Caffeine is the most widely consumed and socially-acceptable of psychoactive drugs. Its arousing properties, both physical and behavioural, have been studied for almost a century, but interest in its effects on cognition is recent. The first tasks employed in cognitive studies were chosen without regard to underlying theory: interpretation of the outcomes therefore remained atheoretical. Four theoretically-based hypotheses, tapping different aspects of cognitive function were, therefore, tested under caffeine at 0, 2 and 4 mg/kg body weight. The paradigms were as follows: a simple information-processing task at two levels of difficulty; the free recall of spoken word lists; a numeric version of the Stroop task; and a lateralized version of the Posner task. A main effect of caffeine was found in the Stroop task where the highest dose caused a significant disruption of response over and above that due to the Stroop effect per se. No other main effects were found, but interactions with the variables of introversion-extraversion and sex were implicated as potent and confounding factors in the effect of caffeine on cognition. An interaction of stimulus type and receptive hemisphere in the Posner task showed that caffeine is able to reverse the normal locus of hemispheric processing: the right hemisphere became more efficient for sequential inputs and the left hemisphere for holistically-analyzed stimuli. It is suggested that a central action of caffeine is to increase stimulus sampling rates, and that its effects on both general arousal and specific information-processing functions follow from this. The general implication of the findings is that the cognitive effects of caffeine are real but subtle, resting on very specific conjunctions of variables, and would thus be more readily demonstrated in uniform groups of individuals, under precisely defined conditions of testing.

Concentration-dependent nicorandil relaxations of rat mesenteric arterial rings, measured in the presence of L-NAME to block NO synthesis, were inhibited either by the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, or by the guanlyl cyclase inhibitor LY 83583. When applied together, glibenclamide and LY 83583 were more effective than either agents alone. Nicordandil was also less potent at inhibiting 60mM K+ concentrations. These results are consistent with nicorandil causing relaxation both by activation of KATP channels and of guanylyl cyclase in this preparation. In whole cell patch clamp experiments, nicorandil activated a K+ current that was inhibited by glibenclamide, but unaffected by the inhibitor of large conductance calcium-activated K+ (BKCa) channels iberiotoxin. Single channels activated by nicorandil, resolved in a whole-cell recording, had a conductance of 31 pS in symmetrical high K+ solutions. Although BKCa channel activation has been reported to be involved in the vasorelaxant action of nitrovasodilators in some studies, iberiotoxin (IbTX) did not affect nicordandil-induced relaxations of the rat mesenteric artery, nor did it inhibit nicorandil-induced K+ currents. Similarly, IbTX and charybdotoxin (ChTX) failed to inhibit relaxations produced by the pure nitrovasodilators glyceryl trinitrate and SNAP. Interestingly, however, in the absence of L-NAME, ChTX significantly inhibited SNAP dose response curves, implying the activation of BKCa channels under these conditions. Thus, in the rat mesenteric arterial preparation used here, the activation of K+ channels by SNAP appears to be dependent on endogenous NO production. In the absence of endothelial NO, the drug produced full relaxation via K+ channel-independent mechanisms. The KATP channel opening ability of nicorandil was postulated to be potentiated by cellular stress because such channels are sensitive to the metabolic state of the cell. In rat mesenteric arteries, the potency of nicorandil was enhanced by metabolic inhibition with carbonyl cyanide m-chlorophenylhydrazone (CCP) and 2-deoxyglucosse. This effect was then studied more fully in pig coronary arteries where metabolic inhibition with CCCP or zero glucose solution, external acidosis, and adenosine receptor activation all increased the potency of nicorandil to cause vasorelaxation.

