The clinical outcome of CYP2C9/2C19 allelic variation on the pharmacotherapy of epilepsy has been investigated; although many new antiepileptic drugs have been launched into the market, carbamazepine, phenobarbital and phenytoin are still the major agents in the pharmacotherapy of epilepsy. Therefore, phenytoin was chosen as a model AED and the effect of CYP2C9/2C19 genetic polymorphism on phenytoin metabolism was further examined. An estimation of the allele prevalence was undertaken for three CYP2C9/2C19 alleles respectively using a meta-analysis of studies that fit the Hardy-Weinberg equilibrium. The prevalence of CYP2C9*1 is approximately 81%, 96%, 97% and 94% in Caucasian, Chinese, Japanese, African populations respectively; the pooled prevalence of CYP2C19*1 is about 86%, 57%, 58% and 85% in these ethnic populations respectively. However, the studies of association between CYP2C9/2C19 polymorphism and phenytoin metabolism failed to achieve any qualitative or quantitative conclusion. Therefore, mephenytoin metabolism was examined as a probe drug for association between CYP2C19 polymorphism and mephenytoin metabolic ratio. Similarly, analysis of association between CYP2C9 polymorphism and warfarin dose requirement was undertaken. It was confirmed that subjects carrying two mutated CYP2C19 alleles have higher S/R mephenytoin ratio due to deficient CYP2C19 enzyme activity. The studies of warfarin and CYP2C9 polymorphism did not provide a conclusive result due to poor comparability between studies. The genetic polymorphism of drug metabolism enzymes has been studied extensively, however other genetic factors, such as multiple drug resistance genes (MDR) and genes encoding ion channels, which may contribute to variability in function of drug transporters and targets, require more attention in future pharmacogenetic studies of antiepileptic drugs.

This project aimed to investigate the main traditional use of the plant Hyptis crenata (HC) and evaluate its activity in a pre-clinical trial. A traditional use survey was carried out in two municipalities in Brazil, interviewing 20 people. The results showed that the main use of HC is for pain relief (19/20). The main method used for its preparation was decoction extract (11/20). The antinociceptive activity of HC decoction extract was evaluated showing that HC 15 mg/kg (p.o.) and HC 150 mg/kg (p.o.) increased delay in withdrawal response in the Hargreaves thermal withdrawal test by 29% and 28% respectively and decreased writhing occurrences induced by acetic acid by 70% and 71% (p<0.05), when compared to vehicle. These treatments were examined in the acetic acid writhing test for their effects on c-fos protein expression. The results showed that HC 150 mg/kg (p.o.) decreased c-fos expression in the hypothalamic paraventricular nucleus by 40% (p<0.05). This study also evaluated the HC doses 1 mg/kg (p.o.), 5 mg/kg (p.o.) and 45 mg/kg (p.o.) to assess the dose-response effect. HC dose- dependently induced antinociception in both animal models from 0 mg/kg (p.o.) to 15 mg/kg (p.o.), until a plateau response occurred between 15 mg/kg and 45 mg/kg (thermal withdrawal test p=0.0002 and writhing inhibition p=0.4725). Then, the decoction extract was fractionated and tested in an attempt to identify which HC compounds were active in inducing the antinociceptive effect. The hexane fraction had the highest activity (per dose) compared to the other fractions, indicating that it was enriched with the active compounds. Also, a preliminary COX inhibition assay was carried out; the results indicating possible COX-2 inhibition for HC treatments. Additionally, an investigation through behavioural analyses of whether HC and its fractions were affecting basal behaviour, such as muscle relaxation and sedation, showed no change of such behaviour. Overall, these data support the antinociceptive effect of Hyptis crenata and that there are specific compounds responsible for this effect.

