Neuropharmacology and Analgesia
Pharmacological characterization of the nociceptin/orphanin FQ receptor non peptide antagonist Compound 24

https://doi.org/10.1016/j.ejphar.2009.04.054Get rights and content

Abstract

Compound 24, 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide was recently identified as a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In this study, the in vitro and in vivo pharmacological profiles of Compound 24 were investigated. In vitro studies were performed measuring receptor and [35S]GTPγS binding and calcium mobilization in cells expressing the recombinant NOP receptor as well as using N/OFQ sensitive tissues. In vivo studies were conducted using the tail withdrawal assay in mice. Compound 24 produced a concentration-dependent displacement of [3H]N/OFQ binding to CHOhNOP cell membranes showing high affinity (pKi 9.62) and selectivity (1000 fold) over classical opioid receptors. Compound 24 antagonized with high potency the following in vitro effects of N/OFQ: stimulation of [35S]GTPγS binding in CHOhNOP cell membranes (pA2 9.98), calcium mobilization in CHOhNOP cells expressing the Gαqi5 chimeric protein (pKB 8.73), inhibition of electrically evoked twitches in the mouse (pA2 8.44) and rat (pKB 8.28) vas deferens, and in the guinea pig ileum (pKB 9.12). In electrically stimulated tissues, Compound 24 up to 1 µM did not modify the effects of classical opioid receptor agonists. Finally in vivo, in the mouse tail withdrawal assay, Compound 24 at 10 mg/kg antagonized the pronociceptive and antinociceptive effects of 1 nmol N/OFQ given supraspinally and spinally, respectively. Under the same experimental conditions Compound 24 did not affect the antinociceptive action of 3 nmol endomorphin-1 injected intrathecally. The present study demonstrated that Compound 24 is a pure, competitive, and highly potent non-peptide NOP receptor selective antagonist.

Introduction

Nociceptin/orphanin FQ (N/OFQ) (Meunier et al., 1995, Reinscheid et al., 1995) modulates several different biological functions via selective activation of the N/OFQ peptide (NOP) receptor (Lambert, 2008). There are numerous studies describing the central and peripheral actions of N/OFQ and selective NOP agonists. In contrast, relatively few studies are available regarding the effects of selective NOP receptor antagonists. This is mainly due to the fact that only few such molecules are described in the literature (Chiou et al., 2007, Lambert, 2008). Moreover not all the described NOP receptor antagonists are commercially available. Nevertheless, there is convincing evidence that blockade of NOP receptors may be beneficial in some conditions or pathological states. For instance, peptide ([Nphe1]N/OFQ(1-13)-NH2 (Calo et al., 2000) and UFP-101 (Calo et al., 2002b)) and non peptide (J-113397 (Ozaki et al., 2000) and SB-612111 (Zaratin et al., 2004)) NOP antagonists were demonstrated to evoke antidepressant like actions in rodents (Gavioli and Calo, 2006, Gavioli et al., 2003, Gavioli et al., 2004, Redrobe et al., 2002, Rizzi et al., 2007). Interestingly and corroborating antagonist studies NOP−/− mice displayed an antidepressant-like phenotype (Gavioli et al., 2003). Moreover, in a rather elegant series of studies, it has been demonstrated that the endogenous N/OFQ–NOP receptor signalling inhibits motor behaviour and that NOP receptor antagonists (UFP-101, J-113397, and Trap-101 (Trapella et al., 2006)) produce beneficial effects in rodent models of Parkinson's disease (Marti et al., 2004a, Marti et al., 2004b, Marti et al., 2005, Marti et al., 2007, Viaro et al., 2008). This indication has also been confirmed in non-human primates (Viaro et al., 2008, Visanji et al., 2008). Very recent findings indicated that plasma N/OFQ levels in sepsis were higher in patients who died within 30 days (Williams et al., 2008) and this parallels the preclinical observation that the NOP receptor antagonist UFP-101 reduces animal mortality in a rat model of sepsis (Carvalho et al., 2008). To be fully validated and firmly attributed to the NOP receptor antagonist class of drugs, these emerging indications should be confirmed in future studies using several chemically unrelated molecules.

A novel NOP receptor non-peptide antagonist, 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide, has been recently identified by Banyu investigators and named Compound 24 (Goto et al., 2006). The synthesis of this novel ligand is relatively easy and the overall yield relatively high (25% in our laboratory, 31% in Goto et al. (2006)). This is particularly true when compared to the synthesis of other non peptide NOP antagonists such as J-113397 and SB-612111 whose synthesis is very difficult and of low overall yield (≈ 1% for both molecules in our laboratories, C. Trapella personal communication).

Thus, the aim of the present study was the synthesis and detailed investigation of the pharmacological profile of Compound 24. The novel ligand was investigated in vitro in receptor binding and [35S]GTPγS experiments performed in CHOhNOP cell membranes, in calcium mobilization experiments performed in CHOhNOP cells expressing the Gαqi5 protein, and in N/OFQ sensitive isolated tissues. Finally, the in vivo actions of Compound 24 were assessed in mice using the tail withdrawal assay.

Section snippets

Cell culture and membrane preparation

CHOhNOP cells were cultured in Dulbecco Minimum Essential Medium (DMEM) and Ham F-12 (1:1) supplemented with 5% foetal calf serum, penicillin (100 IU/ml), Streptomycin (100 µg/ml) and Fungizone (2.5 µg/ml). Stock cultures were further supplemented with geneticin (G418, 200 µg/ml) and Hygromycin B (200 µg/ml) as described previously (McDonald et al., 2003). CHOhmu, CHOhdelta, CHOhkappa and CHOhNOP stably expressing the Gαqi5 protein were generated as previously described (Camarda et al., 2009)

Receptor binding

In receptor binding experiments performed on CHOhNOP cell membranes Compound 24 displaced [3H]N/OFQ in a concentration dependent manner showing subnanomolar affinity (pKi 9.62, Table 1). Under the same experimental conditions, Compound 24 did not bind the delta receptor and showed low affinities for mu and kappa sites (pKi 6.72 and 6.47, respectively). In contrast, the universal opioid receptor ligand naloxone did not bind the NOP receptor while showed the expected rack order of affinity at

Discussion

The present study extend previous findings (Goto et al., 2006) demonstrating that Compound 24 binds with high affinity the NOP receptor and behaves as a pure and potent NOP receptor antagonist showing high selectivity over classical opioid receptor. These pharmacological features of Compound 24 were consistently observed in various assays and preparations expressing the human recombinant as well as the animal native receptors. In addition, the NOP selective antagonist properties of Compound 24

Acknowledgements

We would like to thank Prof. E. Kostenis (Institute for Pharmaceutical Biology, Bonn, Germany) for supplying the plasmid encoding for Gαqi5 protein and Prof T. Costa and his collaborators Dr P Molinari and C Ambrosio (Istututo Superiore di Sanità, Rome, Italy) for generating CHO cells stably expressing the Gαqi5 chimeric protein. This work was supported by funds from the University of Ferrara (FAR grant to SS and GC) and the Italian Ministry of University (PRIN 2006 grant to DR and RG, FIRB

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