Pharmacological characterisation of [(pX)Phe⁴]nociceptin(1-13)amide analogues

Abstract.

Phe⁴ 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 Phe⁴ 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 (CHO_hOP4) and in rat cerebral cortex membranes, [(pF)Phe⁴]NC(1-13)NH₂, [(pNO₂)Phe⁴]NC(1-13)NH₂, and [(pCN)Phe⁴]NC(1-13)NH₂ displayed higher affinity than NC(1-13)NH₂. The affinity of [(pCl)Phe⁴]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 GTPγS binding in CHO_hOP4 cells and rat cerebral cortex membranes and inhibition of cAMP accumulation in CHO_hOP4 cells), the para substituted analogues behaved as full agonists (with the exception of [(pOH)Phe⁴]NC(1-13)NH₂ which acted as a partial agonist in the GTPγS binding assays) with the following rank order potency:

${\rm pF = pNO}_{\rm 2} \ge {\rm pCN} \ge {\rm NC = NC(1 - 13)NH}_{\rm 2} {\rm = pCl} \ge {\rm pBr > pI = pCH}_{\rm 3} {\rm > pOH > pNH}_{\rm 2}$

[(pF)Phe⁴]NC(1-13)NH₂ and [(pNO₂)Phe⁴]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)Phe⁴]NC(1-13)NH₂ which displayed partial agonist/weak antagonist activities. The rank order of potency was similar to that found in the other assays. The effects of all analogues were not modified by naloxone. The selective OP₄ receptor antagonist [Nphe¹]NC(1-13)NH₂, tested in all preparations against one or both of the highly potent derivatives [(pF)Phe⁴]NC(1-13)NH₂ and [(pNO₂)Phe⁴]NC(1-13)NH₂, showed pA₂ values similar to those found against NC, the pA₂ in the GTPγS 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 Phe⁴ 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)Phe⁴]NC(1-13)NH₂ and [(pNO₂)Phe⁴]NC(1-13)NH₂.