Considering the importance of nitrogen-rich heterocycles in drug discovery, a novel strategy towards heterocycle synthesis was envisioned using cyanamide chemistry. Synthesis which involve mild conditions, avoids multi-step sequence and non-toxic reagents are desirable for generation of large combinatorial libraries of drug molecules. We envisaged that the NCN linkage of the cyanamide as well as the concomitant use of the nucleo-and electrophilic centres of the cyanamide could provide a novel synthetic route towards nitrogen heterocycles. The first part (Ch-2) constitute the bulk of the thesis and it focuses on the generation of cyanamide ion and its cyclisative capture with a 1,3-dipole – nitrile oxide in situ. The cycloadduct -1,2,4-oxadiazol-5(4H)-imine was obtained in good yields, which was further transformed into pharmacologically important cores like oxadiazolone and amidines. A library of the different heterocyclic cores was generated, which tolerated a wide variety of functional groups in good to excellent yields. In the second part (Ch-3), we developed a novel protocol for the synthesis of 1,2,4-triazol-3-imine via a formal 1,3-dipolar cycloaddition of in situ generated nitrile imines and cyanamide ion. Further hydrolysis furnished with 1,2,4-triazol-3-one, which is an important core from medicinal chemistry point of view. The concomitant generation and reaction of two reactive species- 1,3-dipoles and cyanamide ion was achieved in a single pot in situ to provide a route towards novel and pharmaceutically important heterocyclic cores. The present work provides a platform for the development of cyanamide derivatives as a ‘single-reagent—diverse-scaffolds’ strategy for time efficient library delivery of structurally diverse molecules.

Poverty stricken areas of the world are affected by Neglected Tropical Diseases, with an estimated 1 billion sufferers. As well as inadequate living conditions and healthcare, there has been very little pharmaceutical incentive to tackle these diseases. As a result, the diseases are still spreading. Drugs available on the market suffer from poor efficacy, high toxicity, increasing resistance and inappropriate dosing for rural treatment. The nature of many NTDs prevents the use of vaccinations. Therefore, more efficacious and safe treatments are sought after. The folate pathway has been extensively studied in a number of organisms, with its essentiality exploited in a number of drugs and drug targets. The same cannot be said for the kinetoplastids. Drug discovery programmes have focused on targeting enzymes of the folate metabolism with very little clinical success. Despite showing significant inhibition of the parasitic enzymes, potency is seen to decrease in cellular and animal models. Understanding how the folate pathway operates in these organisms could provide insight into where and how anti-folate compounds bind. This information could then be used to facilitate better drug treatments for the kinetoplastids. This thesis describes a number of approaches undertaken to better understand folate metabolism in kinetoplastids. Clinical and literature anti-folate compounds were immobilized onto resins, followed by chemical proteomics, utilizing novel techniques (iTRAQ), to allow for target identification. Using competition studies, specific and non-specific targets were identified in parasitic lysate (T. brucei and L. major) for each anti-folate compound. This method was further exploited by creating a folate resin (Folate beads). The resin had the potential to pull down 9 proteins from the “folate-ome”. In future studies, the resin can be used to enrich for the folate proteins in kinetoplastids and related organisms. Alongside the studies of the folate proteins, it was also desired to study proteins involved in the essential pterin pathway. This pathway has not been extensively studied in kintoplastids, with the exception of PTR1 (by-pass protein for DHFR). The failure to synthesise pterin derivatives for bead coupling led to a fragment screening campaign being carried out on QDPR in leishmania major. Working through a triage workflow, two moderately potent fragments were identified, showing inhibition against LmQDPR. Through structure-free optimization strategies, greater than 100 optimized fragments were synthesised in a bid to understand SAR. Although this work remains incomplete, LmQDPR has been successfully crystalized with 23 hit fragments, which are awaiting further biophysical analysis to understand binding.

No description available.