y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain mediating its fast inhibitory action via GABA(_A) receptors. These receptors are implicated in a number of neurological diseases, making GABA(_A) receptor ligands interesting as potential therapeutic agents. The aims of this research project were two-fold: identifying leads for the discovery of new chemical entities that modify GABA(_A) receptor function. The second aim was to increase the understanding of GABAgeric transmission by studying the pharmacological influence of a new interacting protein for the mammalian GABA(_A) receptor, GRIF-1. In the search for novel ligands for GABA(_A) receptor, the pharmacology of three structurally distinct compound classes was investigated. The first class was the NSAID, Mefenamic acid (MFA) and a group of analogues. Results showed that MFA and a series of analogues selectively modulate GABAAR at the agonist binding site, but did not interact with either the picrotoxin or the benzodiazepine sites. Indeed the most significant result of this study was the identification of common active conformers of MFA compound and the differentiation of two analogues based on MFA structure, with an improvement in apparent efficacy. The second compound studied was Octyi-13-Dglucoside, a small molecule congener of a natural fungal metabolite, Caloporoside. These studies demonstrated that Octyi-13-D-glucoside is a positive modulator of GABAA receptor at the channel site demonstrated by its stimulation of specific [(^35)S] TBPS binding. The level of stimulation was similar to that elicited by diazepam and was occluded by GABA. Preliminary structure-activity study showed that the 13-glycosidic linkage and chain length are crucial for the positive modulation of [(^35)S] TBPS binding to the GABAAR by this novel chemical class. The third compound series were essential oils derived from Melissa officinalis and Lavendula angustifolia. These two oils either singly or in combination have been reported to have a significant benefit in the treatment of agitation in dementia. The purpose of this study was to clarify the sedative and calming mechanisms of these two common essential oils by investigating their effects on the GABAAR complex. Melissa and Lavender both singly and in combination inhibit [(^35)S] TBPS binding to the channel site of GABAAR. Melissa oil displayed the higher affinity. Melissa oil alone also showed a stimulatory effect on [(^3)H] muscimol binding. Interestingly, a combination effect on the inhibition of [(^3)H] flunitrazepam binding to the GABAAR has been shown when Lavender and Melissa oils are applied together (50:50), with no effect when applied alone. Neither Melissa nor Lavender oils demonstrated any effect on the binding of [(^3)H] MK-801 to NMDA receptors, or [3H) nicotine to nicotinic acetylcholine receptors. Furthermore, functional studies have demonstrated that both oils (0.01 mg/ml) applied to rat primary cortical neuron cultures, results in a significant reduction in both inhibitory and excitatory transmission, with a net depressant effect on neurotransmission. These data suggests that the calming/sedative effects of Melissa are mediated by multiple mechanisms in the CNS; the net effect is depressant on the overall neuronal network. Finally, a pharmacological study was performed on GRIF-1a, a novel GABAA receptor 132 subunit trafficking protein, to gain further insights into the potential role of this novel protein at the inhibitory synapse. In the present work, evidence was provided that GRIF-1a does not increase a1j32y2 receptor complex numbers, but appears importantly to stabilise the GABAAR in a conformation which facilitates binding to both GABA and benzodiazepines. These findings suggest that GRIF-1 protein may be a novel means of modifying the efficacy of synaptic inhibition. In summary, this thesis provides a clear picture about four novel ways for the modulation of the GABAA receptor inhibitory transmission.

