Background and purpose: Selective cannabinoid CB 2 receptor agonists have demonstrated analgesic activity across multiple preclinical pain models. AM1241 is an indole derivative that exhibits high affinity and selectivity for the CB 2 binding site and broad spectrum analgesic activity in rodent models, but is not an antagonist of CB 2 in vitro functional assays. Additionally, its analgesic effects are m-opioid receptor-dependent. Herein, we describe the in vitro and in vivo pharmacological properties of A-796260, a novel CB 2 agonist. Experimental approach: A-796260 was characterized in radioligand binding and in vitro functional assays at rat and human CB 1 and CB 2 receptors. The behavioural profile of A-796260 was assessed in models of inflammatory, post-operative, neuropathic, and osteoarthritic (OA) pain, as well as its effects on motor activity. The receptor specificity was confirmed using selective CB 1 , CB 2 and m-opioid receptor antagonists. Key results: A-796260 exhibited high affinity and agonist efficacy at human and rat CB 2 receptors, and was selective for the CB 2 vs CB 1 subtype. Efficacy in models of inflammatory, post-operative, neuropathic and OA pain was demonstrated, and these activities were selectively blocked by CB 2 , but not CB 1 or m-opioid receptor-selective antagonists. Efficacy was achieved at doses that had no significant effects on motor activity. Conclusions and implications: These results further confirm the therapeutic potential of CB 2 receptor-selective agonists for the treatment of pain. In addition, they demonstrate that A-796260 may be a useful new pharmacological compound for further studying CB 2 receptor pharmacology and for evaluating its role in the modulation of pain.

BACKGROUND AND PURPOSE Cannabinoid CB2 receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory and neuropathic pain. However, mechanisms underlying CB2‐mediated analgesic effects remain largely unknown. The present study was conducted to elucidate the CB2 receptor expression in ‘pain relevant’ tissues and the potential sites of action of CB2 agonism in rats. EXPERIMENTAL APPROACH Expression of cannabinoid receptor mRNA was evaluated by quantitative RT‐PCR in dorsal root ganglia (DRGs), spinal cords, paws and several brain regions of sham, chronic inflammatory pain (CFA) and neuropathic pain (spinal nerve ligation, SNL) rats. The sites of CB2 mediated antinociception were evaluated in vivo following intra‐DRG, intrathecal (i.t.) or intraplantar (i.paw) administration of potent CB2‐selective agonists A‐836339 and AM1241. KEY RESULTS CB2 receptor gene expression was significantly up‐regulated in DRGs (SNL and CFA), spinal cords (SNL) or paws (CFA) ipsilateral to injury under inflammatory and neuropathic pain conditions. Systemic A‐836339 and AM1241 produced dose‐dependent efficacy in both inflammatory and neuropathic pain models. Local administration of CB2 agonists also produced significant analgesic effects in SNL (intra‐DRG and i.t.) and CFA (intra‐DRG) pain models. In contrast to A‐836339, i.paw administration of AM‐1241 dose‐relatedly reversed the CFA‐induced thermal hyperalgesia, suggesting that different mechanisms may be contributing to its in vivo properties. CONCLUSIONS AND IMPLICATIONS These results demonstrate that both DRG and spinal cord are important sites contributing to CB2 receptor‐mediated analgesia and that the changes in CB2 receptor expression play a crucial role for the sites of action in regulating pain perception.

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Background: Injection of nerve growth factor (NGF) produces mechanical and thermal hypersensitivity in rodents and humans. Treatment with sequestering antibodies demonstrates the importance of NGF in various pain states, with efficacy seen in a number of animal pain models and in painful human conditions. However, these phenomena have not been evaluated in the context of using NGF-induced hypersensitivities as a model of pain. Methods: NGF-induced behaviours were characterized using von Frey filament, pinprick and thermal endpoints and then pharmacologically evaluated with known reference agents. Results: Intraplantar NGF injection produced a dose-dependent increase in thermal sensitivity that lasted through 24 h post-injection and an immediate long-lasting (2 week) increase in mechanical sensitivity at the injection site, with no effects detected at secondary sites. NGF-induced mechanical sensitivity was pharmacologically characterized at 4 h and 1 week post-NGF injection. The nonsteroidal anti-inflammatory drugs (NSAIDs), celecoxib and diclofenac, were minimally effective against both thermal and mechanical endpoints. Gabapenitn and duloxetine were only moderately effective against thermal and mechanical hypersensitivity. Morphine was effective against thermal and mechanical endpoints at every time point examined. Treatment with the transient receptor potential vanilloid 1 (TRPV1) antagonist A-784168 partially attenuated NGF-induced thermal and mechanical sensitivity at all time points examined. Conclusions:The results reported here suggest that effects of NGF on thermal and mechanical sensitivity in rats are similar to those reported in human and are partially driven by TRPV1. The rat NGF model may serve as a potential translational model for exploring the effects of novel analgesic agents.

Studies demonstrating the antihyperalgesic and antiallodynic effects of cannabinoid CB 2 receptor activation have been largely derived from the use of receptor-selective ligands. Here, we report the identification of A-836339 [2,2,3,3-tetramethyl-cyclopropanecarboxylic acid [3-(2-methoxy-ethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]-amide], a potent and selective CB 2 agonist as characterized in in vitro pharmacological assays and in in vivo models of pain and central nervous system (CNS) behavior models. In radioligand binding assays, A-836339 displays high affinities at CB 2 receptors and selectivity over CB 1 receptors in both human and rat. Likewise, A-836339 exhibits high potencies at CB 2 and selectivity over CB 1 receptors in recombinant fluorescence imaging plate reader and cyclase functional assays. In addition A-836339 exhibits a profile devoid of significant affinity at other G-protein-coupled receptors and ion channels. A-836339 was characterized extensively in various animal pain models. In the complete Freund's adjuvant model of inflammatory pain, A-836339 exhibits a potent CB 2 receptor-mediated antihyperalgesic effect that is independent of CB 1 or -opioid receptors.A-836339 has also demonstrated efficacies in the chronic constrain injury (CCI) model of neuropathic pain, skin incision, and capsaicin-induced secondary mechanical hyperalgesia models. Furthermore, no tolerance was developed in the CCI model after subchronic treatment with A-836339 for 5 days. In assessing CNS effects, A-836339 exhibited a CB 1 receptor-mediated decrease of spontaneous locomotor activities at a higher dose, a finding consistent with the CNS activation pattern observed by pharmacological magnetic resonance imaging. These data demonstrate that A-836339 is a useful tool for use of studying CB 2 receptor pharmacology and for investigation of the role of CB 2 receptor modulation for treatment of pain in preclinical animal models.It is estimated that as high as 50% of the population will experience chronic pain during their lifetime, and the prevalence is likely to rise with the continued aging of the population (Markman and Philip, 2007). As a consequence, there exists an ever-growing demand for new therapies to provide safe and effective pain management. Despite intensive research to identify novel therapeutic approaches, there have been few major advances in pain therapy over the past sev-

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