In recent years evidence has accumulated which suggests that prostaglandins are involved in spinal nociceptive processing. Several studies have shown that spinally-administered prostaglandins evoke characteristic pain behaviour in rats. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, inhibit the enzyme cyclooxygenase (COX) and thus prevent the formation of prostaglandins. It has been hypothesised that a component of the analgesic properties of NSAIDs may be due to an action in the spinal cord, a theory which is supported by behavioural and electrophysiological studies. The current study shows by Western blotting that the two isoforms of COX, COX-1 and COX-2, are present in rat spinal cord tissue. Furthermore, using immunocytochemical techniques, we have localised COX-2-like immunoreactivity to regions of the spinal cord associated with nociceptive processing, namely the superficial and deep dorsal horn and around the central canal. Spinal COX-2, but not COX-1 is bilaterally upregulated in a time-dependent manner (< 3 days) following the induction of a unilateral inflammation around the ankle joint. An increase in spinal PGE2 levels occurs over the same time course. The basal release of 4 species of prostaglandins was assessed by superfusing the spinal cord with artificial cerebrospinal fluid and analysing the superfusate by radioimmunoassay (PGD2> PGE2> 6-keto-PGF1> PGF2). Using this superfusion technique and an antibody-microprobe technique we detected increases in the spinal release of PGE2 within 30 minutes following acute inflammation. The increased release of PGE2 was maintained for at least 12 hours. Noxious mechanical stimulation also evoked the spinal release of PGE2. In conclusion, we have shown that COX-2 is present in the spinal cord in locations commensurate with an involvement in nociceptive processing. The increase in spinal COX-2 and PGE2 following inflammation suggests that prostaglandins (possibly synthesised by COX-2) may play an active role in modulating spinal nociceptive processing.

Statins are generally well-tolerated, although statin-related myotoxicity (SRM) has been reported in a considerable number of patients. The risk factors underlying SRM have yet to be fully characterised. This study aimed to further elucidate the risk factors that increase the likelihood of SRM using data generated from cellular and clinical settings. The data from the cellular studies would provide information regarding candidate single nucleotide polymorphisms (SNPs) which could be tested in the clinical setting. In the cellular studies, three model cell lines were used; human proximal tubule (HK-2), rat skeletal muscle (L6) and human muscle cells. Lipophilic statins, simvastatin and atorvastatin, inhibited monocarboxylate transporter 1 (MCT1)-mediated DL-lactate uptake at the same magnitude as phloretin, a well-known MCT1 inhibitor. No significant lactate uptake inhibition was observed with up to 1 mM of hydrophilic statins (pravastatin and rosuvastatin). The magnitudes of inhibition of multidrug resistance-associated protein (MRP)-mediated CMFDA efflux and MDR1-mediated Hoeschst 33342 efflux by the lipophilic statins were lower than that caused by MK571 and cyclosporine A, which are typical inhibitors of MRP and MDR1, respectively. Both hydrophilic statins showed no significant effect on MRPs and MDR1 functions. In the clinical setting, a case-control study (116 cases and 314 controls) of unrelated dyslipidaemic patients was performed to determine the association between 12 SNPs from nine focus genes [i.e., SLCO1B1, ABCC2, ABCG2, CYP3A4 (*22 allele), COQ2, GATM, GPx, SLC16A1, SLC16A3] and SRM. Of the 12 SNPs genotyped, only SNP in SLCO1B1 (rs4149056) appeared to be the most important genetic predictor of SRM (P = 0.059, P = 0.047 in univariate and multivariate analysis, respectively), thus confirming previous findings. The association between rs4149056 and SRM was demonstrated to be independent to the type of administered statins and was likely to be influenced by the patient gender. Further patient recruitment is ongoing to increase study power and to confirm the assumption of the abovementioned association.