Aim: The physical and chemical characterisation of batches of 3,4-methylenedioxymethamphetamine (MDMA) / ‘ecstasy’ tablets seized in Malta were investigated to derive new information to be used for forensic intelligence purposes. Methods: Thirty seizures containing 45 batches of ‘ecstasy’ tablets were investigated for their physical (logo, breakline, colour, shape, mass, diameter, thickness, hardness, friability and disintegration rate) and chemical characteristics over a 5 year period. Impurity profiling was carried out by GC-MS with additional chemical characterization by colour tests, TLC, FT-IR and SEM-EDX. MDMA enantiomer ratio was determined by TFAA derivatisation followed by GC-MS analysis on a chiral column. The effect of UV and fluorescent light, temperature (5, 15, 25, 35 and 40°C) and humidity (33 and 75% RH) on the physical features was assessed. ‘Ecstasy’ tablets from seizures (N = 172) at EDM parties by the police were analysed to determine the psychoactive substance content per tablet. Partygoers at an EDM party were interviewed to determine their drug-use, including ‘ecstasy’ tablets and party behaviour. Results: Of the 45 batches examined 66.7% contained MDMA only as the main active ingredient, 6.7% contained mCPP, 4.4% BZP, 2.2% (1 batch) DPIA, 2.2% methanostenolone and 13% caffeine only. The majority of seized tablets were round in shape (91%) with logo (88.9%). The mean of means for mass of 237.2 mg (RSD 28.9%), diameter 8.09 mm (RSD 12.0%) and thickness 3.86 mm (RSD 19.1%). Of the 4 batches of tablets subjected to photostability testing (white, blue, green and orange colour), only the orange-coloured tablets showed a significant change in colour (p < 0.05, one-way ANOVA). The diameter of tablets stored at 15°C and 75% RH increased by 1.22 mm (p < 0.05, one-way ANOVA) and the thickness of tablets stored at 40 and 15°C respectively and 75% RH increased by 0.14 and 0.12 mm respectively (p < 0.05, one-way ANOVA). Chemical profiling determined that the Leuckart and reductive amination reactions were most probably used to synthesize the racemic MDMA and all tablets had a 50:50 ratio of R and S enantiomers. Lactose was the excipient mostly detected in tablets (40%). ‘Ecstasy’ tablets were the second most commonly confiscated drug at EDM parties (27.9%, n = 48 / 172) (cannabis being the most common), however our interviewed clubbers mostly (98.3%, n = 57) used alcohol. Conclusion: Physical and chemical characterisation can help link or discriminate between batches of ‘ecstasy’ tablets. For intelligence purposes tablets should be stored away from sunlight (visible and UV light) and at low RH (≈ 25%) and temperature (≈ 25°C).

Opioid drugs and their receptors are one of the most extensively studied areas of pharmacology, and in relation to biased agonism in particular. Previous research indicated that biased agonist at the Il-opioid receptor (MOPr) can be detected. Biased agonism theory, when applied to MOPr, offers the possibility to synthesize opioid ligands, lacking such lifethreatening side effects as addiction, tolerance, hyperalgesia and respiratory depression. In the current project the ability of MOPr ligands to activate Extracellular signal-Regulated Kinases 1/2 (ERKl/2), a key mediator of GPCR actions in cells, was studied. The MOPr ligands (DAMGO, morphine, buprenorphine, methadone, oxycodone, etorphine, pentazocine and endomorphin-2) were used that display different degrees of bias between G-protein and arrestin pathways as well as different efficacies for G protein activation. The model system used was HEK293 cells stably expressing MOPr.

Human ether-a-go-go-related gene (hERO) potassium (K+) channels carry the rapid delayed rectifier K+ current (IKr) which contributes to the repolarisation of cardiac action potentials. lKr plays a protective role against premature beats and is highly prone to pharmacological inhibition that can lead to acquired Long QT syndrome. Flecainide (a class le antiarrhythmic agent) is used in the treatment of atrial fibrillation; it inhibits IhERg/IKr at clinically relevant concentration and it has been occasionally associated with life-threatening ventricular tachycardia (Torsade de Pointes). Ivabradine is a specific bradycardic agent with a high selectivity for hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels. However, some evidence has raised the possibility that it may affect IKr. The principal aims of this study were to investigate the mechanism of interaction and the molecular determinants of pharmacological block of hERG by flecainide and ivabradine. In an additional set of experiments, the modulation by extracellular potassium ([K+]o) of the response of hERG to premature stimulations was examined.

