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Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of mortality among infectious diseases. Efforts to combat the disease have been hammered by the emergence of drug resistance as well as drug mismanagement and poverty. In view of the continuous worldwide spread of tuberculosis and declining effectiveness of drugs to fight the disease, there is a need to discover more efficacious anti-tuberculosis agents. InhA, the enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis, is one of the key enzymes involved in the synthesis of mycolic acids. Mycolic acids are known to be important for mycobacterial growth, survival and pathogenicity; thus making the pathway an attractive target for the development of novel anti-tubercular drugs. In this thesis, a combination of several techniques have been utilised to identify potential inhibitors of InhA. The first approach was to carry out a rapid in silico docking study of a library of chemical compounds (approximately 137,000) in order to identify compounds that are most likely to bind InhA. A structure-based virtual screening, using the docking program GOLD, led to the identification of fifteen top compounds which were selected for further experiments on the basis of top fitness scores, good binding mode, interactions and contacts with the protein. To explore further the binding of the selected ligands to InhA, the Molecular Mechanics / Poisson-Boltzmann and Surface Area method was used to estimate the relative binding free energy of the ligands to InhA following molecular dynamics simulations. The binding of all fifteen compounds were found favourable as reflected by negative binding free energy values. To perform the experimental validation of the 'hits’ by steady-state kinetics and inhibition studies, InhA was cloned, expressed, purified and characterised. The compounds were tested "experimentally for inhibitory effect on InhA activity. These studies revealed that seven of the compounds showed more than 20% inhibitory effect against InhA activity, with the highest inhibitory effect observed being 65.7 ± 0.9% inhibition. The compounds that exhibited more than 20% inhibitory activity were further characterised by determining the ICso values and the type of inhibition with respect to InhA substrate 2-£ra/7s-octenoyl-Co-enzyme-A. The most potent compound exhibited an ICso value of 28.06 ± 0.02pM. Submission of the compounds for evaluation as inhibitors of mycobacterial growth in culture revealed that all compounds showed modest antibacterial activity. In addition, crystallographic studies were carried out on InhA mutant and wild-type enzymes. At the time of this thesis work, no crystal structure is available on any InhA mutant ternary complex as well as for one of the reported InhA mutants, Isoleucinel6 to Threonine InhA. Studies were therefore carried out towards obtaining the mutant crystal structures, and on the wild-type enzyme in order to enhance the future structure determination of InhA protein-ligand complex with the identified hits. The mutant proteins were successfully crystallised, as well as the wild-type enzyme which produced a crystal that diffracted to 2.0A

Diclofenac is commonly used 'off-label' for acute pain in children, and it has been shown to be effective for this indication. There is a five-fold range (0.5 to 2.5mg/kg) in dosing of diclofenac for acute pain in paediatric clinical studies, and little published safety information is available. The metabolism of diclofenac to 4'-hydroxydiclofenac is mediated by CYP2C9, the expression of which may differ during development. Three studies have been undertaken to answer the questions: What dose of diclofenac should be given to children with acute pain What are the adverse effects of diclofenac in children treated for acute pain Does the expression of CYP2C9 change with age in children aged one to 12 years The three studies carried out were: A population pharmacokinetic study on a paediatric day surgery ward investigating a new diclofenac oral suspension, results pooled with adult data supplied by the manufacturer and analysed with NONMEM to produce dosing guidelines a clinical safety study to ascertain common adverse reactions of diclofenac in children with acute pain, followed by a systematic literature review to investigate the type and incidence of rare adverse effects and an investigation of the influence of age and CYP2C9 genotype on the formation of 4'- hydroxydiclofenac in children aged one to 12 years using data collected during the pharmacokinetic study. The optimum dose of diclofenac for acute pain in children is lmg/kg. Diclofenac appeared to cause similar types of adverse reactions in children and adults, although the incidence of gastrointestinal bleeding is possibly lower in children. When diclofenac is used as part of the analgesic regimen in the peri-operative period, children suffer less nausea and vomiting, and no increase in operative bleeding. No differences were found in the expression of CYP2C9 estimated using diclofenac 4'-hydroxylation in children aged one to 12 years, which would appear to confirm in vitro findings in paediatric liver samples.

Support vector machines (SVMs) have displayed good predictive accuracy on a wide range of classification tasks and are inherently adaptable to complex problem domains. Structure-property correlation (SPC) analysis is a vital part of the contemporary drug discovery process, in which several components of the search for novel molecular compounds with therapeutic potential may be performed by computer (in silicd). Inferred relationships between molecular structure and biological properties of interest are used to eliminate compounds unsuitable for further development. In order to improve process efficiency without rejecting useful compounds, predictive accuracy of such relationships must remain high despite a paucity of data from which to infer them. This thesis describes the application of SVMs to SPC analysis and investigates methods with which to enhance performance and facilitate integration of the technique into present practice. Overviews of contemporary drug discovery and the role of machine learning place the investigation into context. Computational discrimination between compounds according to their structures and properties of interest is described in detail, as is the SVM algorithm. A framework for the assessment of supervised machine learning performance on SPC data is proposed and employed to assess SVM performance alongside state-of-the-art techniques for in silico SPC analysis on data provided by GlaxoSmithKline. SVM performance is competitive and the comparison prompts adaptations of both data treatment and algorithmic application to explore the effects of data paucity, class imbalance and outlying data. Subsequent work weights the SVM kernel matrix to recognise heavily populated regions of training data and suggests the incorporation of domain-specific clustering methods to assist the standard SVM algorithm. The notion that SVM kernel functions may incorporate existing domain-specific methods leads to kernel functions that employ existing pharmaceutical similarity measures to treat an abstract, binary representation of molecular structure that is not used widely for SPC analysis.