The aim of this study was to study the electrophysiological and mechanical effects of the gap junction uncoupler 1-heptanol in the left ventricle (epicardial surface) of the artificially perfused Langendorff rabbit heart. Specifically, electrical restitution and the dispersion of repolarisation were studied. Methods. Using a single monophasic action potential (MAP) electrode, in the healthy and failing (coronary ligated) heart, the effect of 1-heptanol was studied on rate dependent changes in action potential duration. Dispersion of repolarisation was measured sequentially. A 32 MAP electrode array was developed to simultaneously record dispersion of repolarisation from the epicardial surface of the left ventricle of healthy hearts. Restitution was studied using an extrastimulus protocol that involved electrically stimulating the heart with 16 S1 stimuli (350ms intervals), and an extrastimulus S2. S1-S2 interval was increased progressively from 70 to 600ms. S1-S2 changes of 5ms were made between 70 and 150ms, 10ms between 150 and 350ms, and 50ms between 350 and 600ms. Protocols were run at 37°C, initially in Tyrode's solution, then after addition of 0.3mM 1-heptanol. Results. The single catheter study showed that failure significantly (P<0.05) prolongs MAP duration between cycle lengths of 250ms and 650ms. No base to apex changes, changes in dispersion of repolarisation or ventricular fibrillation thresholds were observed. 1-Heptanol, at cycle lengths above 350ms, significantly (P<0.05) decreased MAP duration in failing and healthy hearts. 1-Heptanol however did not alter the dispersion of repolarisation or ventricular fibrillation threshold in healthy and failing hearts. The last SI MAP in the 16 beat train and the S2 MAP obtained using the 32 electrode array were analysed at 90% repolarisation (MAPD90). S2 MAPD90 increased with S I-S2 interval up to -180ms but decreased at longer intervals. 0.3mM l-heptanol exacerbated this negative slope in the restitution curve from (mean±SEM) -0.031±O.004 in Tyrode's compared to -O.063±O.005 in O.3mM 1-heptanol (P<O.OOI). Dispersion of repolarisation was increased (p>O.05) in the presence of O.3mM l-heptanol. Conduction delay was increased from (mean±SEM) 44.2±0.82ms to 49.2±O.87ms (P<O.OOI). The possibility of an effect on the single cell being the mechanism behind the exacerbation of the negative slope by l-heptanol was investigated in a single cell study. The effect of l-heptanol on single cell fractional shortening and Ca2+ handling was examined. At 1 and 3Hz l-heptanol decreases fractional cell shortening from (mean±SEM): at 1Hz 9.3±0.8% in Krebs to 5.7±O.7% (O.03mM 1-heptanol), 5.6±O.9% (O.lmM l-heptanol) and 3.2±0.8% (O.3mM lheptanol) (P<O.Ol); at 3Hz lO.6±O.8% in Krebs to 6.S±1.4% (O.03mM l-heptanol), 7.7±1.4% (O.lmM l-heptanol) and 4.7±l.S% (O.3mM l-heptanol) (P<O.Ol). The negative inotropic (Le. the reduction in contractility) effect of I-heptanol indicated that this agent is not a specific gap junction uncoupler. SR ci+ release was reduced at 3Hz only by (mean±SEM) 7.8±O.04% (O.03mM l-heptanol), 5.0±0.03% (O.lmM 1-heptanol) and 7.9±O.02% (P<O.OS),indicating an effect on the L-type Ca2+ channel or ryanodine receptor. The effect on the L-type Ca2+ channel was investigated by the use of nifedipine firstly in single cells then in the whole heart. Nifedipine decreased single cell fractional shortening at 1Hz from (mean±SEM) 8.9±O.7% in Krebs to S.8±O.7% (O.lJ,tM nifedipine), 3.4±O.9% (O.l5J,tM nifedipine, P<O.OOI)and O.8±O.2% (O.2JlM nifedipine, P<O.OOl). SR Ca2+ release was also reduced from (mean±SEM) O.75±O.017% in Krebs to O.72±O.024% (O.IJlM nifedipine), O.73±0.024% (O.l5JlM nifedipine), O.71±O.016% (O.2J,tM nifedipine) (P<O.OS). In the whole heart nifedipine did not induce a negative slope in the restitution curve indicating no role of the L-type Ca2+ channel in this phenomenon. Carbenoxolone, a novel specific gap junction uncoupler failed to induce a negative slope in the electrical restitution curve of the whole heart but did increase dispersion of repolarisation (P>0.05) and caused a significant conduction slowing from (mean±SEM) 45.50±2.I2ms in Tyrode to 55.11±2.82ms in carbenoxolone (P<0.05). Carbenoxolone has an inconsistent effect on single cell fractional shortening and Ca2+ handling. Conclusions. The biphasic relationship and the increased dispersion of repolarisation in the presence of 0.3mM I-heptanol may have implications for the development of alternans and/or arrhythmias (Gilmour and Chialvo, 2000). The cause of the negative slope is as yet unknown. but it is likely that it is an effect on the single cell rather than gap junction uncoupling.

