[Phe 1 C(CH 2 -NH)Gly 2 ]NC(1-13)NH 2 has been tested in the electrically stimulated guinea pig ileum and mouse vas deferens, two nociceptin sensitive preparations. The new compound showed per se little or no eect in the two tissues, but it displaced to the right the concentration-response curves of nociceptin in a concentration-dependent manner. Reinscheid et al., 1995) to be the endogenous ligand of the opioid-like orphan receptor (ORL-1). Despite the structural homology of NC and its receptor with the peptides and receptors of the opioid family this peptide/receptor system appears to be pharmacologically distinct from the opioids. During the last two years several papers have described new biological eects mediated by NC both in the periphery and in the central nervous system. Such eects were not modi®ed by naloxone or other more selective opioid receptor antagonists and were considered to be mediated by the activation of a speci®c NC receptor (the ORL-1). Lack of selective NC receptor antagonists prevented a de®nitive pharmacological characterization of ORL-1. In 1997, Kobayashi et al. showed that carbetapentane and rimcazole act as antagonists at the NC receptor. However these compounds showed little anity (IC 50 about 10 mM) and, more importantly, they were found to interact also with other functional sites such as s-, m-, and k-opioid receptors and M 1 -muscarinic receptors. Being non selective, they were therefore considered of little utility for receptor characterization.In the present study, we describe the chemical structure and the in vitro pharmacological eects of [Phe

1 Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a speci®c G-protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identi®cation and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe 1 ]nociceptin(1-13)NH 2 acts as the ®rst truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. ]nociceptin(1-13)NH 2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe 1 ]nociceptin(1-13)NH 2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine-induced analgesia. 4 Collectively our data indicate that [Nphe 1 ]nociceptin(1-13)NH 2 , acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

2 UFP-101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high a nity (pK i 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP-101 competitively antagonizes the e ects of N/OFQ on GTPg 35 S binding in CHO hNOP cell membranes (pA 2 9.1) and on cyclic AMP accumulation in CHO hNOP cells (pA 2 7.1), being per se inactive at concentrations up to 10 mM. 3 In isolated peripheral tissues of mice, rats and guinea-pigs, and in rat cerebral cortex synaptosomes preloaded with [ 3 H]-5-HT, UFP-101 competitively antagonized the e ects of N/OFQ with pA 2 values in the range of 7.3 ± 7.7. In the same preparations, the peptide was inactive alone and did not modify the e ects of classical opioid receptor agonists. 4 UFP-101 is also active in vivo where it prevented the depressant action on locomotor activity and the pronociceptive e ect induced by 1 nmol N/OFQ i.c.v. in the mouse. In the tail withdrawal assay, UFP-101 at 10 nmol produces per se a robust and long lasting antinociceptive e ect. 5 UFP-101 is a novel, potent and selective NOP receptor antagonist which appears to be a useful tool for future investigations of the N/OFQ-NOP receptor system.

Three series of nociceptin (NC)-related peptides were synthesized and their abilities (i) to bind to the NC sites expressed in mouse forebrain membranes, (ii) to inhibit the electrically evoked contraction of the mouse vas deferens, and (iii) to inhibit forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHONCR) were investigated. The compounds of the first series (a series) have an ordinary Xaa1-Gly2 bond, those of the second series (b series) have a Xaa1psi(CH2-NH)Gly2 pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa1-Gly2) structure. The affinity values measured in the binding assay and in the two functional assays with the compounds of the three series showed high levels of correlation. Thus, (I) the compounds of the a series in which Phe1 was substituted with Tyr, Cha, or Leu acted as potent NC receptor agonists; (II) the b series compounds behaved as NC receptor antagonists in the mouse vas deferens and as full agonists in CHO(NCR) cells with different potencies depending on the first amino acid residue, [Phe1psi(CH2-NH)Gly2]NC(1-17)NH2 and [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 being the most potent compounds; (III) the compounds of the third series were all inactive both as agonists and as antagonists with the exception of [Nphe1]NC(1-17)NH2 and [Nphe1]NC(1-13)NH2, which behaved as NC receptor antagonists both in the isolated tissue and in CHO(NCR) cells (pKB 6.1-6.4). In conclusion, this study demonstrates that chemical requirements for NC receptor agonists are different from those of antagonists. Moreover, modifications of the steric orientation of the aromatic residue Phe1 in the NC sequence as obtained with the pseudopeptide bond between Phe1 and Gly2 or with the displacement of the benzyl side chain by one atom, as in Nphe1, lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe1]NC(1-13)NH2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.