The Anxiolytic-Like Effects of the Novel, Orally Active Nociceptin Opioid Receptor Agonist 8-[bis(2- Methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan- 3-ol

Orphanin FQ/nociceptin (OFQ/N) is the endogenously occurring peptide ligand for the nociceptin opioid receptor (NOP) that produces anxiolytic-like effects in mice and rats. The present study assessed the anxiolytic-like activity of 8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510), a novel potent piperidine NOP agonist (EC50 12 nM) that binds with high affinity (Ki 0.3 nM) and functional selectivity ( 50-fold over the -, -, and -opioid receptors). The anxiolytic-like activity and side-effect profile of SCH 221510 were assessed in a variety of models and the benzodiazepine, chlordiazepoxide (CDP), was included for comparison. The effects of chronic dosing of SCH 221510 were also assessed. Furthermore, the specificity of the anxiolytic-like effect of SCH 221510 was investigated with the NOP receptor antagonist 1-[(3R,4R)1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3dihydro-2H-benzimidazol-2-one (J-113397) and the opioid receptor antagonist naltrexone. Like CDP (1–30 mg/kg i.p.), SCH 221510 (1–30 mg/kg p.o.) produced anxiolytic-like effects in the elevated plus-maze (rat and gerbil), Vogel conflict (rat), conditioned lick suppression (rat), fear-potentiated startle (rat), and pup separation-induced vocalization (guinea pig) assays. In the Vogel conflict, the anxiolytic-like effect of SCH 221510 (10 mg/kg) was attenuated by J-113397 (3–10 mg/kg p.o.), but not naltrexone (3–30 mg/kg i.p.). Additionally, the anxiolytic-like effects of SCH 221510 did not change appreciably following 14-day b.i.d. dosing in rats (10 mg/kg). Furthermore, unlike CDP, SCH 221510 (3–30 mg/kg) produced anxiolytic-like activity at doses that did not disrupt overt behavior. Collectively, these data suggest that NOP agonists such as SCH 221510 may have an anxiolytic-like profile similar to benzodiazepines, with a reduced side-effect liability. The nociceptin opioid receptor (NOP) was identified using a human cDNA library on the basis of close homology with the -, -, and -opioid receptors (Bunzow et al., 1994; Mollereau et al., 1994). Subsequently, the endogenous ligand for the NOP receptor, orphanin FQ/nociceptin (OFQ/N), was identified from brain extracts and found to bind with high affinity to the NOP site, but not to -, -, or -opioid receptors (Meunier et al., 1995; Reinscheid et al., 1995). Immunohistochemical studies demonstrated that the mRNA for OFQ/N and its precursor prepronociceptin, as well as immunoreactivity for the NOP receptor, are localized to corticolimbic regions of the central nervous system (CNS). These regions include the amygdaloid complex, septohippocampal regions, periaqueductal gray, locus coeruleus, and dorsal raphe nucleus (Darland et al., 1998). In vitro electrophysiological studies using brain slices have shown that OFQ/N has potent Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.136937. ABBREVIATIONS: NOP, nociceptin opioid receptor; OFQ/N, orphanin FQ/nociceptin; CNS, central nervous system; Ro64-6198, (1S,3aS)-8(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one; EPM, elevated plus-maze; FPS, fear-potentiated startle; CLS, conditioned lick suppression; SCH 221510, 8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol; LMA, locomotor activity; CDP, chlordiazepoxide; CHO, Chinese hamster ovary; GTP S, guanosine 5 -O-(3-thio)triphosphate; VEH, vehicle; J-113397, 1-[(3R,4R)-1cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one. 0022-3565/08/3262-672–682$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 326, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 136937/3367007 JPET 326:672–682, 2008 Printed in U.S.A. 672 at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from inhibitory actions on neurons within the dorsal raphe nucleus, locus coeruleus, periaqueductal gray, and amygdala (Connor et al., 1996; Vaughan and Christie, 1996; Vaughan et al., 1997). The distribution pattern of OFQ/N and the NOP receptor within limbic brain regions, together with the demonstrated inhibitory actions of OFQ/N on neurons within these areas, suggests that the OFQ/N-NOP system may have a modulatory role in the control of several behaviors, including attention, arousal, fear, and anxiety. Indeed, mice with a targeted deletion of the OFQ/N precursor showed an increased susceptibility to a variety of stressors (Köster et al., 1999), and intracerebroventricular injection of OFQ/N in both mice and rats produced behavioral changes consistent with an anxiolytic-like profile (Jenck et al., 1997; Griebel et al., 1999). Furthermore, Jenck et al. (2000) demonstrated that Ro646198, a potent, nonpeptide agonist of the NOP receptor, produced a broad spectrum of anxiolytic-like activity in rats. Ro64-6198 increased exploration in the open arms of a rat elevated plus-maze (EPM), decreased the enhanced startle response in a fear-potentiated startle (FPS) paradigm, and increased the number of punished food deliveries in a GellerSeifter procedure. These effects were similar to those produced by the benzodiazepine alprazolam. Unlike alprazolam, however, the anxiolytic-like effects of Ro64-6198 occurred at doses that did not affect locomotor activity or muscle tone. More recent studies with Ro64-6198 have extended the anxiolytic-like effects to other