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The heptadecapeptide Nociceptin/OrphaninFQ (N/OFQ) is the endogenous ligand for the G-protein coupled receptor, NOP, activation of which is involved in a plethora of physiological functions, including pain. Studies in main are hampered by a relative lack of suitable, well characterised ligands, especially antagonists and an understanding of the effects of prolonged receptor activation.;The N/OFQ sequence can be divided into a message domain, associated with receptor activation and an address domain, associated with receptor binding. In a series of biochemical assays, using Chinese Hamster Ovary (CHO) cells stably expressing human NOP, a number of ligands suitable for in vivo animal testing were characterised including a high affinity, high potency agonist ([(iF)Phe4, Arg14, Lys15]N/OFQ-NH2), a high affinity antagonist ([Nphe 1, Arg114, Lys15]N/OFQ-NH 2 or UFP-101) and several partial agonist molecules.;In a CHO cell line expressing NOP at a range of levels via the ecdysone inducible expression system, and behaviour of the partial agonist [F/G]N/OFQ(1-13)-NH 2 could be modulated to encompass full agonism and antagonism, using the previous assays, underscoring the need to assess activity in a range of models and steps in the signal transduction cascade.;Receptor desensitisation, by prolonged exposure of the inducible cells to N/OFQ, led to a decrease in NOP density, a reduction in N/OFQ stimulated GTPgamma[35S] binding with a reduction in functional potency and a reduction in potency of cAMP inhibition. More interestingly, a reduction in NOP mRNA was also observed.;Collectively this thesis has made a significant contribution to the N/OGQ-NOP field in that: (1) several novel molecules have been characterised and are now available to other researchers; (2) a system in which pharmacological behaviour can be accurately defined has been characterised; (3) the first example of genomic desensitisation of NOP has been described. The N/OFQ-NOP system is ready to leave the pre-clinical laboratory and full clinical evaluation is eagerly awaited.

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Nitric oxide (NO) is involved in numerous biological processes including cancer, where this small diatomic radical can exert both pro- and anti-cancer effects. Two series of novel NO-donors were synthesised in this work, the first representing an extension to the S-nitrosothiols (RSNO) class, with the second utilising the popular 1,2,5-oxadiazole-2-oxide (furoxan) functionality. The oxathiazolylium-5-olates 39a, 39f, 39h-D, were successfully made via improved seven-step synthesis whilst a series of combretastatin-like furoxans 144a-c (NO-hybrids) were generated in a one-pot reaction. All compounds; including byproducts from failed alternative synthetic routes, had their cytotoxic activity evaluated. From this study NO-hybrid 144c showed the most promising biological profile when tested against eight different ovarian cancer cell lines.

Unsaturated carbazole frameworks are found in several important naturally occurring and synthetic biologically active compounds. We envisaged synthesising compounds of this type using an intermolecular Diels-Alder reaction between 3-vinyl-1H-indole and a dienophile, followed by an intermolecular ene reaction between the resultant Diels-Alder adduct (6) and an enophile. This route was tested using tosyl protected 3-vinyl-1H-indole (4) and N-methylmaleimide (5) in a Diels- Alder reaction, followed by ene reactions with a range of enophiles to give unsaturated carbazoles (7) in yields of 50-60% over the two steps. Through utilisation of Soos’s organo-catalyst3, we have been able to control the Diels-Alder reaction between 3-vinyl-1H-indole (8) and N-methylmaleimide (5), to form the Diels-Alder adduct (9) with high enantiomeric excess. The subsequent stereospecific ene reaction then gives unsaturated carbazoles (10) in >96% ee. We then developed a sequential “one-pot” Diels-Alder / ene methodology with an Nprotected 3-vinyl-1H-indole (11), a dienophile and an enophile to make unsaturated carbazoles and pyridazino[3,4-b]indoles (13). This methodological approach gave increased overall yields and reduced purification steps. When Cbz N-protected 3-vinyl-1H-indoles were used the Diels-Alder / ene products (14) could then be deprotected through a PtO2 catalysed hydrogenation reaction to give molecules (15). Several of the synthesised compounds (15) showed moderate biological activity against Escherichia coli, Staphylococcus aureus and Schizosaccharomyces pombe. Compounds (15) are also moderate (μM) inhibitors of several important kinases, including Chk2, Aurora B, Src and JAK2, which are potential targets for cancer treatment.

