The effects of a number of naturally occurring anutrients on various hepatic drug-metabolizing enzyme activities have been studied in rats. These enzyme activities include aromatic hydroxylation (biphenyl-4- and -2-hydroxylases), reduction (p-nitrobenzoic acid reductase), conjugation (4-methylumbelliferone glucuronyl transferase), cytochrome P-450 and cytochrome b[5]. The groups of anutrients studied were terpenoids, alkaloids, flavonoids, esters, ethers, and methylenedioxyaryl compounds. Some of these compounds have shown no immediate effect on the drug-metabolizing enzymes, others have shown a modest induction of some of the above enzymes to the order of 10-15%, a few compounds have shown inhibition. One of the terpenoids, beta-ionone, and two of the methylenedioxyphenyl compounds, safrole and iso-safrole, have exhibited marked induction of all the parameters studied. These three compounds have also been found to decrease the liver glycogen content and to enhance the glucuronic acid pathway of carbohydrate metabolism manifested by an increased excretion of ascorbic acid in the urine. Differences in the enzyme inductive effects of beta-ionone, safrole and iso-safrole dependent on species, age and sex, have also been studied. The enzyme inductive effects of these three compounds have been compared qualitatively and quantitatively to those of phenobarbitone and 3-methylcholanthrene, potent representatives of the two known groups of inducers. The mechanisms of induction of the hepatic drug-metabolizing enzymes by these naturally occurring anutrients have been studied, and in this connection the inhibitors of protein synthesis, actinomycin D (m-RNA synthesis) and thioacetamide (m-RNA release), and of haeme synthesis, 3-amino-1,2,4-triazole, have been used. Other studies have been made, using [3]H-leucine on the rates of synthesis and turnover of proteins in the microsomal and other sub-cellular fractions of the liver. From these experiments it has been shown that beta-ionone, like phenobarbitone, results in a type of enzyme induction which is very largely dependent on the DNA-directed synthesis of new enzyme proteins. Safrole and iso-safrole, on the other hand, are like 3-methylcholanthrene, and result in a type of enzyme induction which is in part dependent on the synthesis of new enzyme proteins and partly results from changes in the conformation of the enzyme or from alterations of its membrane environment. Pretreatment with safrole or iso-safrole has been shown to result in the appearance of a new hepatic microsomal haemoprotein which is normally not detectable in tissue homogenates of untreated animals.

This study has investigated the interactions of a number of methylenedioxy compounds and some substituted benzimidazoles with hepatic microsomal cytochrome P450. Various substituted benzimidazoles were found to bind to rat hepatic microsomal cytochrome P-450, eliciting the formation of different types of binding spectra. A series of 2-n-alkylbenzimidazoles produced either Type I or Type RI binding spectra and these compounds inhibited monooxygenase reactions, Several methylenedioxy compounds elicited the formation of methylenedioxy metabolite-cytochrome P-450 complexes in the presence of either NADPH and oxygen or cumene hydroperoxide in vitro. Methylenedioxy metabolites were bound to the haem iron of cytochrome P-450 and the resultant complexes were less stable in the oxidized (Fe[3+]) than the reduced (Fe[2+]) state. Oxidized isosafrole metabolite-cytochrome P-450 complexes formed in vivo following the administration of isosafrole to rats were readily dissociated in vitro by the addition of certain substrates of the monooxygenase system. Such substrates were typically lipophilic and had a high affinity for the Type I binding site of cytochrome P-450, An apparent increase in cytochrome P-450 levels and enhanced cytochrome P-450-mediated enzyme activities were observed after the time-dependent dissociation had occurred. When hepatic microsomes from rats which received isosafrole in vivo were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis, a protein of 54,000 molecular weight was found to be induced. This protein was identified as a haemoprotein and had an electrophoretic mobility different from the proteins preferentially induced by either phenobarbitone or 20-methylcholanthrene treatment. Both the isosafrole-induced protein and the isosafrole metabolite-cytochrome P-450 complex were observed in microsomal preparations from rats which had received phenobarbitone and isosafrole or 20-methylcholanthrene and isosafrole, Isosafrole-related material was also found to be apparently irreversibly bound to rat and mouse proteins following the administration of [o-[14]C] isosafrole in vivo.

