Lectins are proteins or glycoproteins of non-immune origin, capable of specific interaction with, and reversible binding to carbohydrate moieties of complex glycoconjugates. Lectins are ubiquitous in nature and are present in all living organisms. In nature lectins are implicated in cell recognition and adhesion processes, and have been described as "second generation" mucoadhesives. The aim of this project was to identify lectins that could be incorporated into dosage forms to allow their retention within the oral cavity. This would allow prolonged localised drug delivery. Initial screening studies on human buccal cells, usmg the avidin-biotincomplex/ diaminobenzidine method suggested that all the lectins tested appeared to bind to varying degrees, as visualised by a diaminobenzidine (DAB) precipitate to the oral epithelial surface. The stain intensities were analysed semi-qualitatively with E micro densitometer GN5 (Barr and Stroud). A substantial reduction in lectin binding was observed after exposing buccal cells to a series of lectin solutions pre-treated with secretor and non-secretor saliva. However when bound to the buccal cells, there was little displacement of lectins on exposure to either saliva types. Kinetic binding studies revealed the presence of the lectins from Pisum sativum and Arachis hypogaea, after only 20 s contact. There were some differences in lectin binding evident between rat oral tissue and human buccal cells, but those that bound avidly to both were selected for furthe studies. In order to allow a quantitative assessment of binding, the lectins from Canavali ensiformis, Arachis hypogaea and Triticum vulgaris were labelled with Technetium-99n (Tc-99m) using a cyclic diethylenetriamine pentaacetic acid conjugation technique. In this preliminary study it was found that 1.18 fmoles of the Canavalia ensiformis lectir 3.27 fmoles of the Arachis hypogaea lectin and 11.11 fmoles of the Triticum vulgari lectin bound per buccal cell. This was reduced when Tc-99m-labelled Canavali ensiformis lectin was inhibited by the presence of 40% w/v glucose solution. It was estimated that a therapeutic dose of a potential dosage form could be conjugated to the Triticum vulgaris lectin within the oral cavity, thus demonstrating the feasibility of using this, and possibly other lectins in a drug delivery strategy. Preliminary in vivo studies in the conscious rat model suggested that the Tc-99m-labelled Canavalia ensiformis an Triticum vulgaris lectins were present within the oral cavity, 1 hand 2 h respectively after application, with about 25% of the Canavalia ensiformis lectin being lost into the stomach through swallowing. Transmission electron microscopy studies were initiated to determine the exact sites of lectins binding to the human buccal cell. Fresh, unfixed human buccal cells were incubated in a solution containing 20 nm gold-labelled Canavalia ensiformis lectin. was found that after processing, the lectin bound to the external surface of the buccal cells, or to external mucin. No binding was observed when pre-fixed human buccal cells were used, indicating that fixation affects the glucose/ mannose-containing binding site Canavalia ensiformis lectin binding to the buccal cells was partially inhibited by the presence of glucose, the hapten sugar. It was concluded that lectins will bind to the rat oral epithelial and human buccal cell surfaces to varying degrees, and will persist, even in the presence of saliva. In particular the lectin from Triticum vulgaris was shown to have good stability in the conscious rat model for up to 2 h, and shows great promise for use in a potential lectin-containing drug delivery system.

No description available.

