{2,+} conductance and stimulates K ˆ+ currents. N/OFQ regulates several biological functions both at central (pain, locomotion, memory, emotional responses, food intake) and peripheral (airways, cardiovascular, genitourinary and gastrointestinal systems) sites. Potent and selective NOP ligands are now required for investigating the roles played by NOP receptors in pathophysiological studies and for firmly defining the therapeutic indications of NOP receptor ligands. A novel assay to screen NOP receptor ligands has been validated with a large panel of ligands: the G\alpha_{q,I,5} chimeric protein has been used to force the NOP receptor to signal through the Ca ˆ{2,+} pathway in CHO cells. [Ca ˆ{2,+}]_i levels were monitored using the fluorometer FlexStation II. Data are in general agreement with classical Gi driven assay systems. The NOP peptide partial agonist, ZP120 was extensively characterized in vitro using electrically stimulated isolated tissues (mouse and rat vas deferens) and in vivo with the tail withdrawal assay. The selective involvement of the NOP receptor in the actions of ZP120 has been demonstrated in NOP(-/-) mice studies. A detailed pharmacological characterization of the recently identified non-peptide antagonist Compound 24 has been performed. Moreover in the context of a SAR study on Compound 24, a novel NOP ligand named Compound 35 was identified. Compound 24 and Compound 35 bound the human recombinant NOP receptor expressed in CHO_{h,N,O,P} cell membranes with high affinity (pK_i values 9.62 and 9.14, respectively). Our findings derived from functional studies on CHO_{h,N,O,P} and bioassay studies on native receptors demonstrated that Compound 24 and Compound 35 behave as potent, competitive and selective non-peptide NOP antagonists. Finally, the NOP antagonist properties of Compound 24 have been confirmed in vivo in the mouse tail withdrawal assay.

AstraZeneca for financial support and use of equipment