Opioids are used to alleviate pain however ~10–30% of the Caucasian population obtain ineffective pain relief and/or intolerable side effects. Genetic polymorphisms in opioid pharmacodynamic and pharmacokinetic important genes have been investigated however there has been a lack of conclusive results. Epigenetic gene regulation is another study field of interest. DNA methylation is a gene regulatory mechanism that has been associated with gene repression or expression. Thus it was hypothesised that variable opioid response was influenced by methylation alterations. The promoter region methylation of ABCB1/MDR1, CYP2D6 and OPRM1 genes were analysed by pyrosequencing in smoking, opioid exposed and control pilot populations. Genetic variations within ABCB1/MDR1, COMT, CYP2B6, CYP2D6 and OPRM1 genes were also investigated. Tissue opioid concentrations were determined by HPLCMS/ MS and GC-MS/MS. DNA methylation was influenced by chronic opioid exposure and / or the lifestyle associated with opioid dependency, but not by cigarette smoke exposure. An increase in DNA methylation was observed in the opioid exposed baby population but this was not indicative of development of neonatal abstinence syndrome (NAS). Associations between the CYP2B6*6 genotype and NAS development were found. No relationship was observed between gene methylation and opioid response but variants in OPRM1 and ABCB1/MDR1 exhibited a relationship with opioid response in cancer pain. This investigative pilot research revealed that some genetic variants can be used as diagnostic markers to predict susceptibility of methadone exposed babies to NAS development, and others are associated with variable opioid response in a cancer patient population. Although DNA methylation was not observed to be a contributory factor to opioid response, this research has ascertained the influence of chronic opioid exposure on DNA methylation of various biological samples. This finding is significant for future studies.

The kidney plays a key role in the systemic clearance of new molecular entities (NMEs). Approximately 32% of drugs exhibit significant renal elimination. It is estimated that nephrotoxicity accounts for 8 % of pre-clinical and 9% of clincal safety failures in drug development. Current pre-clinical models used to screen NMEs are poor predictors of human nephrotoxicity. The focus of my project is thus to develop predictive in-vitro rat and human primary proximal tubule cell models as a platform drug transporter and drug-drug interaction (DDI) studies. Primary human and rat proximal tubule cells (PTCs) were isolated from renal cortex using a combination of enzymatic digestion and density centrifugation. The isolation procedure was optimised to maximise cell yield and viability. Human and rat PTCs cultured on Transwell® inserts formed confluent monolayers with low paracellular permeability. Quantitative PCR showed mRNA expression of key renal transporters, OAT1, OAT3, OATP4C1, OCT2, BCRP, MATE1, MATE2-K, MDR1, MRP2, URAT1, NaPi-IIa, NaPi-IIc and PiT2, in human PTC monolayers. Orthologs of these transporters were also detected in rat PTC monolayers. The utility of human and rat PTC monolayers as predictive in-vitro models of proximal tubular drug handling were demonstrated using radiolabeled [3H]-tenofovir (TFV). Human and rat PTC monolayers exhibited a cell-to-media ratio greater than 1, which indicated uptake and accumulation of TFV across the basolateral membrane. We also observed a predominant absorptive pathway of TFV. The transporters mediating the transport of TFV were identified using a cocktail of transporter inhibitors. The basolateral uptake of TFV was mediated by OATP4C1 and OAT1. TFV had low affinities for the apical efflux transporters MRP2, MRP4, MDR1 and BCRP. The novel identification of OATP4C1 as a TFV transporter has led Gilead to develop assays for investigating OATP4C1-mediated DDIs, and the FDA to recognise OATP4C1 as a key renal transporter. The handling of radiolabelled inorganic [32P]-phosphate (Pi) by human and rat PTC monolayers was also investigated. Pi flux measurement revealed a net absorptive pathway of Pi across human and rat PTC monolayers. Uptake of Pi across the apical membrane was sodium dependent, saturable, and inhibited by parathyroid hormone, fibroblast growth-factor 23 and -klotho. Apical uptake of Pi was also inhibited by TFV in a saturable manner. This suggests that the mechanisim of TFVinduced hypophosphatemia is not via TFV-induced nephrotoxicity, but TFV inhibition of Pi reabsorption. The outcomes of this work have initiated a patient clinical trial. This finding could have a large translational impact, as over 14.9 million HIV-patients are pescribed TFV and TFV-related hypophosphatemia affects 30 % of patients. The data highlight the importance of developing holistic cell based models of the proximal tubule. The outcomes of this work demonstrate the power of translational science to have an impact on how the pharmaceutical industry operates.