There is a continuing need to make new antitubercular drugs due to development of resistance towards present drugs. To do this, series of pyruvate hydrazones (PVAHs) and their respective Cu(II) complexes and Zn(II) complexes were synthesized and fully characterised. Variable temperature NMR studies of PVAHs indicate the presence of E and Z isomers in the solution. Determination of the obtained single crystal X-ray structures reveals that Cu(II) ion binds to PVAH ligand in 1:1 ratio resulting in square pyramidal geometry in most of the Cu(II) complexes, whereas Zn(II) ion binds to two PVAH ligands in 1:2 ratio giving rise to octahedral geometry. The electrochemical studies of Cu(II) complexes of PVAH performed using cyclic voltammogram indicate the presence of quasi-reversible behaviour assigned to a Cu2+/Cu1+ peak potential. This indicates structural reorganisation of Cu(II) square pyramidal geometry towards Cu(I) tetrahedral geometry. The tetrahedral geometry of a synthesized Cu(I) complex of PVAHs was confirmed by X-ray crystal structure. The stability studies of selected PVAH ligands and their metal complex indicate that the investigated compounds were stable in extreme basic conditions, but they were unstable in extreme acidic conditions due hydrolysis of azomethine bond. However, stability of these compounds in physiological conditions, i.e. in PBS buffer, reveals that ligand hydrolyses slowly over a period of time, whereas the Cu(II) complex remains quite stable over a monitored period of 120 hours. Interestingly, dihydrazide analogue of PVAH was fairly stable in PBS buffer. EPR studies of investigated Cu(II) complexes in DMSO indicate that PVAHs remains strongly coordinated to Cu(II) centre. The evaluation of the antimycobacterial activity showed that the anionic PVAHs and Zn(II) complexes are essentially inactive. Some of the corresponding neutral Cu(II) complexes, however, exhibit promising antimycobacterial activities if tested under high iron (8 μg Fe per mL) conditions. As observed for the related antimycobacterial agent isoniazid, the activity of the complexes decreases if the M. tuberculosis cells are grown under low iron (0.02 μg Fe per mL) conditions. The Cu(II) complexes may thus have a similar mode of action and may require an iron-containing heme-dependent peroxidase for activation. A series hydrophobic cinnamaldehyde hydrazones (CAHs) and their Cu(II) complexes were also synthesized and tested for their antitubercular activity under similar conditions to that of PVAH series. But they failed to show any inhibitory activity due to their poor cellular uptake owing to their limited solubility in aqueous buffer.

A literature survey has shown that some simple peptides and their derivatives and also some amino acid derivatives have pharmacological activities of interest. The thesis describes the preparation and characterisation of amino acid and thence of peptide derivatives of Aspirin, acetylsalicylic acid, and then of Alclofenac, 4-allyloxy-3-chlorophenylacetic acid. Acetylsalicyloyl-(DL)-alanine, and some amino acid and dipeptide derivatives (and one tripeptide derivative) of Alclofenac were prepared. The compounds have been submitted for pharmacological testing. The results obtained todate indicate that Alclofenac-(DL)-alanine methyl ester and Alclofenac-(DL)-alanyl-(DL)-alanine methyl ester are bioactive compounds. They inhibit the release of histamine from rat peritoneal mast cells in vitro. Further, Alclofenac-glycine and its hydrogenated form, 3-chloro-4-n-propoxyphenylacetyl-glycine, show as much anti-inflammatory activity in vivo as does Alclofenac. Derivatives are named arbitrarily as, e.g. Alclofenac-glycine etc where the first part of such composite names refers strictly to the acyl radical derived from the acid, Alclofenac.

