In HEK cells expressing human and rat isoforms of TRPV1, capsaicin, anandamide (an endocannabinoid), olvanil (a structural analogue of capsaicin) and ethanol displayed TRPV1 activity. in contrast, Delta9-THC (an exocannabinoid) did not. No significant difference occurred between human and rat with respect to capsaicin and anandamide activity at 22ºC or 37ºC. Anandamide displayed partial agonism relative to capsaicin at both temperatures at rat only. Anandamide was also an agonist at cannabinoid CB1 receptors. Capsazepine, a competitive TRPV1 antagonist, inhibited capsaicin, olvanil and anandamide response; was not temperature-dependent but displayed a 6-fold higher potency at human TRPV1.;The first neuronal model to express recombinant human TRPV1 was produced by sub-cloning and transfection into the neuroblastoma SH-SY5Y cell-line, a more physiologically relevant system. Pharmacological characterisation confirmed expression of TRPV1 with comparable pharmacology to non-neuronal models in this thesis. For example, capsaicin pEC50/capsazepine pKB in HEk (human TRPV1) and SH-SY5Y were 6.77/6.75 and 6.63/7.44 respectively. In SH-SY5Y cells, the agonist resiniferatoxin displayed the highest potency (pEC50 9.03). Iodo-RTX, an antagonist, revealed a higher affinity than capsazepine. Capsaicin-mediated increases in intracellular calcium (pEC50 6.63) in SH-SY5Y cells were sufficient to sustain [3H]noradrenaline release (pEC50 9.21). In a perfusion system, it was shown that the two events were temporally linked.;Overall, these findings have: 1. expanded current knowledge of TRPV1 pharmacology. 2. established a neuronal model for further studies, especially TRPV1 desensitisation.

The endogenous cannabinoid anandamide (AEA) can modulate the excitability of sensory neurones; this may be of benefit in the treatment of pain disorders. In this study, the whole cell patch clamp technique and fura-2 calcium imaging have been used to investigate the actions of AEA in cultured dorsal root ganglion (DRG) neurones with particular reference to modulation of ion conductances, and activation of TRPV1 receptors. AEA inhibited voltage-activated Ca²⁺ currents independently of CB₁ receptor activation. AEA had dual effects on depolarisation-evoked Ca²⁺ flux in distinct subpopulations of DRG neurones; the inhibitory effect was CB₁ independent, while the enhancing effect was CB₁ mediated but pertussis toxin insensitive, indicating activation of multiple signalling pathways of AEA. AEA both inhibited voltage-activated K⁺ currents and slowed the recovery phase of depolarisation-evoked Ca²⁺ transients, both effects being insensitive to pertussis toxin treatment. These effects were also attenuated by the PMSF, an inhibitor of AEA hydrolysis, and mimicked by arachidonic acid, a primary metabolite of AEA, suggesting that production of active metabolites contributes to the observed effects of AEA. AEA elicited TRPV1 mediated responses with less efficacy than capsaicin. When applied directly to the intracellular environment of DRG neurones, the ability of AEA to evoke TRPV1 responses was significantly enhanced as compared with extracellular application. In neurones treated chronically with nerve growth factor (NGF), previously shown to sensitise TRPV1 receptors, responses to AEA and capsaicin were affected differentially. Further investigations revealed a cannabinoid receptor mediated inhibition of TRPV1 activity in low LGF neurones, which was disrupted by chronic NGF treatment.

Enhancement of the endocannabinoid (EC) system is analgesic in many models of acute and chronic pain. Current knowledge of EC biosynthetic pathways and their roles in normal physiology and pathophysiology is limited. EC lipids can be measured using LC-MS/MS but there are very few analytical techniques available to measure their biosynthetic precursors and intermediates. This study sought to develop a novel LC-MS/MS to measure EC biosynthetic components and describe their contribution to pain pathophysiology. LC-MS/MS methodology capable of measuring ECs, their biosynthetic precursors and intermediates was developed and applied to rat tissues involved in nociceptive processing. This method measured N-acyl phosphatidyl ethanolamine (NAPE) precursors of the N-acyl ethanolamine (NAE) ECs in rat brain, spinal cord and hind paw skin tissue. Additionally, glycerol-phospho-NAE (GpNAE) components of the GDE1 alternative synthetic pathway were also detected in these tissues demonstrating the functional presence of this pathway. An important finding is the drastic effects of anaesthesia in EC, EC precursor and intermediate levels in the rat spinal cord whereby reductions compared to stunning and decapitation were measured. In the MIA model of OA, EC synthesis is unlikely to contribute'to disease pathology in the spinal cord due to the comparable measurements of EC biosynthetic lipids in both diseased and sham rats. Intraplantar injection of carrageenan reduced EC levels and their related GpNAE intermediates in the rat hindpaw skin demonstrating a close relationship of this biosynthetic pathway and the NAE products in this model. Modulation of the PEA generating NAPE suggests a neuroprotective role following stimulation in Page 11 Understanding the Role of the Endocannabinoid Biosynthet ic Pathways in Mediat ing Analgesia areas of nociceptive processing. NAPE-PLD, the most commonly accepted NAE biosynthetic enzyme, was visualised in spinal cord neuronal cells and hindpaw skin of this model by a newly developed selective immunohistochemistry technique. NAPE-PLD expressing cells were increased in the dorsal horn of the spinal cord following carrageenan injection. Collectively this thesis presents the development and application of novel methodology to understanding the role of EC synthesis in pain pathology.

