| Summary: | Chronic pain is a debilitating condition that is poorly treated in the clinic as conventional analgesics often lack efficacy and therapeutic doses can be limited by adverse side effects. The endocannabinoid (EC) system has been implicated in the modulation of pain and the antinociceptive effects of ECs can be enhanced by inhibiting catabolic enzymes. The main aims of this thesis were to investigate the roles that EC metabolism play in nociceptive behaviour. Theses aims were addressed by measuring tissue levels of ECs in inflammatory conditions, probing the mechanisms underlying EC mediated antinociception using pharmacological approaches and developing a sensitive analytical method to facilitate the analysis of ECs in microdialysate samples. t The key pharmacological tools used in the studies were the fatty acid amide hydrolase (FAAH) inhibitor URB597, the selective COX2 inhibitor nimesulide and the putative monoacylglycerol lipase (MAG lipase) inhibitor URB602. Intraplantar injection of URB597 (25 Ng), nimesulide (50 pg) or URB602 (70 pg) was analgesic in the carrageenan model of inflammatory pain, but only COX2 inhibition was shown to be antiinflammatory. A higher dose of URB597 (100 pg), was not, however, found to be analgesic in this model. Inhibition of metabolising enzymes resulted in changes in levels of ECs in spinal and paw tissue in the presence of carrageenan induced inflammationThe analgesic effects of URB597 (25 pg) and nimesulide (50 pg) were associated with an increase in peripheral levels of the EC-like acylethanolamine palmitoylethanolamide and were found to be mediated by the peroxisome proliferator-activated receptor alpha (PPARa). The higher dose of URB597 (100 pg) was associated with an increase of the EC anandamide, which has been shown to be a ligand for the pro-nociceptive transient receptor potential channel type V1 (TRPV1). However, the disappearance of an analgesic response at the higher dose of URB597 was independent of TRPV1 activation. Treatment with URB602 (70 pg) resulted in an increase in all ECs and related compounds ipsilateral hindpaw tissue suggesting that the inhibitor blocks FAAH as well as MAG lipase activity and so is not a selective inhibitor. Two high performance liquid chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) methods, nano-HPLC-MS/MS and micro- HPLC-MS/MS, were developed to quantitatively analyse ECs and related compounds in low fmol concentrations. The micro-HPLCMS/ MS was then used to assess the time-related effects of systemic URB597 treatment on EC levels in the hippocampus of the awake, freely-moving rat and post-mortem EC levels in hippocampal tissue. Whilst intraperitoneal injection of URB597 (1 mg kg-1) appeared to reduce release of ECs in the hippocampus compared to vehicle controls, a positive correlation between dialysate and tissue EC levels was seenThe results of these studies show that EC metabolism is a key limiting factor in the analgesic action of the EC system in inflammatory pain and highlights EC metabolising enzymes as potential targets for new analgesic drugs
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