Chronic diseases such as cardiovascular diseases and colorectal cancer are the leading cause of mortality worldwide. Commonly used drugs such as aspirin and warfarin are shown to effective at reducing the risk of chronic diseases but have a narrow therapeutic window and are associated with adverse drug reactions, particularly, hemorrhage. Identification of pharmacogenetic markers such as single nucleotide polymorphisms (SNPs) that could help deliver personalized dose could help improve the risk-benefit ratio. Furthermore, development of a rapid point of care genotyping device consisting of a pharmacogenetic SNP panel for aspirin and warfarin could help implement personalized medicine in the clinical setting. Analysis of candidate SNPs in aspirin’s pharmacokinetic and pharmacodynamic pathways was carried out to explain variation in aspirin’s colorectal chemopreventive efficacy using two large population based case-control datasets. Associations and interactions were tested using logistic regression models and meta-analysis of the 2 datasets. A novel sitespecific association for rs1799853 (OR=0.73, 95% CI=0.60-0.90, P=0.003) and rs1105879 (OR=1.16, 95% CI=1.02-1.32, P=0.03) with colon cancer risk was observed. Furthermore, stratification by aspirin use showed increased risk of colorectal cancer in aspirin users but not in non-users carrying variant allele of the SNPs rs4936367 and rs7112513 in PAFAH1B2 gene and rs2070959 and rs1105879 in UGT1A6 gene (Pinteraction < 0.05 for all). These results provide insight into aspirin’s differential chemopreventive efficacy and the neoplastic transformation of cells in colon and rectum. Utility of clinically validated pharmacogenetic dosing algorithms consisting of three warfarin dose associated SNPs from the European population needs to tested in the Gujarati Indians, an Indian sub-population. Dose prediction accuracy of the algorithms was compared between Gujarati Indian and European population. Mean squared difference of both pharmacogenetic algorithms was higher in Gujarati Indian compared to European population (Klein et al 2009, 216.3 v/s 160.7, P=0.05; Gage et al 2008, 170.6 v/s 143.2, P=0.07). Poor prediction accuracy could be explained by the presence of study subjects requiring dose for target INR range 2.5-3.5 and low frequency of the VKORC1 rs9923231 variant, which is the most important genetic determinant of warfarin dosing in Europeans. Therefore, the SNP panel and dosing algorithms developed from European populations cannot be assumed to have utility in the Gujarati Indian population. Finally, to help develop a rapid, point-of care, silicon nanowire (SiNW) based SNP genotyping device, a panel of isothermal melting probes were designed to genotype three warfarin dose associated SNPs. Testing of hybridization and washing conditions to have optimal hybridization kinetics between the probe and target DNA and high target sequence specificity was carried out using custom designed microarray platform. Accurate genotype calls for all 3 SNPs in 2 anonymised samples using empirically optimized hybridization and washing conditions was carried out successfully. Current work highlighted associations between probe characteristics and hybridization parameters, which would be useful in designing and testing probes on the SiNW platform. Identification, validation and testing of clinical utility of population specific pharmacogenetic markers along with development and deployment of ultra-rapid point of care genotyping technologies could help deliver personalized risk-benefit ratio for aspirin and warfarin.

Alterations in cardiovascular parameters are common during general anaesthesia, owing to the combined effects of anaesthetic drugs, surgical stimuli, changes in intravascular volume and baroreflex activity. In healthy patients, these changes may be well tolerated with minimal morbidity. However, in patients with limited cardiovascular function, hypotension, hypertension and tachycardia may lead to cardiac arrhythmias, myocardial ischaemia and infarction. Certain phases of anaesthesia and surgery are associated with particular increased risk for cardiovascular instability, especially induction of anaesthesia, instrumentation of the airway, skin incision, emergence and awakening from anaesthesia. Therefore specific measures may be required to attenuate cardiovascular responses to noxious stimuli. Remifentanil is a new opioid introduced into clinical practice in 1996, whose pharmacokinetic and pharmacodynamic properties suggested it might be useful to attenuate adverse autonomic and cardiovascular responses. This thesis describes studies of these possible effects of remifentanil. Remifentanil attenuated the cardiovascular and plasma catecholamine responses to laryngoscopy and tracheal intubation in healthy young adults. However, bradycardia and hypotension were observed in some individuals, and further studies confirmed that lower doses than originally recommended were effective. Remifentanil was also effective in elderly patients, and hypertensive patients, but observations of myocardial ischaemia confirmed that these groups are at risk. There was significant inter-individual variation in cardiovascular responses, confirming that remifentanil and other drugs used during anaesthesia should be titrated to effect. Remifentanil administered as a bolus attenuated the cardiovascular responses to emergence from anaesthesia and tracheal extubation, without compromising clinical recovery. Future studies should also address its place as an analgesic/sedative outside the operating room, for example in Intensive Care. This thesis details the effects of remifentanil on responses to noxious stimuli during anaesthesia and adds to our collective understanding of cardiovascular responses during anaesthesia in different groups of patients.