Cannabinoid receptors play an important role in regulating bone mass and bone turnover. Studies in our laboratories have shown that young mice lacking type 1 cannabinoid receptor (CNR1-/-) had increased bone mass and were resistant to ovariectomy-induced bone loss. Other workers have reported that type 2 cannabinoid receptor knockout mice (CNR2-/-) develop age-related osteoporosis. The aim of this PhD thesis was to further investigate the role of CNR2 in bone metabolism in vitro and in vivo, using genetic and pharmacological approaches. This study showed that CNR2-/- mice had normal bone mass and bone turnover at 3 months of age, but following ovariectomy, CNR2-/- mice were partially protected from bone loss, because of a mild defect in osteoclast formation and bone resorption. In keeping with this, studies in vitro showed that RANKL-stimulated bone marrow cultures from CNR2-/- mice had fewer osteoclasts than cultures from wild type littermates. The CNR2-selective antagonist/inverse agonist AM630, inhibited osteoclast formation in wild type bone marrow cultures in vitro and prevented ovariectomy-induced bone loss in wild type mice in vivo. In contrast, osteoclast cultures from CNR2-/- mice were resistant to the inhibitory effects of AM630 at low concentrations and CNR2-/- ovariectomised mice did not respond to its protective effects at low doses, consistent with a CNR2- mediated effect. These results indicate that CNR2 regulates bone loss under conditions of increased bone turnover, such as ovariectomy, by affecting osteoclast differentiation and function. CNR2-deficient mice developed accelerated age-related osteoporosis and by 12 months of age they had a significant reduction in osteoblast numbers and bone formation, whereas osteoclast numbers remained comparable to wild type littermates. In agreement with this, osteoblasts derived from bone marrow of CNR2-/- mice had reduced PTHstimulated alkaline phosphatase activity and ability to form bone nodules, when compared with wild type cultures. The CNR2-selective agonist, HU308, stimulated bone nodule formation in wild type calvarial osteoblast cultures in vitro and reversed ovariectomy-induced bone loss in wild type mice in vivo. HU308 had blunted effects on bone nodule formation in cultures from CNR2-/- mice and no significant effects on ovariectomy-induced bone loss in CNR2-/- mice, indicating a CNR2-mediated effect. These studies demonstrate that CNR2 protects against age-related bone loss by mainly enhancing osteoblast differentiation and bone formation. In conclusion, type 2 cannabinoid receptors protect from bone loss by maintaining bone remodelling at balance. In addition, type 2 cannabinoid receptor agonists show evidence of anabolic activity, whereas antagonists/inverse agonists show evidence of antiosteoclastic activity in vitro and in vivo.

Ochratoxins are a group of nephrotoxins produced by a variety of moulds and were first described in 1965 [1]. Dietary exposure to ochratoxins represents a serious health issue and has been associated with several human and animal diseases including porcine nephropathy, Human Endemic Nephropathies and urinary tract tumours in humans. More than 20 years ago, ochratoxin A (OTA) - the most prominent member of this toxin family - was shown to be carcinogenic in rodents and since then extensive research has been performed in order to investigate whether OTA acts by a DNA reactive mode of action. Only recently, this theory has been conclusively disproven and a non-genotoxic mechanism is currently widely accepted for renal toxicity and carcinogenicity of OTA. The work presented in this thesis contributed to this field of science in various ways. First of all, new renal in vitro models were established and existing models were improved for the investigation of renal ochratoxin toxicity. Using these models, the in vitro effects of various OTA exposure scenarios were investigated and endpoints included amongst others general cytotoxicity, cell cycle analysis, protein binding and toxin transport. In all of these studies, distinct species- and sex-differences were observed which mirrored the observed effects in vivo described in literature. Furthermore, the differential toxicity of ochratoxin group members was investigated, the results of which inferred a need for specific detection of OTA and OTB. Due to the lack of such a detection tool, a new, highly specific and sensitive OTB-ELISA was successfully developed based on an OTB-specific monoclonal antibody produced and characterised in our laboratory. This tool allows screening of OTB in food and feed products, which will further improve detection and risk assessment. All of these studies contributed to the elucidation of the underlying mechanisms of ochratoxin toxicity. Therefore, this thesis can be seen as a fundamental part of a paradigm change on our understanding of how ochratoxins exert their harmful long-term effects in humans and animals, thus elucidating the ochratoxin enigma.

No description available.

