Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys10,14]N/OFQ(1-14)NH2 and c[Nphe1,Cys10,14]N/OFQ(1-14)NH2.

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys(10,14)]N/OFQ(1-14)NH(2) (c[Cys(10,14)]) and its [Nphe(1)] derivative c[Nphe(1),Cys(10,14)]N/OFQ(1-14)NH(2) (c[Nphe(1),Cys(10,14)]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation. In competition binding studies of rat, mouse and human NOP the following rank order pK(i) was obtained: N/OFQ(1-13)NH(2)(reference agonist)>N/OFQ=c[Cys(10,14)]>>c[Nphe(1)Cys(10,14)]. In GTPgamma(35)S studies of Chinese hamster ovary cells expressing human NOP (CHO(hNOP)) c[Cys(10,14)] (pEC(50) 8.29) and N/OFQ(1-13)NH(2) (pEC(50) 8.57) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. Following 30 min pre-incubation c[Nphe(1)Cys(10,14)] competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pA(2) and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys(10,14)] (pEC(50) 9.29, E(max) 102% inhibition of the forskolin stimulated response), N/OFQ(1-13)NH(2) (pEC(50) 10.16, E(max) 103% inhibition) and c[Nphe(1)Cys(10,14)] (~80% inhibition at 10 microM) displayed agonist activity. In the mouse vas deferens c[Cys(10,14)] (pEC(50) 6.82, E(max) 89% inhibition of electrically evoked contractions) and N/OFQ(1-13)NH(2) (pEC(50) 7.47, E(max) 93% inhibition) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. c[Nphe(1)Cys(10,14)] (10 microM) competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pK(B) of 5.66. In a crude attempt to assess metabolic stability, c[Cys(10,14)] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH(2)). In summary, we report that c[Cys(10,14)] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe(1)Cys(10,14)] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTPgamma(35)S assay and agonist in cAMP assay) dependent activity.