Pharmacological characterisation of [(pX)Phe4]nociceptin(1-13)amide analogues -: 1.: In vitro studies

Phe(4) in the nociceptin (NC) sequence has been identified as the most critical residue for receptor interaction. In the present study. we investigated the pharmacological activity of a series of NC(1-13)NH2 analogues, in which the hydrogen atom in the para position of Phe(4) was substituted with F. NO2. CN, Cl, Br, I, CH3, OH or NH2. In receptor binding studies, performed using CHO cells expressing the recombinant human NC receptor (CHOhOP4) and in rat cerebral cortex membranes, [(pF)Phe4]NC (1-13)NH2, [(pNO(2))Phe(4)]NC(1-13)NH2, and [(pCN)Phe(4)] NC(1-13)NH2 displayed higher affinity than NC(1-13)NH2. The affinity of [(pCl)Phe(4)]NC(1-13)NH2 was essentially identical to that of NC(1-13)NH2, while the remaining compounds displayed reduced affinity. In a series of functional assays (stimulation of GTPgammaS binding in CHOhOP4 cells and rat cerebral cortex membranes and inhibition of cAMP accumulation in CHOhOP4 cells). the para substituted analogues behaved as full agonists (with the exception of [(pOH)Phe(4)]NC(1-13)NH2 which acted as a partial agonist in the GTPgammaS binding assays) with the following rank order potency: pF = pNO(2) greater than or equal to pCN greater than or equal to NC = NC(1-13)NH2 = pCl greater than or equal to pBr > pI = pCH(3) > pOH > pNH(2) [(pF)Phe(4)]NC(1-13)NH2 and [(pNO(2))Phe(4)]NC(1-13)NH2 were either inactive or displayed micromolar potencies in cAMP accumulation experiments performed on cells expressing classical opioid receptors. All compounds were full agonists in isolated tissues from various species (guinea pig ileum, mouse colon and mouse/rat vas deferens) with the exception of [(pOH)Phe(4)]NC(1-13)NH2 which displayed partial agonist/weak antagonist activities. The rank order of potency was simdar to that found in the other assays. The effects of all analogues were not modified by naloxone. The selective OP4 receptor antagonist [Nphe(1)]NC(1-13)NH2, tested in all preparations against one or both of the highly potent derivatives [(pF)Phe(4)] NC(1-13)NH2 and [(pNO(2))Phe(4)]NC(1-13)NH2, showed pA(2) values similar to those found against NC, the pA(2) in the GTPgammaS binding/rat cerebral cortex assay being much higher (ca. 7.5) than in the other functional assays (ca. 6). This study further supports the notion that Phe(4) of NC is the critical residue for receptor occupation and activation. Moreover, as part of this study, we have identified two novel, highly potent and selective agonists for the OP4 receptor, [(pF)Phe(4)]NC(1-13)NH2 and [(pNO(2))Phe(4)]NC (1-13)NH2.