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NEUROPHARMACOLOGY

Modification of Nociception and Morphine Tolerance by the Selective Opiate Receptor-Like Orphan Receptor Antagonist (–)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111)

GlaxoSmithKline Pharmaceuticals, Departments of Neurobiology Research (P.F.Z., G.P., M.S., M.G., C.F., P.P., M.A.S.) and Medicinal Chemistry (S.R., G.A.M.G.), Milan, Italy; and Neurology Centre of Excellence for Drug Discovery, New Frontiers Science Park, Harlow, United Kingdom

Received June 17, 2003; accepted October 16, 2003.

	Abstract

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(–)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) is a novel human opiate receptor-like orphan receptor (ORL-1) antagonist that has high affinity for the clonal human ORL-1 receptor (hORL-1 K_i = 0.33 nM), selectivity versus µ-(174-fold), -(6391-fold), and (486-fold)-opioid receptors and is able to inhibit nociceptin signaling via hORL-1 in a whole cell gene reporter assay. SB-612111 has no measurable antinociceptive effects in vivo in the mouse hot-plate test after intravenous administration but is able to antagonize the antimorphine action of nociceptin [ED₅₀ = 0.69 mg/kg, 95% confidence limit (CL) = 0.34–1.21]. SB-62111 administration can also reverse tolerance to morphine in this model, established via repeated morphine administration. In addition, intravenous SB-612111 can antagonize nociceptin-induced thermal hyperalgesia in a dose-dependent manner (ED₅₀ = 0.62 mg/kg i.v., 95% CL = 0.22–1.89) and is effective per se at reversing thermal hyperalgesia in the rat carrageenan inflammatory pain model. These data show that an ORL-1 receptor antagonist may be a useful adjunct to chronic pain therapy with opioids and can be used to treat conditions in which thermal hyperalgesia is a significant component of the pain response.

Although nociceptin initiates signaling via the ORL-1 receptor in a manner that is similar to activation of the classical µ-, -, and -opioid receptors, its pharmacological effects can differ significantly. Nociceptin can have an antiopioid action and when administered into the brain of mice potently antagonizes analgesia induced by systemic and intracerebroventricular (i.c.v.) morphine (Mogil et al., 1996). At the spinal level, intrathecal injection of low concentrations of nociceptin in mice causes allodynia and hyperalgesia (Hara et al., 1997), whereas at higher concentrations this peptide can cause analgesia (King et al., 1997; Tian et al., 1997). These effects are mediated at both a presynaptic site of action in afferent nerve terminals, and at a postsynaptic site of action on spinal interneurons (Neal et al., 1999). Ko et al. (2002) have further demonstrated a peripheral nervous system role of ORL-1 receptors in regulating thermal antinociception in primates.

These different activities of nociceptin suggest the existence of multiple activation pathways, making it difficult to predict the overall effects of systemically available ligands that can activate or antagonize the ORL-1 receptor. ORL-1 receptor knockout mice display the same nociceptive threshold as control mice in acute pain models (Nishi et al., 1997), suggesting that the ORL-1 receptor system is activated primarily in evoked pain states. In normal mice, a reduction in morphine-induced analgesia occurs in response to chronic nociceptin treatment (Mitchell et al., 2000), whereas ORL-1 receptor knockout mice maintain a higher level of sensitivity to morphine analgesia (Ueda et al., 2000). These data suggest that activation of the ORL-1 receptor system is involved in development of the reduced response to morphine that accompanies chronic opiate administration.