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There is considerable interest in the synthesis of K-antagonists as therapeutic agents but also as a means of further understanding the role of the K-opioid receptor. In the search for irreversible and selective reversible K-opioid antagonists, it was decided to modify the structures of the two most well-known K-antagonists, GNTI and norBNI. In particular, two main approaches have been used for the design of novel ligands; these explored the introduction of electrophilic (isothiocyanate) or lipophilic (substituted/unsubstituted benzyl) groups onto the guanidinium moiety of GNTI or at the pyrrolic nitrogen of norBNI. In the first series of compounds, jp-hydroxy-, w-hydroxy-, p-methoxy-, jp-methyland 3,4-dichlorobenzylGNTI analogues have been prepared. Binding and functional studies of /p-hydroxy- and m-hydroxy-derivatives have confirmed the results of molecular modelling studies, which had suggested that the phenolic group of the former should mimic the second phenolic group of norBNI that was previously shown to be crucial for k - antagonist selectivity. Electrophilic ligands modelled on GNTI have also been prepared and N'-(5-isothiocyanatopentyl)GNTI has been sent for biological evaluation, while the successful synthesis of N'-(4-aminobutyl)GNTI will allow the preparation of the corresponding isothiocyanate to be achieved. Modification of norBNI followed previous studies where the duration of action of naltrindole was extended by indolic N-benzylation. In-vivo, BnorBNI was a p-agonist when administered sc and an irreversible K-antagonist when administered icv. This led us to prepare 17,17'-di-NMe derivatives of norBNI and BnorBNI as potential mixed pagonist/K-antagonist ligands. Binding and functional studies of these analogues showed that replacement of the cyclopropyl methyl groups with methyl groups led to a decrease in K-antagonist potency and p-agonist potency with concomitant increase in p-agonist efficacy. We have also prepared isothiocyanates modelled on BnorBNI, with the electrophilic moiety attached directly to the benzyl group of BnorBNI or linked by a methylene spacer. At the time of submission, pharmacological evaluation of these ligands was still outstanding. Finally, unexpected reactions with norBNI have led us to investigate whether the pphthalimidobenzyl group can be used as a general protecting group for indolic and pyrrolic nitrogens. Evaluation on carbazole, tetrahydrocarbazole and an indolomorphinan has shown this is not the case.

Introduction: This dissertation investigates the current management of cancer pain with particular emphasis on the World Health Organization analgesic ladder. This was considered necessary because published studies examining the adequacy of cancer pain control have suggested that the efficacy of the WHO ladder may have been overestimated and because the place of morphine as the 1st line opioid at Step III of the ladder has been challenged. The dissertation also investigates whether an alternative approach might be superior and further explores the patient barriers to the use of opioids. Methods: The studies incorporated within this dissertation include an observational pain study examining pain control in 242 patients under the care of specialist palliative care teams, a systematic review and meta-analysis of 4 trials investigating oxycodone in cancerrelated pain, a pilot study for a randomised controlled trial of an experimental 2-step analgesic ladder versus the traditional 3-step approach and a qualitative study exploring patients' views and concerns when offered opioids for the treatment of pain caused by cancer. Results: The observational study showed that pain was not well controlled for the majority (79.3%: C. I. 74.1% to 84.4%); the systematic review showed that there was no difference in efficacy and tolerability between oxycodone and morphine; and the 2-step trial showed that earlier use of Step III opioids within the novel 2-step approach might result in better pain control. However, the qualitative study showed that patients associate morphine and other Step III opioids with death and therefore they reject them as useful means of controlling pain. Conclusion: Morphine and other opioids currently remain our best means of managing pain caused by cancer, but that both professionals and patients require ongoing education, so that we can break down the barriers that still inhibit their use.