This thesis investigates the role anaesthetics play in the depression of the acute hypoxic and hypercapnic ventilatory response. Particular attention is paid to TASK (TWIK related acid sensitive potassium) channels that are known to be sensitive to both hypoxia and the volatile anaesthetics. After chapters relating to a general introduction and methods, chapter 3 describes studies into the hypoxic and hypercapnic responses of mice with genetic ablation of TASK 1 and/or TASK 3. By using whole animal unrestrained plethysmography, it was demonstrated that any loss of TASK channels resulted in a depression of ventilatory responses to hypoxia and hypercapnia. In isocapnic hypoxia, there is a genotype specific depression by anaesthetics of the remaining AHVR, depending on whether halothane or isoflurane is inspired. Chapter 4 establishes that propofol depresses the chemosensory responses of carotid body glomus cells. Using ratiometric flurimetry to measure the intracellular calcium concentration in isolated neonatal rat glomus cells, it was demonstrated that propofol depresses the normal cellular response to both hypoxia and hypercapnia. Using pharmacological modulators of GABA and nAChR, and single channel patch clamp electrophysiology of TASK, it is apparent that this depression is independent of these candidate mechanisms. Chapter 5 describes the effects of the anaesthetic dexmedetomidine at two levels of the hypoxic chemoreflex arc in rodents. Neither isolated rat glomus cells, nor unrestrained mice show significant depression of hypoxic responses when dexmedetomidine is present. Chapter 6 illustrates the potential role for specific antagonism of TASK channels in stimulating the carotid body. Two TASK channel blockers, A1899 (TASK 1 antagonist) and PK-THPP (TASK 3 antagonist) are shown to cause a significant influx of calcium into isolated glomus cells, and an augmentation in the glomus cell response to hypoxia, which persists in the presence of isoflurane. Overall, this thesis provides evidence that both TASK 1 and TASK 3 channels are essential for normal hypoxic chemosensitivity. It has extended the knowledge that anaesthetics depress glomus cell chemosensation, by demonstrating that propofol too causes this effect. It also proposes that dexmedetomidine may have greater utility in sparing of ventilatory reflexes. By selectively blocking TASK channels, glomus cells can be stimulated, and exploiting this mechanism would be useful in minimising anaesthetic-induced depression of ventilation.

Inductively coupled plasma mass spectrometry (ICP-MS) has been widely used for environmental and trace analysis since its introduction in the early 1980s. This thesis describes an exploration of the potential of HPLC-ICP-MS within the pharmaceutical industry. Determination of platinum in the anticancer drug ZD0473 was performed in comparison with conventional HPLC-MSMS, investigating limit of detection, linearity and reproducibility on spiked samples. Both methods were capable of providing accurate and precise results with samples from rats dosed intravenously 0.5 mg/kg and orally at 6 mg/kg, but the HPLC-ICP-MS Pt method had extended linear range and superior sensitivity, providing a limit of quantification of 0.1 ng/mL compared to 5 ng/mL by HPLC-MSMS. Impurity and metabolite profiles for ZD0473, using platinum as a marker with HPLC-ICP-MS, were compared to profiles from 14C-labelled compound with radioactivity detection, showing that the compound was converted to 2-picoline. Since the number of compounds containing platinum found in the pharmaceutical industry is limited ICP-MS was then evaluated for other elements. The detection of carbon was investigated, since this is present in all organic molecules, and a limit of detection of 0.47 mumol of carbon was achieved for sulphanilamide using superheated water as the mobile phase. Isotopically enriched solvents (12C-methanol 99.95 atom %) were used as organic modifier to aid chromatography. Detection limits of 86 mumol for 13C-triple-labelled caffeine and 79 mumol 13C-double-labelled phenacetin. Halogen (Br, I and Cl) detection was investigated. Metabolite profiling and excretion balance studies were carried out using these elements after dosing suitable model compounds (substituted anilines and benzoic acids) to rats. Limits of detection for Br and I were measured down to 0.1 mM. Profiling for 2-, 3-, 4-bromobenzoic acids showed glycine and glucuronide metabolites, in different proportions dependant on the position of the Br. This was also seen for the 2-, 3-, 4-iodobenzoic acids. Sulphur and phosphorous containing drugs were analysed using the reaction cell of the ICP-MS to chemically enhance the signal by reacting the element with oxygen (e.g. to give SO[+]), moving the detection away from a region of isobaric interference. Metabolite profiling of omeprazole was performed, with limits of detection of 800 pg of sulphur on column (an improvement of 100 fold in sensitivity from detection without oxygen). Similar studies with phosphorus containing drugs also showed a significant increase in sensitivity following reaction with oxygen compared to conventional analysis by ICP-MS.The studies undertaken here demonstrate the significant potential of HPLC-ICP-MS as a contributor to the analysis of drugs and metabolites in the pharmaceutical industry.

