The Opioid Receptor Like-1 Receptor Agonist Ro 64-6198 (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) Produces a Discriminative Stimulus in Rats Distinct from That of a , , and Opioid Receptor Agonist Cue

Male Wistar rats were trained to discriminate either the opioid receptor like (ORL)-1 receptor agonist Ro 64-6198 (1S,3aS-8– 2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one) or morphine from saline using a twochoice, food reinforced, operant procedure. Acquisition of Ro 64-6198 discrimination was relatively slow (mean trials to criterion 113 6), and a final 4 mg/kg dose (initial training dose 2 mg/kg) was required to establish appropriate stimulus control. In comparison, a separate group of rats attained a morphine (2 mg/kg) discrimination in 44 4 trials. In tests of substitution, Ro 64-6198 produced a dose-related generalization to its own cue (ED50 of 1.1 mg/kg i.p.), yet only weakly generalized to the morphine cue (19% at 10 mg/kg i.p.). In contrast, morphine generalized completely to the morphine cue (ED50 of 0.7 mg/kg s.c.), yet only partially generalized to the Ro 64-6198 cue (40% at 6 mg/kg s.c.). The opioid receptor agonist U50,488 [trans-3,4-dichloro-N-methyl-N(2-[1pyrrolidinyl]cyclohexyl) benzeneacetamide methanesulfonate] (0.3–6 mg/kg s.c.) and the opioid receptor agonist SNC-80 [( )-4-[( R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3methoxybenzyl]-N,N-diethylbenzamide] (0.3–6 mg/kg i.p.) failed to evoke significant generalization to either cue. The opioid receptor agonists codeine (0.3–20 mg/kg) and buprenorphine (0.01–1 mg/kg) completely generalized to the morphine cue, but only buprenorphine partially generalized to the Ro 64-6198 cue. Naloxone pretreatment completely blocked the morphine cue (ED50 of 0.005 mg/kg s.c.), yet only weakly attenuated the Ro 64-6198 cue at 0.3 mg/kg. Finally, the selective ORL-1 antagonist J-113397 [1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one] completely blocked the Ro 64-6198 cue at a dose (30 mg/kg i.p.) that had no effect against the morphine cue. The present studies demonstrate that rats may be trained to discriminate Ro 64-6198 from saline, and the pharmacological characteristics of this cue are most consistent with ORL-1 receptor activation. Opioid receptors are currently classified into four subclasses, the opioid peptide receptor, opioid peptide receptor, opioid peptide receptor, and the nociceptin/orphaninFQ peptide receptor (also termed ORL-1) subclass (Calo et al., 2000; Snyder and Pasternak, 2003). The ORL-1 receptor represents the most recent addition to this family, identified by various laboratories during efforts to clone subtypes to the , , or receptor subclasses (for recent reviews, see Calo et al., 2000; Mogil and Pasternak, 2001). The ORL-1 receptor has a pharmacology that is clearly distinct from the other opioid subclasses. For example, naloxone, a relatively nonselective antagonist at , , and receptors, has low affinity for the ORL-1 subtype (Calo et al., 2000; Mogil and Pasternak, 2001). Furthermore, nonpeptide ligands apparently selective for the ORL-1 receptor have been identified, notably the agonist Ro 64-6198 (Jenck et al., 2000), and the antagonist J-113397 (Ozaki et al., 2000). Ro 64-6198 seems to function as a full agonist, equivalent to the likely endogenous agonist orphaninFQ/nociceptin (OFQ/N; Calo et al., 2000) in cell-based systems expressing the human ORL-1 receptor, and with at least 100-fold selectivity versus Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.071423. ABBREVIATIONS: ORL-1, opioid receptor like; Ro 64-6198, 1S,3aS-8–2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one; OFQ/N, orphaninFQ/nociceptin; U50,488, trans-3,4-dichloro-N-methyl-N(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide methanesulfonate; SNC-80, ( )-4-[( R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; J-113397, 1-[(3R,4R)1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one; CPP, conditioned place preference; MOR, morphine group; RO, Ro 64-6198 group. 0022-3565/04/3112-652–658$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 311, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 71423/1174027 JPET 311:652–658, 2004 Printed in U.S.A. 652 at A PE T Jornals on Sptem er 6, 2017 jpet.asjournals.org D ow nladed from other opioid receptors (Jenck et al., 2000; Hashiba et al., 2002). One of the most compelling demonstrations of the apparent in vivo selectivity of Ro 64-6198, at least in the mouse, is the demonstration that many neurological effects produced by this drug, e.g., hypolocomotion, ataxia, and hypothermia, are abolished in ORL-1 receptor-deficient mice (Higgins et al., 2001). Furthermore, various studies have now documented the in vivo effects of Ro 64-6198 in a variety of assays designed to