Estrone sulfatase (STS) has been shown to play a major role in the biosynthesis of estrogens [in particular estrone (E 1) which subsequently leads to the biosynthesis of estradiol (E2)] and therefore the initiation and progression of hormone-dependent breast cancer in post menopausal women. Recently, a range of thiosemicarbazone-based compounds have been reported as inhibitors of STS, however, due to the mode of action of these inhibitors, no detailed structure-activity relationship (SAR) was offered. In an effort to investigate the SAR within the thiosemicarbazone-based compounds, the current project considered the synthesis and biochemical evaluation of a range of novel thiosemicarbazone-based compounds. The target compounds were synthesised through a reaction between a carbonyl containing starting material (benzaldehyde- or acetophenone-based starting material) with N-cyclohexylhydrazinecarbothioamide using ethanol as the reaction solvent. When benzaldehyde was used for the reaction to produce benzaldehyde N-cyclohexylthiosemicarbazone-based compounds, the target compounds were obtained in good to excellent yield -the target compounds were obtained in a range of 60% [for 1-(4-methylphenyl) ethan-1-one Ncyclohexylthiosemicarbazone (218)] to 92% [for benzaldehyde Ncyclohexylthiosemicarbazone (201)]. However, when acetophenone-based starting material was used, for example 1-phenylethan-1-one Ncyclohexylthiosemicarbazone- based compounds, the target inhibitors were obtained in poor yield (30% or lower)- it was postulated that steric hindrance was the major factor in the reduced yield, indeed, in the most hindered case no product was obtained. Attempts to increase the yield, involving the use of a higher boiling point solvent such as toluene resulted in the production of a by-product, namely, N,N -dicyclohexylhyd razine-1 ,2 -d icarbothioam ide(200). The biochemical evaluation (using STS from rat liver) of a small range of the thiosemicarbazone-based compounds showed that these compounds are weak inhibitors of STS in comparison to the sulfamate-based standard compounds, namely COUMATE and EMATE, however, the evaluation of these compounds suggested that hydrogen bond interaction between the inhibitor and the binding site was important in the inhibition of STS. A range of sulfonate-based potential inhibitors were also synthesised using a multistep synthetic route with the initial synthesis of a range of 4-hydroxyphenyl ketone-based compounds. The phenyl ring was then derivatised involving the synthesis of the 3,5-dibrominated derivatives which were subsequently derivatised to both the methanesulfonate- or the trifluoromethanesulfonatederivatives. These compounds were produced without any major difficulties (except for the dibromination step where the larger alkyl chain containing compounds could not be synthesised in sufficient quantity to proceed to the sulfonate derivatives). The sulfonate-based compounds were not evaluated against STS due to the relocation of the research group, however, they are currently undergoing biochemical evaluation at the University of the West of Scotland.

Nucleic acid therapies may have significant potential for effectively treating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be effective, the restorative pDNA or inhibitive siRNA must access the viable skin layers and cells in a stable and functional form, preferably without painful administration. Microneedles are able to penetrate the stratum corneum skin barrier in a minimally invasive manner to allow targeted delivery of therapeutic macromocules. To date, there are limited studies reporting the delivery of nucleic acids, particularly siRNA, to the skin using microneedle devices. A range of in vitro, ex vivo and in vivo skin models was developed to characterise nucleic acid delivery and functional response. In vitro studies conducted in both continuous and primary keratinocyte cultures provided proof-of-concept of efficient and non-toxic cell uptake and gene silencing with siRNA and moderately efficient gene expression with pDNA. In initial studies, pDNA and siRNA was pre-complexed with lipid-based transfection reagents, however, in the case of siRNA, coating of the lipoplexes onto microneedles resulted in a reduction in functionality. Hence, modified self-delivery (sd) siRNA formulations were used in subsequent microneedle delivery experiments. Stainless steel microneedles coated with reporter pDNA or sd-siRNA were successful in penetrating the stratum corneum barrier of ex vivo viable human breast skin. It was difficult to demonstrate equivocally both plasmid gene expression and functional gene silencing in the skin explants, which only remain viable for short periods. Delivery of pDNA and sd-siRNA to in vivo mouse skin however, resulted in demonstrable gene expression and gene silencing, particularly evident at the protein level, in an established transgenic reporter mouse skin model. Overall, these investigations generally support the use of the coated steel microneedles for the simple and potentially self-administrable delivery of nucleic acids to the skin.

No description available.