Evidence from clinical and experimental research suggests that exposure to commonly used inhaled anaesthetics during early development may lead to neurobehavioral deficit later in life: However, exposure to these drugs may be unavoidable as exposure to pain and distress may lead to worse outcome. It is possible that co-administration of xenon, a rare anaesthetic gas, with proven neuroprotective properties, may reduce exposure to inhaled anaesthetics in neonates and provide a method to avoid these long term sequelae. Research in these areas relies on neonatal rodent models which contain confounding variables; many linked to the physiological derangement caused by the dosage of inhaled anaesthetic that has been chosen. This dissertation describes a new method to measure a sub-anaesthetic potency of sevoflurane and xenon in neonatal rats on postnatal days seven and nine: The effective inhaled concentration preventing cold-stimulated vocalisation in 50% (EiC50 CSV). The method, unlike other anaesthetic dose-response studies, uses randomisation of groups of rats to a range of concentrations and three durations of exposure. Logistic regression, with absence of vocalisation as the dependent variable and concentration as the explanatory variable is then performed and concentration-response curves with confidence intervals can be plotted. Additional explanatory variables can then be inserted into the model to assess the effect on the EiC50 CSV: Adequacy of exposure duration can be assessed with this approach. Models and equipotent sub-anaesthetic concentrations of sevoflurane and xenon in normothermic neonatal rats are presented. Maintenance of cardio-respiratory function and equal suppression of single channel electroencephalogram is demonstrated with exposure to these concentrations. Finally, neonatal models of anaesthesia are proposed in rats, pigs and humans.

Opioid analgesics are the leading class of prescription drugs that cause unintentional overdose deaths via respiratory depression. Poly-drug use is a common cause of death, with ethanol detected in approx.. 50% of heroin-related cases. Tolerance can be reversed by low concentrations of ethanol in locus coeruleus neurones and tolerance in these neurones occurs through a PKC-dependant mechanism. One possible explanation for the correlation between simultaneous opiate and alcohol use, and the likelihood of fatality, is that alcohol has a direct effect on PKC activity causing a reversal of opioid induced tolerance. This thesis aims to elucidate the mechanisms involved in the development and maintenance of opioid tolerance and the potential drug interactions that may affect this phenomenon. The effect of ethanol on PKC activity was investigated using a non-radioactive PKC detection assay and a fluorescent DAG-reporter expressed in HEK293 cells. The results from the in vitro assay did not demonstrate a direct effect of ethanol on PKC activity. In HEK293 cells ethanol reduced the increase in fluorescence associated with increased DAG activity. Therefore ethanol may indirectly inhibit PKC activity by inhibiting DAG. The role of PKC in morphine-induced desensitisation was investigated in brain slices containing LC neurones. PKC was shown to play two different but important role in desensitisation. At the lower basal levels of PKC in ex vivo brain slices, PKC inhibitors enhanced desensitisation, which was subsequently reversed in the presence of both the GRK and PKC inhibitors. Following preactivation of PKC with oxo-M, inhibitors caused reduction in the extent of desensitisation, together suggesting that more than one kinase may be involved in MOPr desensitisation. Using whole body plethysmography, the PKC inhibitor, GF109203x, reversed tolerance to morphine-induced respiratory depression. Tamoxifen (also reported to inhibit PKC activity) also reversed tolerance to respiratory depression. Therefore it is likely that PKC plays a role in opioid-induced tolerance to respiratory depression and this may be important in the increased risk of overdose in polydrug use. Data in this thesis provides evidence that PKC plays a pivotal role in the development of both desensitisation and tolerance. However there may be . additional signalling pathways involved, such as GRK. Furthermore, ethanol may inhibit DAG activation, thus indirectly affecting PKC activity; however a direct ethanol-PKC interaction was not demonstrated. Further elucidating the mechanisms involved in opioid tolerance will allow more effective advice to be given to opioid users at risk.

No description available.

No description available.