Previous work on the unconditioned effects of amphetamine in rats has examined qualitative changes in behaviours which become stereotyped and quantitative changes in locomotion. Stereotyped behaviours have been adopted as a model of raised caudate-putameri function whilst locomotion has been adopted as a model of raised mesolimbic dopamine function. These models have been used to study drugs which are effective in the treatment of schizophrenia. Only locomotion is reliably antagonised by all classes of antipsychotic drugs, although it has been hypothesised that, under some doses of amphetamine, locomotion may also become stereotyped. The Lyon-Robbins hypothesis of the behavioural effects of amphetamine predicts competition between the output of the mesolimbic and caudate-putamen, and would predict that stereotyped locomotion represents a 'blending' of mesolimbic and caudate-putamen behavioural output. An experiment was conducted to test the Lyon-Robbins hypothesis using contrast-based image analysis to determine the spatio-temporal characteristics of open-field locomotion. A further four experiments examined the effects of a classic antipsychotic (haloperidol), the atypical antipsychotics (clozapine and sulpiride) and a putative antipsychotic (a 5-HT3 antagonist, ondansetron) on open-field locomotor routes taken by rats following treatment with 3.5mg/kg amphetamine. Measures of stereotyped locomotion derived from image analysis were supported by a novel form of behavioural analysis based on multi-dimensional scaling which provided an integrated analysis of behavioural change following drug treatment. Haloperidol blocked locomotion and stereotyped behaviours including stereotyped locomotion, whereas clozapine, sulpiride and ondansetron blocked locomotion but not stereotyped locomotion and in some cases increased stereotyped behaviours. This suggests that stereotyped locomotion represents synergistic functioning of both mesolimbic and caudate-putamen systems, when the output from the caudate-putamen is insufficient to over-ride that of the mesolimbic system. Antagonism of a 5-HT3 enhancement of mesolimbic locomotor activity by ondansetron allowed latent 5-HT and dopamine mediated behaviours to be expressed. This effectively mimicked a leftwards shift of the amphetamine dose response curve, hypothesised as amplification of the caudate-putamen output. These findings lend support to the Lyon-Robbins hypothesis of the behavioural effects of amphetamine.

No description available.