The concept of collecting Adverse Events (AEs) arose with the advent of the Thalidomide incident. Prior to this the development and marketing of drugs was not regulated in any way. It was the teterogenic effects which raised people's awareness of the damage prescription drugs could cause. This thesis will begin by describing the background to the foundation of the Committee for the Safety of Medicines (CSM) and how AEs are collected today. This thesis will investigate survival analysis, discriminant analysis and logistic regression to identify prognostic indicators. These indicators will be developed to build, assess and compare predictor models produced to see if the factors identified are similar amongst the methodologies used and if so are the background assumptions valid in this case. ROC analysis will be used to classify the prognostic indices produced by a valid cut-off point, in many medical applications the emphasis is on creating the index - the cut-off points are chosen by clinical judgement. Here ROC analysis is used to give a statistical background to the decision. In addition neural networks will be investigated and compared to the other models. Two sets of data are explored within the thesis, firstly data from a Phase III clinical trial used to assess the efficacy and safety of a new drug used to repress the advance of Alzheimer's disease where AEs are collected routinely and secondly data from a drug monitoring system used by the Department of Rheumatology at the Haywood Hospital to identify patients likely to require a change in their medication based on their blood results.

Valproic acid (VPA) is extensively used for the treatment of epilepsy. This drug also functions as an inhibitor of histone deacetylase enzymes and causes the expression of growth arrest genes. It has been reported that mild to moderate cognitive impairments occur in adult patients taking VPA. This investigation aimed to examine the relationship between cognition and changes in cell proliferation within the rat hippocampus, a brain region where continued formation of new neurons is associated with learning and memory. Also, the antiproliferative function of VPA was investigated in rat and mouse hippocampal neural stem cells (NSCs) and cancer cell lines in vitro. The cognitive and antiproliferative effects of VP A were determined in adult Hooded Lister rats treated with VP A (300mg/kg) by intraperitoneal injection twice daily for 10 days. Cognition was assessed by the Novel Object Location (NOL) and contextual fear conditioning tests. Cell proliferation within the subgranular zone (SGZ) of the dentate gyrus was determined by immunostaining for Ki67. Additionally, levels of the brain-derived neurotrophic factor (BDNF), doublecortin (DCX) and the receptor Notch1 expression were measured by Western blotting. The results showed that animals treated with VPA had a hippocampal specific cognitive impairment as shown by NOL, test but not contextual fear conditioning. This was linked to a significant reduction in cell proliferation within the SGZ. Moreover, VPA treatment statistically significantly decreased levels of BDNF and Notch1, but not DCX within the hippocampus. The antiproliferative effect of VPA treatment (0.3 and 1 mM) for 24 hours was investigated in hippocampal neural stem cells (NSCs) in vitro. The numbers of neurosphere and cell proliferation assessed in VPA-treated rat and mouse hippocampal NSCs showed significantly decreased in a concentration-dependent manner. Levels of Notch1 , Sox2, nestin and c-Myc gene expression were quantified using qPCR. This revealed that 1 mM VPA reduced expression of Notch1 , Sox2 and nestin but not c-Myc. However, there were no changes in levels of these gene expressions in 0.3 mM VPA. The influence of VPA on cancer cell proliferation was examined in human Epn1, Med1 and SHSY5Y cell lines by treating with 1,2 and 3 mM VPA for 72 hours. The data of cell proliferation showed that VPA produced a significant reduction of tumour cell growth in a dose-dependent manner in all three cell lines. VPA (1, 2 and 3 mM) induced levels of Notch1 expression in SHSY5Y cell lines. Additionally, the number of dividing cells of Epn1 and Med1 treated with 2.5 mM VPA was significantly decreased compared to untreated cells using Ki67 immunostaining. However, Notch1 expression was not detected in either Epn1 or Med1 cell lines. These results indicate that VPA treatment induced cognitive deficits in rats and that this was associated with a reduction in hippocampal NSCs both in vivo and in vitro. In addition, VPA reduced BDNF and Notch1 expression in the hippocampus. Moreover, VPA induced a decrease of levels of Notch1, Sox2 and nestin gene expression in hippocampal NSCs. The present study also reveals that antiproliferative effect of VPA was associated with an increase of Notch1 expression in SHSY5Y. However, this action of VPA appeared to have no link with Notch1 expression in Epn1 and Med1 cell lines.