BACKGROUND AND PURPOSENeuropeptide FF (NPFF) behaves as an endogenous opioid-modulating peptide. In the present study, the opioid and NPFF pharmacophore-containing chimeric peptide BN-9 was synthesized and pharmacologically characterized. EXPERIMENTAL APPROACHAgonist activities of BN-9 at opioid and NPFF receptors were characterized in in vitro cAMP assays. Antinociceptive activities of BN-9 were evaluated in the mouse tail-flick and formalin tests. Furthermore, its side effects were investigated in rotarod, antinociceptive tolerance, reward and gastrointestinal transit tests. KEY RESULTSBN-9 acted as a novel multifunctional agonist at μ, δ, κ, NPFF1 and NPFF2 receptors in cAMP assays. In the tail-flick test, BN-9 produced dose-related antinociception and was approximately equipotent to morphine; this antinociception was blocked by μ and κ receptor antagonists, but not by the δ receptor antagonist. In the formalin test, supraspinal administration of BN-9 produced significant analgesia. Notably, repeated administration of BN-9 produced analgesia without loss of potency over 8 days. In contrast, repeated i.c.v. co-administration of BN-9 with the NPFF receptor antagonist RF9 produced significant antinociceptive tolerance. Furthermore, i.c.v. BN-9 induced conditioned place preference. When given by the same routes, BN-9 had a more than eightfold higher ED 50 value for gastrointestinal transit inhibition compared with the ED 50 values for antinociception. CONCLUSIONS AND IMPLICATIONSBN-9 produced a robust, nontolerance-forming analgesia with limited inhibition of gastrointestinal transit. As BN-9 is able to activate both opioid and NPFF systems, this provides an interesting approach for the development of novel analgesics with minimal side effects.

The cannabinoid system has been demonstrated to modulate the acute and chronic pain of multiple origins. Mouse VD-hemopressin (a) [(m)VD-Hpa], an 11-residue a-hemoglobin-derived peptide, was recently reported to function as a selective agonist of the cannabinoid receptor type 1 (CB 1 ) in vitro. To characterize its behavioral and physiological properties, we investigated the in vivo effects of (m)VD-Hpa in mice. In the mouse tail-flick test, (m) VD-Hpa dose-dependently induced antinociception after supraspinal (EC 50 5 6.69 nmol) and spinal (EC 50 5 2.88 nmol) administration. The antinociceptive effects of (m)VD-Hpa (intracerebroventricularly and intrathecally) were completely blocked by N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; CB 1 antagonist), but not by 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl (4-methoxyphenyl)-methanone (AM630; CB 2 antagonist) or naloxone (opioid antagonist), showing its selectivity to the CB 1 receptor. Furthermore, the central nervous system (CNS) effects of (m)VD-Hpa were evaluated in body temperature, locomotor activity, tolerance development, reward, and food intake assays. At the highly antinociceptive dose (3 Â EC 50 ), (m)VD-Hpa markedly exerted hypothermia and hypoactivity after supraspinal administration. Repeated intracerebroventricular injection of (m)VD-Hpa resulted in both development of tolerance to antinociception and conditioned place aversion. In addition, central injection of (m)VD-Hpa dosedependently stimulated food consumption. These findings demonstrate that this novel cannabinoid peptide agonist induces CB 1 -mediated central antinociception with some CNS effects, which further supports a CB 1 agonist character of (m)VD-Hpa. Moreover, the current study will be helpful to understand the in vivo properties of the endogenous peptide agonist of the cannabinoid CB 1 receptor.

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Neuropeptide FF (NPFF) belongs to an opioid-modulating peptide family. NPFF has been reported to play important roles in the control of pain and analgesia through interactions with the opioid system. However, very few studies examined the effect of supraspinal NPFF system on analgesia induced by opiates administered at the peripheral level. In the present study, intracerebroventricular (i.c.v.) injection of NPFF (1, 3 and 10 nmol) dose-dependently inhibited systemic morphine (0.12 mg, i.p.) analgesia in the mouse tail flick test. Similarly, i.c.v. administration of dNPA and NPVF, two agonists highly selective for NPFF(2) and NPFF(1) receptors, respectively, decreased analgesia induced by i.p. morphine in mice. Furthermore, these anti-opioid activities of NPFF and related peptides were blocked by pretreatment with the NPFF receptors selective antagonist RF9 (10 nmol, i.c.v.). These results demonstrate that activation of central NPFF(1) and NPFF(2) receptors has the similar anti-opioid actions on the antinociceptive effect of systemic morphine.