The Rutaceae family consists of 150 genera and 1,500 species, which are herbs, shrubs, and trees. The members of genus Glycosmis and Clausena are aromatic (contain volatile oils) and traditionally used for fever, swollen spleen, digestion, topical infections, skin itch, scabies, boils and ulcers. Accordingly from this family, Tetractomia roxburghiana, Glycosmis calcicola, and Clausena excavata were selected for systematic biological screening to exploit and identify compounds which may serve as subsequent leads for the treatment of skin diseases. While the initial aim of the programme had been to characterise these barely studied plants, the programme was subsequently extended to study their biological activity in order to justify their traditional use as medicines. During the course of this study, supporting analytical methodologies were used extensively and ultimately the evaluation of these methodologies contributed a significant proportion of the overall research programme. Initially crude extract was subjected to column chromatography and the compounds were isolated by overloading an analytical HPLC column. By the end of the programme, crude extracts were being analysed directly by gradient reversed-phase HPLC with subsequent direct scale up to preparative isolation on a (250 ´ 22) mm id column. Analytical reversed phase high performance liquid chromatography (HPLC) of the crude methanolic leaf extracts of T. roxbhurghina, G. calcicola, and C. excavata was carried out in order to qualitatively assess the number of constituents present in each fraction. Separation was achieved by using ACE-5-C18 (250 ´ 4.6 mm) with a flow rate of 1.5 ml/min, with the UV detection at 254 nm. Semi-preparative and preparative HPLC were also carried out in order to isolate components of these mixtures. Using spectral analysis, as swertisin, gallic acid, α-asarone and angelicin (furanocoumarin) were identified. In the same way, angelicin was identified from the methanolic leaf extract of Glycosmis calcicola and preparative HPLC of the methanolic leaf extract of Clausena excavata afforded three compounds, namely 2-(3,4-dimethoxy-phenyl)-5,7-dimethoxy-chromen-4-one, 2-(3,4-dimthoxy-phenyl) 3,7-dihydroxy-5-methoxy-chromen-4-one and 5,7-dihydroxy-2-phenyl-chromen-4-one (chrysin), which were confirmed by spectroscopic methods. Micellar electrokinetic chromatography (MEKC) was evaluated as a possible high-resolution technique for checking the purity of fractions isolated from preparative RP-HPLC. However it proved more effective to exploit orthogonal ( to RP-HPLC) modes of LC by using –NH2 and –SCX ion-exchange HPLC columns and/or, if resolution on analytical RP-HPLC was possible, structural elucidation was carried out using LC-NMR-MS. With respect to biological activity, a range of procedures that had been established at the University for checking activity against skin diseases was used. Free radical induced lipid peroxidation model has been selected for evaluation of antioxidant activity of the extract. The anti-oxidant activity of these extracts and compounds were assessed by free radical induced lipid peroxidation model. The results indicated that the methanolic leaf extract of Tetractomia roxburghiana showed marked anti-oxidant activity whereas methanolic leaf extract of Glycosmis calcicola and Clausena excavata showed moderate anti-oxidant activity. The IC50 value of the methanolic leaf extract of Tetractomia roxburghiana was found to be 201.3 µg/ml; whereas those for Glycosmis calcicola and Clausena excavata were found to be 450.6 µg/ml and 1106 µg/ml respectively. For all the extracts, no anti-bacterial activity was found against Staphylococcus aureus, Streptococcus pyogenes, Propionibacterium acne. Also no anti-fungal activity against Candida albicans was found. A total of three crude methanolic plant extracts, four isolated compounds and eleven semi-purified fractions were tested for in-vitro efficacy, using an agar incorporation method to determine the minimum inhibition concentration (MIC), against the dermatophyte species; Trichophyton rubrum, Trichophyton mentagrophytes and Epidermatophyton floccusum. The MIC value for crude methanolic extracts of Tetractomia roxbhurghiana and Glycosmis calcicola was found to be 62.5 µg/ml and 31.2 µg/ml against T.rubrum and T.mentagrophytes, whereas the methanol extract of Clausena excavata did not show any activity against dermatophytes. In conclusion, the anti-oxidant activity of Tetractomia roxburghiana was found to be comparable with that of propylgallate, which was used as a standard drug thus confirming, as anticipated, that Tetractomia roxburghiana might be a good source of anti-oxidant drugs. The extended degree of anti-oxidant activity displayed by methanolic extract of Tetractomia roxburghiana could be contributed to the presence of swertisin, gallic acid and angelicin, which are proven anti-oxidants. The anti-fungal activity of Glycosmis calcicola could be partly due to the presence of angelicin.

The importance of electronic healthcare has caused numerous changes in both substantive and procedural aspects of healthcare processes. These changes have produced new challenges to patient privacy and information secrecy. Traditional privacy policies cannot respond to rapidly increased privacy needs of patients in electronic healthcare. Technically enforceable privacy policies are needed in order to protect patient privacy in modern healthcare with its cross organisational information sharing and decision making. This thesis proposes a personal information flow model that specifies a limited number of acts on this type of information. Ontology classified Chains of these acts can be used instead of the "intended/business purposes" used in privacy access control to seamlessly imbuing current healthcare applications and their supporting infrastructure with security and privacy functionality. In this thesis, we first introduce an integrated basic architecture, design principles, and implementation techniques for privacy-preserving data mining systems. We then discuss the key methods of privacypreserving data mining systems which include four main methods: Role based access control (RBAC), Hippocratic database, Chain method and eXtensible Access Control Markup Language (XACML). We found out that the traditional methods suffer from two main problems: complexity of privacy policy design and the lack of context flexibility that is needed while working in critical situations such as the one we find in hospitals. We present and compare strategies for realising these methods. Theoretical analysis and experimental evaluation show that our new method can generate accurate data mining models and safe data access management while protecting the privacy of the data being mined. The experiments followed comparative kind of experiments, to show the ease of the design first and then follow real scenarios to show the context flexibility in saving personal information privacy of our investigated method.

