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Nociceptin orphanin FQ (N/OFQ) is the 17 amino acid endogenous ligand for the Gi-coupled N/OFQ-receptor (NOP). In vivo administration produces a wide range of physiological responses including; analgesia, hyperalgesia and anti-opioid actions.;In a series of in vitro assays including [leucyl -3H]N/OFQ binding, GTPgamma[35S] binding and inhibition of cAMP formation the following linked studies were performed; (1)N/OFQ structure-activity relationships (SAR) in cells (CHO) stably expressing human NOP (2)evaluation of receptor density on efficacy using the ecdysone inducible expression system and native tissues, (3)an investigation of NOP/G-protein coupling efficiency.;SAR studies can be summarized as combining arginine14, lysine15 repeat in N/OFQ (increase affinity/potency) with C-terminal [F/G]N/OFQ(1--13)NH2([F/G]) and [Nphe1]N/OFQ(1--13)NH 2([Nphe1]) modifications (reduce/eliminate efficacy). Arg14/Lys15 increased the affinity (pKi:10.31--11.16) and potency (pEC50:8.98--9.85) of N/OFQ. [F/G] and [Nphe 1] reduced the efficacy of N/OFQ from 1.0 to 0.44 and 0. Combination of Arg14/Lys15 and [Nphe1] in UFP-101 produced the highest affinity peptide antagonist available (pA 2:9.13, [Nphe1] alone:7.54). Using the ecdysone inducible expression system it was possible to vary the efficacy of [F/G], between antagonist (alpha:0 upstream/low density), partial agonist (alpha:0.3--0.75) and full agonist (alpha:1). In the mouse vas deferens and colon [F/G] displayed varying degrees of partial agonism possibly indicating differences in NOP receptor density. The binding of GDP and GTP in CHOhNOP was to high and low affinity sites. The fraction of GDP binding to the high affinity site was reduced by N/OFQ (77%, under basal to 32%). The reduction in high affinity binding of GDP appeared dependent on the efficacy of the ligand.;This thesis has identified several new peptides of varying efficacy/potency for use in defining the pathophysiological role(s) of N/OFQ-NOP. Efficacy is not just a property of the ligand but of the assay systems and its input receptor density. A cautious approach to new ligands characterized at a single endpoint is advocated.

Opioids drugs are a mainstay for the treatment of acute and chronic pain, however their use is limited by potentially fatal depression of breathing. In this thesis we have set out to identify networks of respiratory control and to investigate the effects of the opioid drug remifentanil upon areas of the brain that control breathing, using functional magnetic resonance imaging (FMRI) in healthy human volunteers.

is postulated that dynorphin inhibits the cocaine-induced increase in striatal dopamine levels, that may contribute to dynorphin's ability to prevent cocaine induced conditioned place preference and to attenuate an increase in locomotor activity. In this thesis interactions between dynorphin and the dopaminergic system in cocaine addiction have been addressed using preprodynorphin gene knockout mice treated with a chronic "binge" cocaine protocol.

