The CB 1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ 9 -tetrahydrocannabinol and other CB 1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB 1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB 1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB 1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [ 3 H]CP55,940 to the CB 1 receptor as well as enhancing AEA-stimulated [35 S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB 1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB 1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB 1 PAM and provide the first proof of principle that CB 1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.

BACKGROUND AND PURPOSESince monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compound, KML29 (1,1,1,3,3,[1,3]dioxol-5-yl)(hydroxy)methyl)piperidine-1-carboxylate). EXPERIMENTAL APPROACHIn the present study, we tested KML29 in murine inflammatory (i.e. carrageenan) and sciatic nerve injury pain models, as well as the diclofenac-induced gastric haemorrhage model. KML29 was also evaluated for cannabimimetic effects, including measurements of locomotor activity, body temperature, catalepsy, and cannabinoid interoceptive effects in the drug discrimination paradigm. KEY RESULTSKML29 attenuated carrageenan-induced paw oedema and completely reversed carrageenan-induced mechanical allodynia. These effects underwent tolerance after repeated administration of high-dose KML29, which were accompanied by cannabinoid receptor 1 (CB1) receptor desensitization. Acute or repeated KML29 administration increased 2-AG levels and concomitantly reduced arachidonic acid levels, but without elevating anandamide (AEA) levels in the whole brain. Furthermore, KML29 partially reversed allodynia in the sciatic nerve injury model and completely prevented diclofenacinduced gastric haemorrhages. CB1 and CB2 receptors played differential roles in these pharmacological effects of KML29. In contrast, KML29 did not elicit cannabimimetic effects, including catalepsy, hypothermia and hypomotility. Although KML29 did not substitute for Δ 9 -tetrahydrocannabinol (THC) in C57BL/6J mice, it fully and dose-dependantly substituted for AEA in fatty acid amide hydrolase (FAAH) (−/−) mice, consistent with previous work showing that dual FAAH and MAGL inhibition produces THC-like subjective effects. CONCLUSIONS AND IMPLICATIONSThese results indicate that KML29, a highly selective MAGL inhibitor, reduces inflammatory and neuropathic nociceptive behaviour without occurrence of cannabimimetic side effects. LINKED ARTICLESThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx

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BACKGROUND AND PURPOSE Inhibition of diacylglycerol lipase (DGL)β prevents LPS-induced pro-inflammatory responses in mouse peritoneal macrophages.Thus, the present study tested whether DGLβ inhibition reverses allodynic responses of mice in the LPS model of inflammatory pain, as well as in neuropathic pain models. EXPERIMENTAL APPROACHInitial experiments examined the cellular expression of DGLβ and inflammatory mediators within the LPS-injected paw pad. DAGL-β (À/À) mice or wild-type mice treated with the DGLβ inhibitor KT109 were assessed in the LPS model of inflammatory pain. Additional studies examined the locus of action for KT109-induced antinociception, its efficacy in chronic constrictive injury (CCI) of sciatic nerve and chemotherapy-induced neuropathic pain (CINP) models. KEY RESULTSIntraplantar LPS evoked mechanical allodynia that was associated with increased expression of DGLβ, which was co-localized with increased TNF-α and prostaglandins in paws. DAGL-β (À/À) mice or KT109-treated wild-type mice displayed reductions in LPSinduced allodynia. Repeated KT109 administration prevented the expression of LPS-induced allodynia, without evidence of tolerance. Intraplantar injection of KT109 into the LPS-treated paw, but not the contralateral paw, reversed the allodynic responses. However, i.c.v. or i.t. administration of KT109 did not alter LPS-induced allodynia. Finally, KT109 also reversed allodynia in the CCI and CINP models and lacked discernible side effects (e.g. gross motor deficits, anxiogenic behaviour or gastric ulcers). CONCLUSIONS AND IMPLICATIONSThese findings suggest that local inhibition of DGLβ at the site of inflammation represents a novel avenue to treat pathological pain, with no apparent untoward side effects.Abbreviations 2-AG, 2-arachidonoylglycerol; ABHD6, α β hydrolase domain-containing protein 6; CCI, chronic constriction injury; CINP , chemotherapy-induced peripheral neuropathy; DGL, (also known as DAGL), diacylglycerol lipase; F AAH, fatty acid amide hydrolase; MGL, monoacylglycerol lipase; THC, Δ 9 -tetrahydrocannabinol

Background.-Isovaleric Acid (IVA) is a 5-carbon branched chain fatty acid present in fermented foods and produced in the colon by bacterial fermentation of leucine. We previously reported that the shorter, straight chain fatty acids acetate, propionate and butyrate, differentially affect colonic motility; however, the effect of branched chain fatty acids on gut smooth muscle and motility is unknown. Aims.-To determine the effect of IVA on contractility of colonic smooth muscle. Methods.-Murine colonic segments were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with acetylcholine (ACh) and the effects of IVA on ACh-induced contraction were measured in the absence and presence of tetrodotoxin (TTx) or inhibitors of nitric oxide synthase (L-N-nitroarginine (L-NNA)) or adenylate cyclase (SQ22536). The effect of IVA on ACh-induced contraction was also measured in isolated muscle cells in the presence or absence of SQ22536 or protein kinase A (PKA) inhibitor (H-89). Direct activation of PKA was measured in isolated muscle cells. Results.-In colonic segments, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion; the IVA response was not affected by TTx or L-NNA but inhibited by SQ22536. Similarly, in isolated colonic muscle cells, AChinduced contraction was inhibited by IVA in a concentration-dependent fashion and the effect blocked by SQ22536 and H-89. IVA also increased PKA activity in isolated smooth muscle cells. Conclusions.-The branched chain fatty acid IVA acts directly on colonic smooth muscle and causes muscle relaxation via the PKA pathway.