There is significant inter-individual variation in response to morphine in terms of analgesia and side-effects. In recent years there has been growing interest in the possibility that genetic factors may play a role in variability in morphine response. The aims of this thesis were to develop a clinically relevant method of defining response to morphine, to investigate how multiple clinical and genetic factors may interact together to influence response to morphine and to explore constipation as a common side-effect of morphine. Clinical and biological data were collected as part of two clinical trials: 1) A prospective observational study which included patients taking oral morphine for cancer pain (N=298) and 2) A prospective follow-up randomised controlled trial of oral morphine versus oral oxycodone for cancer pain (recruitment ongoing). Symptom complexes were examined using Principal Components Analysis. Genetic association testing was carried out using both the candidate gene approach (sequence-specific primers with polymerase chain reaction) and genome-wide assays. Multivariate regression analyses were used to explore gene-gene and gene-environment interactions. Preliminary testing of a constipation assessment tool was performed. Analgesic response and central side-effects appear to be distinct components of morphine response. The genetic and clinical factors associated with these clinical outcomes and with daily morphine dose requirements are markedly different. There is inter-individual variation in bowel function in cancer patients on oral morphine. Constipation is a common symptom and is generally poorly managed. It is too early to be able to apply the results of genetic association studies of morphine response in cancer pain to clinical practice. Response to morphine is complex, both in terms of clinical confounders and pharmacogenetics. Challenges for future research include carrying out carefully designed studies of adequate power, standardising outcome measures of response to morphine and expansion of the genetic association testing.

L'aspirine est l'anti-plaquettaire le plus largement prescrit à la phase aiguë de l'accident vasculaire cérébral (AVC) ischémique. Cependant, la survenue de récidives, malgré cette prescription, est fréquente. La description de l'effet de l'aspirine sur l'activité plaquettaire durant cette phase n'a jamais été réalisée. Elle pourrait mettre en évidence une moindre réponse plaquettaire et aider à établir de nouvelles stratégies thérapeutiques. Cinquante patients, ont reçu par voie orale 300 mg d'aspirine, suite à un AVC ischémique. Ensuite, des prélèvements sanguins ont été réalisés : entre 2 et 3 heures (T1), entre 23 et 24 heures (T2) après la prise d'aspirine et, pour des patients déjà traités quotidiennement par une dose inférieure, avant la prise d'aspirine (T0). Les concentrations sériques de thromboxane (TX) B2 ont été mesurées, ainsi que les agrégations induites par l'acide arachidonique, par le collagène à la concentration de 2µg/L (Col2) et 20 µg/L (Col20). Afin de diminuer l'effet des variations de condition d'expérience, les résultats pour Col2 ont été rapportés à ceux pour Col20 (Col2/20). Tous les patients ont présenté une réponse à l?aspirine visible à T1 avec de plus, des concentrations de TXB2 abaissées en comparaison à T0. Il existe une récupération de l'activité plaquettaire à T2 comparée à T1, montrée par les concentrations de TXB2 et le rapport Col2/20. La dose orale de 300 mg d'aspirine, donnée à la phase aiguë de l'AVC, entraîne une inhibition plaquettaire, mais avec une récupération visible sur 24 heures. Pour les patients déjà traités quotidiennement par une dose inférieure, elle permet de compléter l'inhibition de la voie TXA2 dépendante / Aspirin is the most commonly used antiplatelet treatment during the acute phase of cerebral ischemic events. But, despite this protection, early ischemic recurrences are frequent, and considered as clinical failures of this therapy. We studied laboratory parameters of the first 300 mg oral dose of aspirin given, within 48 hours, after ischemic cerebral event. Fifty patients were included. For all patients, two blood sampling were performed, the first, during the third hour after aspirin intake (T1) and the second during the twenty-fourth hour (T2). For patients already treated with a daily dose of aspirin, a supplementary withdrawn was done before aspirin intake (T0). Platelet reactivity was studied on the basis of serum thromboxane (TX) B2 levels and light transmission aggregometry after stimulation of platelet-rich plasma by acid arachidonic and collagen 2µg/mL reported to results with collagen 20 µg/mL (ratio Col2/20). Inhibition of platelet activity was observed, at T1, for all patients. There is a significant increase of TXB2 values, and of relative values of the ratio Col2/20, at T2 as compared to T1. For already aspirin treated patients, there is a significant decrease of TXB2 levels at T1 as compared to T0. There is a platelet reactivity recovery within 24 hours, following the first 300 mg oral dose of aspirin, during the acute phase of a cerebral ischemic event, and demonstrated by TXB2 levels and ratio Col2/20. This fact would favour early ischemic recurrences. However, this dose is able to complete the inhibition of the TXA2 pathway for already aspirin treated patients