Halichlorine 1 is a marine spirocyclic alkaloid, which has shown to exhibit anti-inflammatory properties.1 Due to the complexity of this structure, and the low abundance in nature, the development of total and partial syntheses of this compound have become of interest to the organic chemist. This project aimed to evaluate the therapeutic potential of this class of compounds by producing a library of simplified halichlorine derivatives by addition of Grignard reagents onto a key spironitrone that maps onto the core structure of halichlorine and thence to monitor potential bioactivity by conducting a series of biological assays to determine what effects these compounds have on human U937 cells. Addition of a wide range of Grignard reagents to spironitrone 128 was successful and generally proceed with high diastereoselectivity. In addition, reductive cleavage of the resulting N-hydroxyspirocycles with Zn/AcOH provided a host of N-acetyl-C7-substiuted spirocyclic derivatives 167-172. Reduction with indium provided free amines 173-181. As additions to spironitrone 128 proceeded with undesired stereoselectivity attempts were made to access O-protected spironitrone 204 by oxidation of spiroamines such as 199. This strategy was unsuccessful. In order to explore alternative spirocyclic derivatives, synthetic studies were also directed in attempts to access un-substituted derivatives by ring closing metathesis (RCM) of diene precursors 222-224. While RCM substrates were accessed cyclisation of these did not proceed. It was discovered that heating 6,5-spiroisoxazolidine 102 under pressure in a microwave reactor provided access to the corresponding 6,6-isomer 164 which maps onto the core structure of the amphibian toxin histrionicotoxin (HTX). Oxidation to 6,6-spironitrone 192, as followed by conversion to cycloadducts 193-195, which represent new analogues of the HTX family of alkaloids. Grignard additions to this nitrone, did not proceed in general. Biological screenings using undifferentiated and LPS activated U937 cells helped to identify a number of biologically active derivatives, when tested in the NO and growth and viability assays. The NO assay using LPS activated cells, identified that the adducts containing larger alkyl or aryl chains, particularly the pentyl, hexyl and benzyl adducts, expressed significant differences in NO inhibition at both 10-4 M and 10-5 M concentrations tested, compared to the untreated cells.

No description available.

Aims: Proteinase-activated receptor 2 (PAR2), a GPCR subtype with a novel activation mechanism, has recently received increasing interest due to its potential neuroprotective role in CNS diseases. Investigating the role and properties of PAR2 in CNS was previously made difficult by the limited selectivity and potency of available activators. Recently however, novel PAR2 activators have been developed allowing further characterisation and investigation. Methods: Ca2+ imaging was performed on rat primary hippocampal cultures used at 10-14 DIV. Internalisation studies were performed using PAR2 transfected tsA-201 cells. Mice pup organotypic hippocampal slices were prepared with protection against kainite (KA)-induced neurotoxicity investigated from 15 DIV. Finally, effects of PAR2 activation were assessed in vivo on cardiac function, on behaviour such as locomotion and anxiety and in the EAE mouse model of MS. Statistical analysis were performed using one or two-way ANOVA and Dunnett’s or Bonferroni post hoc tests for multiple comparisons, with p < 0.05 considered as significant. Results: In this report we have been able to study the effects of PAR2 activation in CNS preparations where we monitored comparable increases in Ca2+ concentration to those seen in previous studies following application of peptide-based activators. Furthermore, the proposed PAR2 antagonist GB88, while not inducing changes in Ca2+ concentrations, led to PAR2 internalisation, therefore suggesting it is a biased agonist. Additionally, we have shown in an ex vivo model of excito-toxicity, consistent and long lasting beneficial effects of PAR2 activation. Finally, in the EAE model of CNS inflammation, we have detected beneficial effects induced by PAR2 activation including decreases clinical signs intensity as well as in the relapses occurrence and intensity furthermore, looking at the cytokine profiles, we were able to identify correlations between PAR2 activation and IL-6 production. Conclusions: The results presented in this thesis establish PAR2 activation as neuroprotective in vitro and in vivo in the context of neurodegeneration, thus constituting a solid foundation to establish PAR2 as an intriguing potential target for CNS drug discovery and suggest that modulation of its expression or function may be a viable strategy in the treatment of a large range of CNS diseases.

The legislative environment in India underwent changes in 2005 leading to product patentprotection for pharmaceuticals. Equipped with superior process capabilities acquired forgeneric manufacturing, the pharmaceutical sector embarked on a journey to undertake radicalinnovation. The aim of the research is to explore how changes in the national environmentand asset profile of firms have influenced openness for research of novel drugs. The reviewof literature, synthesis of conceptual framework and data analysis is shaped by the theoreticalapproaches of national innovation systems (NIS), dynamic capabilities and open innovation. The theoretical lens of national innovation systems enables our understanding of what shapesinnovation behaviour of a firm, open innovation approach provides a description of howfirms adopt open innovation and dynamic capabilities concept supports the interpretation ofwhy firms differ in opening up their boundaries. The answers are examined through ninecases of established and start-up pharmaceutical firms in India. New drug research is challenging and requires collaborative effort predominantly at the drugdiscovery stage. However, in the Indian setting, research networks are minimal and there islittle cohesion within the ecosystem among firms, research institutes and universities. Firmsare opening up their R&D innovation process to foreign partners through open innovationstrategies; in-licensing, out-licensing and collaborative innovation that vary by stage of drugresearch. The open innovation pathways adopted by firms are influenced by four 4Rs -resource supplementation and risk mitigation that initiate open innovation, retention ofcontrol and revenue maximization that impel closed innovation. The insights into the strategictrends of these firms unveil an open innovation framework that is relevant to the openinnovation theory and praxis.