Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript> and c[Nphe<Superscript>1</Superscript>,Cys<Superscript>10,14</Superscript>]N/OFQ(1–14)NH<Subscript>2</Subscript>

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys^10,14]N/OFQ(1–14)NH₂ (c[Cys^10,14]) and its [Nphe¹] derivative c[Nphe¹,Cys^10,14]N/OFQ(1–14)NH₂ (c[Nphe¹,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)NH₂(reference agonist)>N/OFQ=c[Cys^10,14]>>c[Nphe¹Cys^10,14]. In GTP³⁵S studies of Chinese hamster ovary cells expressing human NOP (CHO_hNOP) c[Cys^10,14] (pEC₅₀ 8.29) and N/OFQ(1–13)NH₂ (pEC₅₀ 8.57) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. Following 30 min pre-incubation c[Nphe¹Cys^10,14] competitively antagonised the effects of N/OFQ(1–13)NH₂ with a pA₂ and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys^10,14] (pEC₅₀ 9.29, E_max 102% inhibition of the forskolin stimulated response), N/OFQ(1–13)NH₂ (pEC₅₀ 10.16, E_max 103% inhibition) and c[Nphe¹Cys^10,14] (~80% inhibition at 10 M) displayed agonist activity. In the mouse vas deferens c[Cys^10,14] (pEC₅₀ 6.82, E_max 89% inhibition of electrically evoked contractions) and N/OFQ(1–13)NH₂ (pEC₅₀ 7.47, E_max 93% inhibition) were full agonists whilst c[Nphe¹Cys^10,14] alone was inactive. c[Nphe¹Cys^10,14] (10 M) competitively antagonised the effects of N/OFQ(1–13)NH₂ 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-NH₂).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[Nphe¹Cys^10,14] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTP³⁵S assay and agonist in cAMP assay) dependent activity.Presented in part to The British Pharmacological Society at the Brighton, UK Meeting January 2003     

UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPγ<Superscript>35</Superscript>S binding studies

Studies of the pharmacology of nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) have been hampered by the lack of a range of high potency antagonists. In this study we have examined the effects of a novel N/OFQ analogue [Nphe(1),Arg(14),Lys(15)]N/OFQ NH(2) hereafter referred to as UFP-101. [(3)H]N/OFQ competition binding and GTPgamma(35)S binding assays were performed using CHO cells expressing the human NOP receptor (CHO(hNOP)). UFP-101 (pK(i) of 10.14+/-0.09) and a range of NOP selective agonists displaced [(3)H]N/OFQ binding with the following rank order of affinity: [Arg(14),Lys(15)]N/OFQ>[( pF)Phe(4)]N/OFQ(1-13)NH(2)>N/OFQ(1-13)NH(2)>UFP-101>N/OFQ>Ro64-6198>[Nphe(1)]N/OFQ(1-13)NH(2). N/OFQ, N/OFQ(1-13)NH(2), [( pF)Phe(4)]N/OFQ(1-13)NH(2), [Arg(14),Lys(15)]N/OFQ and Ro64-6198 also produced a concentration dependent (pEC(50) values of 8.75+/-0.11, 9.28+/-0.15, 9.69+/-0.04, 9.12+/-0.11 and 8.09+/-0.07 respectively) and saturable stimulation of GTPgamma(35)S binding and all were full agonists. UFP-101 did not stimulate GTPgamma(35)S binding per se, but produced a concentration dependent and parallel rightward shift in the concentration response curves to all agonists. UFP-101 yielded pA(2) values in the range 8.4-9.0. For comparison a pA(2) for [Nphe(1)]N/OFQ(1-13)NH(2) (the template for UFP-101) against N/OFQ of 7.33+/-0.08 was obtained. Slope factors for the Schild regression lines were approximately 1 indicating competitivity. When UFP-101 is compared with its template molecule [Nphe(1)]N/OFQ(1-13)NH(2), Arg(14),Lys(15) substitution produced approximately 1 log greater potency. We suggest that due to its high potency UFP-101 should prove a further useful tool in the evaluation of the N/OFQ-NOP receptor system.     

Interactions of orphanin FQ/nociceptin (OFQ/N) system with immune system factors and hypothalamic–pituitary–adrenal (HPA) axis

BackgroundBrain–immune system interactions and neurohormonal changes which are induced by psychophysiological factors are growing areas of scientific interest. Central (CNS) and autonomic nervous-endocrine-immune system pathways are connected with a number of behavioral and physiological factors which may be linked to disease susceptibility and progression.MethodsIn this paper, influence of orphanin FQ/nociceptin receptor (OFQ/N) on the hypothalamic–pituitary–adrenal (HPA) axis and their influence on the immunological system was reviewed.ConclusionsThe neuroendocrine system, in particular the hypothalamic–pituitary–adrenal (HPA) axis, is closely connected with the cytokines. HPA axis activation by cytokines, via the release of glucocorticoids has, in turn, been found to play a critical role in restraining and shaping immune responses. Investigation of the OFQ/N system and G-proteins suggests a role for this receptor as a down-regulator of cytokine, chemokine and chemokine receptor expression.