Previous research has provided pre-clinical validation for inhibition of the enzyme N-myristoyltransferase (NMT) target as a novel treatment of fungal and parasitic infections. This thesis describes the discovery of novel NMT inhibitor series derived from high-throughput screens (HTS) of the Pfizer compound collection against NMTs from both Leishmania donovani and Plasmodium falciparum. The further development of two series using structure-based drug design will be discussed, culminating in a series of compounds with efficacy in a mouse malaria model. Following the initial HTS, the hits were triaged based on the orthogonal attributes of structural diversity of each series of analogues; synthetic ease; orthologue selectivity; and estimated ligand efficiency. Iterative screening resulted in a master set that was further triaged through dose-response assays, including selectivity screening against both human NMTs. Eight novel series of NMT inhibitors, half selective for Leishmania and half for Plasmodium NMTs were approved for disclosure by Pfizer. One Leishmania-selective series, based on a thienopyrimidine template, was selected for further development based on a physicochemical hypothesis for achieving cell-based activity. Synthesis of a close analogue of the HTS hit provided validation of the series, which was further developed through the iterative synthesis and testing. The key objective of greatly improved activity in the Leishmania cell-based assays was not achieved. Development of a Plasmodium-selective series of aminomethylindazoles was more successful. Co-crystallisation of a close analogue of the screening hit demonstrated an orthogonal binding mode to the previously published quinoline series. Use of a fragment-linking approach enabled the design and synthesis of novel, highly potent inhibitors with balanced activity against three Plasmodium spp. NMTs, while retaining selectivity over both human NMTs. Excellent translation into cell-based activity was achieved, resulting in several lead compounds with efficacy in a mouse malaria model following both intraperitoneal and oral dosing.

Transcription factors are important control proteins in cells that bind to their cognate DNA sequences in the promoter regions of genes, either up-regulating or down-regulating protein expression. In many cancer types, transcription factors are up-regulated and promote the expression of genes important in survival and metastasis. For this reason, transcription factors are good targets for novel anticancer agents. The STAT family of transcription factors (seven are now acknowledged) recognize and bind to a ~10 base pair sequence of DNA in the promoter region of a number of genes, enhancing the expression of oncogenic proteins such as Survivin, Cyclin D1, Bcl-2 and VEGF. There are currently no small-molecule STAT3 inhibitors in clinical use, so there is a need for the development of assays that can be used to screen molecules to identify lead compounds. The main focus of this project has been to develop an in vitro homogenous time resolved FRET (HTRF) assay that can be used in low-, medium- and high-throughput modes for the discovery of novel inhibitors. The project started with the cloning, production and purification of recombinant STAT3βTC, which is a homodimeric protein. This was challenging and time-consuming as initial solubility and stability issues were encountered. However, experimental conditions were eventually established that allowed useful quantities (i.e.10 mg batches) of purified and stable protein to be obtained. As part of the optimization process, the STAT3βTC was re-cloned into a HIS-Tag vector which facilitated purification using affinity (Ni2+) chromatography along with size exclusion chromatography to produce pure monomeric STAT3βTC. This could be dimerised to provide pure STAT3βTC homodimer. The pure protein was used to develop a HTRF assay by first labelling the STAT3βTC with Europium. Next, the cognate DNA recognition sequence in the form of an 18-mer duplex oligonucleotide was biotinylated and joined to the second fluorophore label (D2) via a streptavidin linkage. The strength of the FRET signal between these two components could then be used to measure the interaction between them. As part of a multi-well system, this could then be used to screen for small molecules capable of disrupting the protein/DNA complex. The assay was validated using unphosphorylated STAT3 that does not form the biologically-relevant homodimer, and non-biotinylated DNA, which would not form the active FRET pair. Further validation of the assay was carried out using known STAT3 inhibitors such as the peptidomimetics PYLKTK and YLPQTV, and the small-molecule inhibitors STA-21 and Stattic. It was then used to screen a 40-membered library of novel SH2-targeted molecules produced in-house, in which it successfully identified six “hit” molecules with low micro molar activity. These were further evaluated by establishing IC50 values in a number of cell lines including MDA-MB231, HELA, A4 and NCI-H1975. These studies revealed a correlation between the FRET assay results and the cytotoxicity of the molecules in the STAT3-dependent cell lines. The molecules were also studied in cellular experiments to establish their effect on STAT3-regulated genes such as Cyclin D1 and Survivin, in which a correlation was also observed. As a result, these molecules are now in further development. Finally, the assay has been modified for high-throughput use in a 384-well system, and will be used for robotic screening in the future.