models, including rat conditioned lick suppression (CLS), separation-induced vocalization assays in both rat and guinea pig pups, mouse marble burying, and the mouse Geller-Seifter assay (Varty et al., 2005; Nicolas et al., 2006). Again, these studies suggested that, although there were mechanism-based side effects with NOP receptor agonists, such as sedation, motor impairments, and hypothermia (Higgins et al., 2001; Varty et al., 2005), there seemed to be a significant therapeutic window between efficacious and adverse effects. Therefore, collectively, these data suggest that nonpeptide NOP receptor agonists have the potential to serve as a novel class of antianxiety agents with a reduced propensity to produce unwanted side effects. The present study has expanded this work further by testing an orally active compound unrelated to nociceptin or Ro64-6198. Specifically, we investigated the anxiolytic-like activity of a novel piperidine NOP receptor agonist, SCH 221510 (Fig. 1). The anxiolytic-like effects of SCH 221510 were assessed primarily in rats using EPM, Vogel conflict, CLS, and FPS assays; however, SCH 221510 was also tested in a gerbil EPM and a separation-induced vocalization assay in guinea pig pups to assess cross-species effects. Furthermore, to confirm the mechanism of action by which SCH 221510 produced its anxiolytic-like actions, antagonism studies were conducted using both the NOP receptor antagonist J-113397 (Ozaki et al., 2000) and the opioid antagonist naltrexone. Furthermore, to test for any tolerance to the anxiolytic-like effects, SCH 221510 was dosed for 14 days and tested in the rat Vogel conflict assay. Finally, SCH 221510 was assessed in spontaneous locomotor activity (LMA) and rotarod assays to assess any effects of motor function and coordination. It is noteworthy that the effects of SCH 221510 were compared with those of the benzodiazepine anxiolytic chlordiazepoxide (CDP) to provide a better understanding of the potential advantage an NOP receptor agonist may offer over benzodiazepine anxiolytics. Materials and Methods Opioid Receptor Binding Assays. NOP receptor binding assays were performed as described by Fawzi et al. (1997) and Corboz et al. (2000). In brief, CHO cell membranes expressing human NOP receptors were incubated with I-[Tyr14]-N/OFQ (PerkinElmer Life and Analytical Sciences, Waltham, MA) and increasing concentrations of compound in binding assay buffer containing 50 mM HEPES, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM NaCl, 0.025% bacitracin, and 0.1% bovine serum albumin. Assays were performed at room temperature for 60 min and were terminated by rapid filtration over GF/B membranes (PerkinElmer Life and Analytical Sciences). Radioactivity retained on filters was determined in a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences). All assays were performed in duplicates. Total and nonspecific binding was determined in quadruplicates. Opioid receptor binding assays ( -, -, and -opioid receptors) were performed on CHO cell membranes expressing the human opioid receptors (Receptor Biology, Beltsville, MD) as described by Corboz et al. (2000). In brief, CHO cell membranes were incubated with [H]diprenorphine (PerkinElmer Life and Analytical Sciences) and increasing concentrations of compounds in binding assay buffer for 60 min at room temperature. Assays were terminated by rapid filtration over GF/B membranes, and radioactivity retained on filters was counted in a TopCount microplate scintillation counter. Ki values (n 4–6/receptor) were determined using GraphPad Prism curve-fitting and data analysis software (GraphPad Software Inc., San Diego, CA). Opioid Receptor Functional Assays. [S]GTP S binding to recombinant CHO cell membranes was performed according to the method of Fawzi et al. (1997), with some minor modifications. CHO cell membranes expressing human NOP receptors (4 g) or human -, -, and -opioid receptors (5 g) (Receptor Biology, Beltsville, MD) were incubated for 30 min at room temperature with 100–300 pM [S]GTP S (PerkinElmer Life and Analytical Sciences) in an assay mixture (200 l) containing 50 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mg/ml bovine serum albumin, 120 mM NaCl, 0.2 mM EGTA, and 1 M GDP. Assays were terminated by rapid filtration over GF/B unifilter plates (PerkinElmer Life and Analytical Sciences) (presoaked for 30 min in 10 mM K2HPO4) and washed seven times with 2 ml of cold (4–10°C) buffer containing 20 mM Tris-HCl, pH 8.0, 20 mM MgCl2, and 100 mM NaCl. Filter-bound radioactivity was quantified on a TopCount (PerkinElmer Life and Analytical Sciences). Nonspecific binding was determined by performing the assay in the presence of 10 M GTP S. For the determination of the agoniststimulated increase in the binding of [S]GTP S, membranes were preincubated with SCH 221510 for 60 min before the initiation of the assay. EC50 values were determined using GraphPad Prism software. Subjects. Male CD and Wistar (only for FPS studies) rats (200– 500 g) and female Mongolian gerbils (30–50 g) were obtained from Charles River Laboratories (Kingston, NY). Male and female Dunkin Hartley guinea pig pups (100–250 g) were used for the vocalization studies (Charles River Laboratories). Pups were 2 to 3 days of age upon arrival and were used up to 18 days of age. Pups were housed three per cage with the dam, and rats and gerbils were housed three N CH3