In the 1980s a group of vasodilator drugs were found to possess a common mechanism which involved opening KATP channels located in the cell membrane of smooth muscle cells, leading to vasorelaxation via membrane hyperpolarisation. A number of additional K+ channels have been identified in vascular smooth muscle cells and one of these channels, the large conductance calcium-activated K+ channel (BKCa), is a promising therapeutic target.;This thesis uses the various configurations of the patch-clamp technique to examine the effects of NS1619, nitric oxide (NO) and compounds resulting from the activation of the cGMP signalling pathway by NO, on the activity of ion channels.;Using single smooth muscle cells enzymatically isolated from the rat basilar artery, NS1619 was confirmed as an effective BKCa channel opener and hyperpolarising agent. Studies of the mechanism of action of NS1619 concluded that it has a direct effect on the BKCa channel itself or an associated regulatory site, possibly leading to an increase in the Ca2+-sensitivity of the BKCa channel. NS1619 also blocked at least two other channels, voltage-activated K+ channels and DHP-sensitive Ca2+ channels, and this latter effect almost certainly explains the functional vasorelaxation produced by NS1619 actions which will certainly limit the use of NS1619 in defining physiological roles for BKCa channels.;Nitric oxide activated whole cell currents when applied to single smooth muscle cells, which were blocked by specific BKCa channel blockers. Additional experiments determined that this action of NO could not be explained by a direct effect on BKCa channels but probably occurred by a mechanism involving a phosphorylation reaction catalysed by cGMP-dependent protein kinase. This stimulatory effect of cGMP-dependent protein kinase on BKCa channels may be involved in the vasorelaxation produced by nitric oxide and nitrovasodilators.

Steroidal neuromuscular blocking drugs (NMBDs) produce a range of effects on the cardiovascular system corresponding both bradycardia and tachycardia. Logical targets, include the muscarinic receptor in the heart (M2) and/or modulation of noradrenergic (NAdr) transmission in this tissue. In an attempt to address these two possible "side effect target sites" this thesis represents a detailed and systematic examination of the effects of a range of steroidal neuromuscular blocking drugs on recombinant human muscarinic receptors (assessed in [3H]N-methyl scopolamine (NMS) binding studies using Chinese hamster ovary cells transfected with M1-M5 muscarinic receptors and cAMP/[Ca2+]i measurements) and noradrenaline uptake-release mechanisms in simple cellular systems (using [3H]NAdr in SH-SY5Y neuroblastoma cells and HEK293 cells expressing uptakei). Four main NMBDs have been chosen; pancuronium (an agent that produces a tachycardia), vecuronium (an agent that products a bradycardia), rocuronium (an agent with variable effects) and gallamine, a well documented muscarinic antagonist (an internal positive control). [3H]-NMS binding was displaced in a concentration dependent manner from m1-m5 receptors by all NMBDs. Further kinetic studies with pancuronium, vecuronium and gallamine indicated an allosteric action at M2 receptors. Methacholine inhibition of cAMP formation at the M2 receptor was reversed by pancuronium. Rocuronium and vecuronium produced a small but significant shift in the methacholine response. At clinically achievable concentrations no NMBD affected [Ca2+]i. Collectively these data conclusively identify pancuronium (at clinical concentrations) as a moderately selective M2 receptor antagonist with an additional allosteric mode of action. Studies such as these are important to enable clinicians to make rationale evidence based choices in their use of neuromuscular relaxants.