The field of pharmacogenetics has grown rapidly over the last few years yet, with a couple of exceptions, pharmacogenetic diagnostics are not yet used in a clinical setting. Progress in the field is discussed and the current state of pharmacogenetic research is assessed in a literature review. This thesis describes studies to investigate the pharmacogenetics of the anti-epileptic drugs phenytoin, carbamazepine and levetiracetam, using data obtained from the routine clinical use of these drugs, and the pharmacogenetics of the beta-blocker bucindolol in a clinical trial setting. Evidence is presented of a common polymorphism in the SCN1A gene that is associated with the clinical use (dosing) of both phenytoin and carbamazepine. Preliminary results are also presented concerning genetic variation in the SV2A and SV2C genes which may influence response to levetiracetam. For this study genetic variation was represented using the tagging SNP method applied to HapMap data. Genetic variation in ADRB2 may influence response to bucindolol yet the results are not conclusive. More importantly however, they do provide an illustration of how differences in polymorphism frequencies among populations could account for average differences in drug response among populations. It is also shown that there are substantial genetic differences within self-identified racial groups within the context of a clinical trial. The implications of pharmacogenetics for different racial or ethnic groups are discussed in a separate chapter. In conclusion, common variation in obvious candidate genes does influence drug response, however, rigorous clinical study is required before the use of pharmacogenetic variants to guide choice or dose of drugs becomes part of clinical practice.

Carbon monoxide (CO) is commonly considered to be toxic due to the propensity of this gaseous molecule to bind to ferrous iron in haemoglobin and cytochromes, thereby inhibiting respiration and the transport of oxygen around the body. However, CO is produced endogenously by haem oxygenases and has various cytoprotective functions including being vasodilatory, anti-inflammatory and anti-apoptotic. The development of CO-RMs (Carbon Monoxide-Releasing Molecules), which are generally transition metal carbonyls that release CO under certain conditions, has facilitated research into the physiological effects of CO and the potential use of CO as a therapeutic agent. Furthermore, CO-RMs have been found to reduce significantly the viability of various Gram-positive and Gram-negative bacteria, which is thought to be caused in part by the binding of CO from CO-RMs to the terminal oxidases of aerobic respiratory chains. Interestingly, CO-RMs are known to elicit many effects distinct from those of CO gas, including acting as more potent bactericidal agents. This thesis aims to increase the current knowledge of the antibacterial effects of CORMs, with a particular focus on the interactions with respiration, oxidases and thiol compounds. In contrast to CO gas, CORM-3 was not preferentially inhibitory to respiration at low oxygen tensions; however, in accordance with the relative resistance of cytochrome bd-I to CO gas, this oxidase was found to be the most resistant of E. coli to respiratory inhibition by CORM-3, and possession of this oxidase conferred some protection against growth inhibition in the presence of this CO-RM. Inhibition of E. coli respiration by CORM-3 was photosensitive and light reduced significantly the toxic effects of this compound, suggesting that CO from CORM-3 binds to ferrous haems in a classical, light-sensitive manner. This supports the hypothesis that the binding of CO from CORM-3 to haemoproteins is largely responsible for killing by these compounds. However, in opposition to this hypothesis, the non-haem oxidase AOX from Vibrio fischeri was found to be hypersensitive to inhibition by CORM-3, but not to CO, emphasising the complex effects of these compounds. Data are presented to show that thiol-containing compounds, which have been widely reported to abolish the biological effects of CO-RMs, substantially reduce the uptake of ruthenium-containing CO-RMs. The generation of reactive oxygen species by CO-RMs is also demonstrated and investigated. Finally, the generation and preliminary characterisation of CO-RM-resistant E. coli mutants is described. This work was done with the aim of revealing previously unappreciated bacterial targets for CO-RMs. Sugar-transporting phosphotransferase systems were identified as a possible means of CO-RM entry into the bacterial cell.