The kidney plays a vital role in the elimination of many endogenous metabolites and xenobiotics. Drug transporters expressed in the proximal tubule cells are key factors in the ability of the organ to successfully carry out its function. Previously, primary human proximal tubule cells have been shown to retain a remarkable degree of differentiation in culture and provide a realistic model of the proximal tubule. To address the challenge of extrapolation of drug transporter data from animal and human, this project was set out to develop a parallel rat proximal tubule cell model. This would allow direct comparison of the handling of candidate drugs in both species, and provide better understanding of the mechanisms of drug transport. A technique to isolate primary rat proximal tubule cells (PTCs) was successfully developed using a collagenase digest/Percoll gradient approach. Rat PTCs cultured for 6 days were shown to exhibit cobberstone morphology, typical of many epithelial cells. A range of transport proteins including Mdr1a/b, Bcrp, Mrp2, Oat1, Oct2, Oatp4c1, Slc2a9, Urat1, Mate1, and Mct1 were detected at the mRNA level in these cells. Functional expression of Mdr1a/b, Bcrp, Mrp2, Oct2 and Mct1 was also detected using fluorescence substrate retention assays. In addition, Mdr1a/b, Bcrp and Mrp2 transporters were found localised on the apical membrane of polarised rat PTC monolayer, and Oct2 was found on the basolateral membrane. The handling of urate by rat PTC monolayers was investigated. The monolayers showed absorptive and secretory pathways for urate, although the absorptive pathway was 3.2-fold higher in magnitude. Similarly, 3.4-times more urate was predominant across the apical than across the basolateral membrane. Oat1 and Bcrp were deduced as the transporters responsible for the secretory pathway, and Urat1 and Slc2a9 in the absorptive pathway. This was in accordance with the human PTC monolayers, and both models were representative of urate transport in vivo. Digoxin transport exhibited a net absorptive flux in rat PTC monolayers; absorptive flux was 1.7-fold higher in magnitude than the secretory flux. In contrast, in human PTC monolayers, digoxin secretory flux was 4.2-fold higher than the absorptive flux. In human PTC monolayers, digoxin secretion consisted of OATP4C1-mediated digoxin uptake by the basolateral membrane and MDR1-mediated efflux across the apical membrane. In rat PTC monolayers in addition to these pathways, a significant Oatp-mediated absorptive flux of digoxin located on the apical membrane of rat PTC monolayer was identified as the difference between rat and human digoxin handling, resulting in a dominant absorptive flux of digoxin in rat compared to net secretion in human PTC monolayers. These data alone highlight the importance of developing realistic in vitro human and rat PTC models to understand species difference in renal drug handling.

The pharmacological effects of a number of centrally acting drugs have been compared in euthyroid mice and mice made hyperthyroid by pretreatment with sodium-1-thyroxine. The potencies of two barbiturates, pentobarbitone and thiopentone - as indicated by the duration of their hypnotic actions and their acute toxicities - are increased in hyperthyroid mice. An acutely active uncoupler of phosphorylative oxidation is 2, 4-dinitrophenol, an agent which proved to be a potent hypnotic when administered intracerebrally. An attempt has been made to relate the mechanism of action of the barbiturates to the uncoupling effects of thyroxine and 2, 4-dinitrophenol. The pharmacological effects of chlorpromazine, reserpine and amphetamine-like drugs have also been studied in hyperthyroid mice. After pretreatment with thyroxine, mice show a reduced tendency to become hypothermic after chlorpromazine or reserpine; in fact, under suitable laboratory conditions these agents produce a hyperthermic effect. Yet their known depressant effects upon locomotor activity were not substantially altered. Thus it appeared that depression of locomotor activity and hypothermia are not necessarily correlated, an observation at variance with previously held opinion. These results have been discussed in the light of our knowledge of the role of the thyroid gland in thermoregulation. The actions of tremorine and its metabolite, oxotremorine, have also been examined. Hyperthyroid animals are less susceptible to both the hypothermia and tremor produced by these agents. An attempt is made to explain these observations, in view of the known mechanism of action of oxotremorine and the tremorgenic actions that thyroxine may have. A number of experimental methods have been used to study the anti-nociceptive (analgesic) effects of drugs in euthyroid and hyperthyroid mice. The sites and mechanisms of action of these drugs and the known actions of thyroxine have been discussed.