The aim of the study was to ascertain the capabilities of the graphite furnace atomic absorption spectroscopy (OF AAS) system to discriminate between the two separate ecstasy batches according to the differing concentrations of trace metals. Previous studies have utilised rcp techniques such as rCP-AES and rCP-MS to determine trace metal concentrations in ecstasy tablets with particular interest in potential synthesis marker metals such as Pt, B and Hg. Five metals: Cu, Ba, Ni, Cr and Ph were quantified with good repeatability and reproducibility. The selection of elements comprises of metals that may be present due to additives, dyes, reducing agents, catalysts and elements recorded to be commonly present in ecstasy tablets, for example Ba. Elements Cu, Cr and Pb have been observed previously to be more homogenous within batches than elemental markers for synthesis routes suggesting these trace metals will be of use for indicating linkages between batches. Two separate batches of seized ecstasy tablets were analysed by GF AAS following digestion with nitric acid and hydrogen peroxide. Ecstasy tablets were acquired from the TICTAC unit at St George's Hospital London Tooting. HPLC analysis conducted at St. George's indicated that Batch 21573 of 20 tablets contained significant quantities of MDMA while Batch 26237 of 20 tablets contained predominantly caffeine. The small sample size has allowed for careful interpretation of intra-batch variation and inter-batch variation in the metal concentrations determined for each ecstasy batch. A previous study has used a similar sample size. The current study has successfully demonstrated the quantitation of five trace metals in ecstasy tablets for the first time via OF AAS. Large intra-batch variations were fOlU1d as expected for tablets produced in clandestine laboratories. Nickel in the MDMA containing batch 21573 was present in the range 0.47-13.1 ppm and in the caffeine containing batch 26237 in the range 0.35-9.06 ppm, very similar to previous reports of 1- 25 ppm and 0.3-16 ppm. The Cr levels detenmined in the current study (MDMA batch: 0.12-3.16 ppm, caffeine batch: 0.10-1.36 ppm) are lower than reported previously (0.7-34 ppm). The range ofPb concentrations (MDMA batch: 0.1 1-3.81 ppm, caffeine batch: 0.12- 4.91 ppm) are similar to previously reported (0.02-10 ppm). The copper levels in these samples were found to be particularly high (MDMA batch: 4-2379 ppm, caffeine batch: 17-1851 ppm) compared with previous studies using ICP techniques which fOlU1d ranges of 1-19 ppm and 0.8-38 ppm in ecstasy tablets. Barium concentrations were relatively similar within each batch with significantly higher concentrations found in the caffeine containing batch 26237 (rv1DMA batch: 0.19-0.66 ppm, caffeine batch: 3.77-5.47 ppm). Barium was the only element to offer discrimination between the two batches of tablets. AAS systems are well established, cheaper to install and run, and require less user skill as well as less sample volume. The addition that the current study offers in light of previous literature in the area is evidence of the capabilities of GF AAS to perform analyses on ecstasy tablets, with good detection limits, good precision and small sample volume, but with a technique that is well establish, fmancially less challenging and readily available in forensic laboratories in comparison to the more powerful ICP-MS technique

Background: Opioids are used to treat moderate to severe pain, including that associated with surgery, cancer and infection. Findings from a range of species and methodologies have demonstrated that whereas some opioids are immunoneutral (e.g. buprenorphine), others can be immunosuppressive (e.g. morphine) or immunostimulatory (e.g. tramadol). This study compared the effects of commonly used opioids on the potential to mount protective immunity against bacterial infections (neutrophil/monocyte phagocytosis and oxidative burst responses) and cancer (NK cell cytotoxicity/activation), as well as T cell activation and cytokine production. Methodology: Peripheral blood was obtained from healthy volunteers and the influence of clinically relevant concentrations of morphine, tramadol, fentanyl, buprenorphine, methadone, oxycodone, diamorphine and codeine on neutrophil and monocyte phagocytosis of E.coli and oxidative burst responses to fMLP, PMA and E.coli were determined by flow cytometry. Their effects on NK cell cytotoxicity in IL-2 stimulated, platelet-free peripheral blood mononuclear cells (PBMCs) and NK and T cell activation in IL-2 and anti-CD3/28 mAb stimulated PBMCs were also assessed. Cytokine production by anti-CD3/28 mAb and IL-2 stimulated PBMCs was determined using a cytometric bead array technique. Results: The only consistent, statistically significant effect was that methadone, oxycodone and diamorphine inhibited the production of IL-6 in IL-2 stimulated PBMCs, which could impair the acute phase response. There was however marked variability between individuals, both in their baseline and the effect of opioids, this was especially evident for morphine, tramadol, fentanyl and buprenorphine in the phagocytic and oxidative burst response to E.coli. Conclusion: These findings suggest that opioid choice could influence the susceptibility to bacterial or fungal infection, but have little effect on anti-cancer or anti-viral protection. Clinical studies aimed at assessing the in vivo effects of opioid administration on immune function in relevant patient groups are required in order to assess the clinical significance of opioid choice.

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