The liver has a wide range of capabilities and is the key organ for drug metabolism, detoxification and elimination. Molecules are absorbed from the GI tract into the bloodstream and transported to the liver through the portal vein circulation system. Most of the metabolic functions within the liver are processed by hepatocytes. The project aim was to test two drugs (5-Fluorouracil (5-FU) and diclofenac) with different metabolic pathways, interpret their toxic effects in hepatocytes and relate these to mechanical and morphological alterations in the cells. The findings of this project thereby allow development of a cell mechanics model for toxicity profiling and drug efficacy at a subcellular level. Hepatocellular carcinoma (HCC) is the second most common cancer in the world and the most frequent type of liver cancer. Therefore, a great amount of effort has been aimed at the discovery of anticancer compounds to treat it. The majority of conventional chemotherapeutic drugs work on the principle of halting DNA synthesis, and 5-FU follows this principle. This drug is also commonly used for treatment for most gastrointestinal tract cancers. When 5-FU is administered, the toxic adverse effects need to be considered as there is a possibility of severe side effects. To characterise toxic changes in hepatocytes, an Atomic Force Microscope (AFM) was used, which can produce high-resolution images by probing the surface of the cell, to provide information on the cell’s mechanical properties (such as Young’s modulus). However, with the complexity of this technique it has proved to be challenging to measure relative Young’s modulus values that minimise artefacts which affected the image quality of the cells. Using 5-FU as a model drug, apoptosis was detected by relating surface morphology and mechanical measurements. The surface morphology of HepG2 cells was examined with AFM, and the images produced showed cells exhibiting networking lines of a fibrous nature on the cell surface and protrusions from the cell membrane after the application of 5-FU. This is thought to be related to apoptotic behaviour occurring within the cell but this is not conclusive and further investigations need to be conducted. Mitochondria are the main source of adenosine triphosphate (ATP), which is vital for energy, and therefore control all active processes within the cell. Mitochondrial injury often occurs due to drug toxicity, causing altered metabolic function within the cells. Diclofenac is a widely prescribed NSAID, which may cause serious hepatotoxicity, and is thought to be a mitochondrial toxicant. When altering the physiological conditions from glucose containing medium to galactose containing medium, it was shown that the growth and metabolic function of HepG2 cells decreased. Diclofenac caused a depletion of ATP within the cells. When imaging the cell with AFM, after treatment with diclofenac, there were alterations at surface of HepG2 cells. When the cantilever was separated from the cell surface, the retraction curves showed intermolecular interactions occurring, after treatment with diclofenac which were not observed if the cells were untreated. Microscopic evidence suggested apoptosis may have occurred and it is proposed that the changes in the cell surface reflect this. Liposomes consisting of lipid bilayers can encapsulate a wide range of drugs, and their behaviour can be controlled by modifying their surface properties. This research also studied the production of liposomes in order to understand their interaction with cells. Liposomal delivery systems are used to improve the bioavailability of drugs and can reduce toxic effects. The liposomes were shown to engage with the cell surface by use of AFM but did not influence the cells viability, suggesting that they had potential as a non-toxic delivery system. This thesis has produced initial data to suggest that changes in cell mechanical properties can be used to detect changes in cell behaviour, such as apoptosis, but the method is still fraught with complexity.

This thesis is concerned with an investigation of the thermodynamic parameters of complexation of calix[4]resorcarene and calix[4]arene with metal cations and with anions, aiming at the removal of polluting ions from contaminated sources (such as the re-use of processed water) and for the extraction of heavy metals from aqueous solutions. Synthesis of 5, 11, 17, 23 ethylthiomethylated calix[4]resorcarene (L2), 5, 17 ethylthiomethylated calix[4]resorcarene (L3) and 25, 27-bis[N-(2-hydroxy-1,1 bis-hydroxymethyl-ethyl)aminocarbonylmethoxy] calix[4]arene-26, 28-diol (L4) and their characterisation by different techniques such as NMR and elemental analysis was accomplished. Partition experiments were carried out to determine that L2 and L3 are predominantly in their monomeric state in the solvents in interest. NMR technique was used for the characterisation of the ligands based on calixarene and resorcarene and for the investigation of the active site of complexation between the ligand and metal cations or anions in different solvents at 298 K. From the 1H NMR in MeCN, it can be inferred that the resorcarene ligand (L2) is interacting with Hg2+, Pb2+ and Ag+ while L3 showed interaction with Hg2+. In MeOH, both L2 and L3 showed interaction with Hg2+ and Ag+. As far as L4 is concerned, it was inferred that L4 interacts with alkaline-earth metal cations, Zn2+ and Pb2+ between the cations and with fluoride and dihydrogen phosphate in acetonitrile. Conductometric titrations in solution were carried out to determine the composition of the chemical species formed. Thermodynamic parameters of complexation of L2 and L3 with the appropriate cations reveal that L2 is selective for Ag+ and L3 possesses high stability with Hg2+. X-ray crystallography of the L2-Hg2+ complex reflects the decomposition of the ligand and the formation of polymers containing mercury and sulphur chains. Thermodynamic parameters of complexation of L4 with metal cations and anions of interest reveal the following selectivity trends at 298.15 K. L4: Mg2+ > Zn2+ > Ca2+ > Sr2+ > Pb2+ > Ba2+ (MeCN) L4: F-> H2PO4- (MeCN, DMF) Finally, conclusions and suggestions for further work in the area are given.