The coherent body of research described in the existing published work is concerned with new assay method development and validation using novel systematic approaches for pharmaceutical and diagnostic compounds. The first stage of the research was to study how analytical method development and validation are typically carried out at present and to formulate this into a simple step-by-step approach. Such a template and protocol was not only used as the foundation of this research programme but could also serve as a simple systematic guide for other practitioners and those new to the field. Furthermore, it was recognised that this protocol should satisfy the requirements of the most strategically important regulatory agencies. The second stage of this research involved evaluation and application of the above validation approach to new methods that were developed for a diverse range of analytes and samples. A new purity assay for 1,10-phenanthroline-5,6- dione and 4,7-phenanthroline-5,6-dione using high-performance liquid chromatography (HPLC) was developed and validated. Impurities in these compounds were identified by liquid chromatography-mass spectrometry (LCMS). Best practice in method development and validation is equally important in the analysis of both active components and excipients in formulated products. In the first case, a liquid chromatography assay method for determining the content of 2-(diethylamino)-N-(2,6-dimethylphenyl) acetamide in a gel formulation was developed and validated. In the second case, the individual contents of three phydroxy benzoic acid ester preservatives in a complex multi-component sample were determined following the development and validation of a liquid chromatography method. Finally, the validation approach was evaluated as applied to another analytical technique. Here, gas chromatography (GC) successfully used to develop a novel assay for p-cymene in tea tree oil formulations presented different analytical problems because of the very complex nature of this natural product. Stability study information to increase the shelf life of the product and validation data for the analytical method for p-cymene content was critically evaluated. iv In essence, the critical review of the requirements for method validation for various agencies and the subsequent preparation of guidelines on how to go about method validation have had a significant impact on how analytical practitioners worldwide go about method development and, more importantly, method validation. Further it was possible to apply these guidelines to conduct a series of effective, successful method validation for assays involving a range of typical pharmaceutical samples.

The use of API interfaces with quadrupole mass spectrometers has been shown to give rise to good sensitivity, selectivity, and robustness for the interfacing of LC to MS. Since their introduction in the 1990s the technique has rapidly become widespread, but at the outset of this research programme, there were still a number of problems associated with it, particularly when dealing with complex sample matrices. The aim of this research programme was to study illustrative examples of the kinds of problems associated with the analysis of biological samples using LC-API-MS in an attempt to arrive at strategies which could be employed to eliminate, or at least compensate for, the problems. Commonly reported problems include the occurrence of matrix effects - a change in response of the target analyte(s) as a result of the presence in the samples of co /late eluting interferences. An investigation which compared ESI with APCI ionisation illustrated a significant drawback in the accepted methodology for the elimination of matrix effects. Optimal LC conditions for a number of assays may use non-MS-friendly mobile phases. A simple and convenient solution to this problem was found to be the post column addition of organic modifier, which reproducibly and reliably enhanced sensitivity. This approach was initially used for a range of dihydropyridine calcium channel blockers and was subsequently applied to a range of chiral compounds from different therapeutic groups to illustrate that this was applicable as a generic technique for increasing sensitivity (typically by around an order of magnitude) in low organic mobile phases. Strategies to develop and validate methods for the determination of endogenous analytes in a biological fluid were investigated. This involved the use of a surrogate matrix, to develop a method for the determination of endogenous testosterone in human serum and the use of non-matrix calibration standards for the successful development and validation of a method for the analysis of indolyl 3 acryloylglycine (IAG) in human urine. As a result of observations suggesting promotion of ionisation of deltamethrin in liver tissue sample extracts, it was postulated that this was due to the presence of high concentrations of surfactants. After confirming the effect, a series of systematic investigations were performed to attempt to understand the mechanism to be able to utilise this as a general method for the enhancement of signal with low sensitivity analytes. It was found that the type of surfactant and concentration used was directly associated with an increased (or decreased) response. Although there remain a number of problems associated with the use of LC-API-MS, the work undertaken for this thesis has successfully demonstrated a number of techniques that can be applied to overcome these problems. Knowledge of the nature of the sample undergoing analysis, the required analytical conditions, and where required careful application of one of the techniques described will ensure that a robust method can be readily developed.