investigate the anxiolytic, cognitive, and abuse liability of this drug class (Jenck et al., 2000; Higgins et al., 2002; Le Pen et al., 2002). Clinically, many opioid drugs are traditionally associated with their subjective effects, which can be investigated experimentally in various animal species through drug discrimination procedures (Stolerman et al., 1987; Dykstra et al., 1997). Among the opioid subclass, the discriminative stimulus effects of -, -, and -selective compounds have been intensively investigated in multiple species, including rats and primates. Morphine, U50,488, and SNC-80 have each been used as training drugs to establish and characterize , , and opioid receptor cues, respectively (Shannon and Holtzman, 1976; Picker et al., 1990; Broqua et al., 1998; Brandt et al., 1999; Stevenson et al., 2002). Such studies suggest that each receptor subtype is associated with a pharmacologically distinct cue, consistent with a clinical literature that describes a unique subjective state induced at least by and receptor-selective ligands (Smith and Beecher, 1962; Kumor et al., 1986; Dykstra et al., 1997; Walsh et al., 2001). As yet, there is little or no information relating to whether animals can be trained to an ORL-1 receptor discriminative stimulus, and if so, how it may compare with other opioid receptor cues. Accordingly in the present article, we trained of a group of Wistar rats to discriminate Ro 64-6198 from saline. Once discriminative control had been attained, we first established that the cue is clearly distinct from a morphine ( ) cue, and the pharmacology is most consistent with ORL-1 receptor activation. Second, because Ro 64-6198 has at least 100-fold selectivity over other opioid receptors, notably (Jenck et al., 2000), we trained a separate group of rats to a morphine cue to examine the effect of Ro 64-6198 in these animals. These studies were also of interest, given recent reports that OFQ/N pretreatment may block the acquisition of a morphine conditioned place preference (Murphy et al., 1999; Ciccocioppo et al., 2000), thus indicating a potential interaction between ORL-1 receptor agonism and a morphine-discriminative stimulus. Furthermore, because ORL-1 receptor agonists are considered to have therapeutic potential in indications such as anxiety (Jenck et al., 2000; Smith and Moran, 2001), a comparison between the Ro 64-6198 and morphine cue may provide insight into any potential abuse liability that selective ORL-1 agonists may have in humans (Mansbach et al., 2003). Materials and Methods Animals and Housing. Fourty-eight male Wistar rats (starting weight 200–250 g; Charles River Laboratories, Inc., Wilmington, MA) were randomly divided into two groups of 24. One group was assigned to receive either morphine or saline, the other Ro 64-6198 or saline (see below for details). The animals were grouped in pairs, in plastic bottomed cages containing sawdust bedding in a holding room controlled for temperature (22 1°C) and humidity (45%). The animals were placed on restricted diet (ad libitum food 1 h/day) presented as a single meal at the end of the day. All testing was conducted during the light phase of the animals light/dark cycle (lights on 7:00 AM–7:00 PM). Animal housing and subsequent experimentation was conducted in accordance with the National Institutes of Health Guide for the Health and Care of Laboratory Animals. A local Animal Users Committee also served to ensure effective implementation of standards. Drugs and Injections. Drug form and source were as follows: morphine sulfate, naloxone HCl, codeine sulfate, and buprenorphine HCl were all from Sigma-Aldrich (St. Louis, MO); Ro 64-6198 and J-113397 were synthesized in-house by Drs. Ginny Ho and Deen Tulshian (Department of Chemistry, Schering-Plough, Kenilworth, NJ); and U50,488 and SNC-80 were both from Tocris Cookson Inc. (Ellisville, MO). Morphine, codeine, naloxone, U50,488, and buprenorphine were each dissolved in saline and injected subcutaneously at 1 ml/kg. SNC-80 was prepared in saline with drops of 0.1 M HCl and injected intraperitoneally at 3 ml/kg. Ro 64-6198 was suspended in 0.3% Tween 80 and injected intraperitoneally at 5 ml/kg. J-113397 was dissolved in 1% Tween 80 solution and injected intraperitoneally at 2 ml/kg. All drugs were injected 30 min before testing with the exception of naloxone, which was injected 45 min before testing. All drug doses refer to base. Apparatus. Test apparatus consisted of 12 standard operant boxes (33 25 33 cm, length width height; MED Associates, St. Albans, VT), each equipped with a central food hopper dispensing 45-mg food pellets (Bio-Serve, Frenchtown, NJ). Two retractable levers were located 7.5 cm either side of the food tray. The equipment was under the control of Kestrel software (Conclusive Solutions,