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BACKGROUND AND PURPOSE The amphibian peptide Bv8 induces potent nociceptive sensitization in rodents. Its mammalian homologue, prokineticin 2 (PROK2), is strongly up-regulated in inflamed tissues and is a major determinant in triggering inflammatory pain. Bv8 and PROK2 activate two closely related GPCRs, PK1 and PK2, in a relatively non-selective fashion. To characterize better the roles of the two receptors in hyperalgesia and to obtain ligands whose binding affinity and efficacy differed for the two receptors, we modified the Bv8 molecule in regions essential for receptor recognition and activation. EXPERIMENTAL APPROACH We modified the Bv8 molecule by substituting Trp in position 24 with Ala (A-24) and compared it with Bv8 for binding and activating PK1 and PK2 receptors in cell preparations and in affecting nociceptive thresholds in rodents. KEY RESULTS A-24 preferentially bound to PK2 receptors and activated them with a lower potency (5-fold) than Bv8. When systemically injected, A-24 induced Bv8-like hyperalgesia in rats and in mice, at doses 100 times higher than Bv8. Locally and systemically injected at inactive doses, A-24 antagonized Bv8-induced hyperalgesia. In rat and mouse models of inflammatory and post-surgical pain, A-24 showed potent and long-lasting anti-hyperalgesic activity. Unlike Bv8, A-24 increased β-endorphin levels in mouse brain. CONCLUSIONS AND IMPLICATIONS A-24 induced its anti-hyperalgesic effect in rodents by directly blocking nociceptor PK1 receptors and by activating the central opioid system and the descending pain control pathway through brain PK2 receptors.

Background and purpose: N-arachidonoyl-serotonin (AA-5-HT) is an inhibitor of fatty acid amide hydrolase (FAAH)-catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA-5-HT antagonizes the transient receptor potential vanilloid-1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia. Experimental approach: AA-5-HT was tested in vitro, on HEK-293 cells overexpressing the human or the rat recombinant TRPV1 receptor, and in vivo, in rats and mice treated with formalin and in rats with chronic constriction injury of the sciatic nerve. The levels of the endocannabinoids, AEA and 2-arachidonoylglycerol, in supraspinal (periaqueductal grey, rostral ventromedial medulla), spinal or peripheral (skin) tissues were measured. Key results: AA-5-HT behaved as an antagonist at both rat and human TRPV1 receptors (IC50=37-40 nM against 100 nM capsaicin). It exerted strong analgesic activity in all pain models used here. This activity was partly due to FAAH inhibition, elevation of AEA tissue levels and indirect activation of cannabinoid CB1 receptors, as it was reversed by AM251, a CB1 antagonist. AA-5-HT also appeared to act either via activation/desensitization of TRPV1, following elevation of AEA, or as a direct TRPV1 antagonist, as suggested by the fact that its effects were either reversed by capsazepine and 5′-iodo-resiniferatoxin, two TRPV1 antagonists, or mimicked by these compounds administered alone. Conclusions and implications: Possibly due to its dual activity as a FAAH inhibitor and TRPV1 antagonist, AA-5-HT was highly effective against both acute and chronic peripheral pain. British Journal of Pharmacology (2007) 150, 766–781. doi:10.1038/sj.bjp.0707145

Background and purpose: We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity. Experimental approach: The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-γ-S binding to cell membranes, and their action on nociception in vivo was determined. Key results: CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50∼20 nM, max. stimulation=48%), behaving as an inverse CB1 agonist, but it stimulated GTP-γ-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50=100 nM, max. stimulation=48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50=1600 nM, max. inhibition=68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50=450 nM, max. inhibition=40%) and hCB1-CHO cells (EC50=2600 nM, max. inhibition=62% of CP-55,940 effect), and stimulated GTP-γ-S binding to mouse brain membranes (EC50=11 nM, max. stimulation∼16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-γ-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception. Conclusions and implications: CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors. British Journal of Pharmacology (2006) 149, 431–440. doi:10.1038/sj.bjp.0706888