Several small molecule antagonists of the ORL-1 receptor have been described and used in studies aimed at clarifying possible physiological roles of the ORL-1 receptor. JTC-801 is a 4-aminoquinoline derivative described by Yamada et al. (2002) as having affinity (K_i = 45 nM) for the ORL-1 receptor and ability to antagonize the nociceptin-mediated inhibition of cAMP production (IC₅₀ = 2.58 µM). After systemic administration, JTC-801 blocks the allodynic effect of nociceptin and is active per se in the mouse hot-plate and rat formalin tests, both models of an acute pain response. Muratani et al. (2002) further showed that JTC-801 can block pronociceptive effects caused by low doses of nociceptin in the formalin test but is unable to alter its antinociceptive effects at high doses. The benzimidizole analog J-113397 has been reported by Ozaki et al. (2000) as having high affinity and selectivity for the ORL-1 receptor (K_i = 1.8 nM), antagonist activity in a test of nociceptin-stimulated [³⁵S]guanosine 5'-O-(3-thio)triphosphate binding (IC₅₀ = 5.3 nM), and ability to inhibit the activity of nociceptin in the mouse tail-flick test (Ozaki et al., 2000).

In this study, we present results describing (–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) as a high-affinity and broadly selective ORL-1 receptor antagonist in vitro and provide evidence as to its antinociceptive activity in vivo. We show that SB-612111 can reverse pain signaling in models of both evoked and subchronic pain response but is unable to reverse an acute pain response. Importantly, we further demonstrate that SB-612111 can resensitize mice to morphine in animals that had been chronically treated with opiate, suggesting utility of this class of ORL-1 receptor antagonist in prolonging the analgesic action of morphine.

	Materials and Methods

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Materials
The following drugs and chemicals were obtained by the source indicated: media and sera (Invitrogen, Carlsbad, CA); [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin ([³H]DAMGO, 60 Ci/mmol) and [³H][D-Ala²,D-Leu⁵]-enkephalin (55 Ci/mmol) (New England Nuclear, Bruxelles, Belgium); [³H]U-69593 (60 Ci/mmol) and [³H]nociceptin (156 Ci/mmol) (Amersham, Italia Srl, Milan, Italy); naloxone hydrochloride and morphine hydrochloride (S.A.L.A.R.S., Como, Italy); [D-Pen²,D-Pen⁵]-enkephalin (DPDPE) and DAMGO (Sigma, Milan, Italy); BRL 52656 and U-69593 were purchased from Sigma/RBI (Natick, MA); and nociception was from Saxon Biochemical GmbH (Hannover, Germany). All other compounds used have been synthesized in-house.

In Vitro Pharmacological Assays
Cell Culture. Clonal cell lines stably expressing the human ORL-1, µ-opioid (hMOR), -opioid (hDOR), and -opioid receptor were established to allow the assessment of ligand binding affinities in radioreceptor assays specific for each receptor. Cell cultures were routinely maintained at 37°C in a humidified atmosphere containing 5% CO₂. Chinese hamster ovary (CHO) cells [317, obtained from ETCC (European Collection of Cell Cultures, Salisbury, UK)] were grown in suspension in 1017S03 culture medium (GlaxoSmithKline, Harlow, UK) containing 10% (v/v) fetal bovine serum (FBS) and 0.05% (v/v) pluronic acid (F68). Human embryonic kidney (HEK) 293 cells (85120602, obtained from ETCC) were grown in monolayer in Eagle's minimal essential medium supplemented with 10% (v/v) FBS and 2 mM L-glutamine. hORL-1, hDOR, and hMOR receptor cDNAs were expressed in CHO cells and hKOR receptor cDNA in HEK293 cells, after integration into a pCDN expression vector (GlaxoSmithKline). Subclones stably expressing receptor were selected by growth in the absence of nucleosides (CHO) or by resistance to G-418 (HEK293). Subclones expressing high levels of radioligand binding were selected for further characterization.

Membrane Preparation. Membranes were prepared for use in radioligand binding assays by lysis in hypotonic phosphate buffer using a modification of the method described by Scheideler and Zukin (1990). Cells were harvested in phosphate-buffered saline (approximately 30 x 10⁶ cells/30-ml tube), collected by centrifugation (1200 rpm, ca. 800g, 5 min), resuspended in 10 mM potassium phosphate buffer, pH 7.2 (buffer A), and centrifuged at 40,000g, 10 min. The pellets obtained were resuspended in the same volume of buffer A, incubated on ice for 20 min, and centrifuged at 1200 rpm, 5 min, saving the supernatants. The low-speed pellets were resuspended in buffer A again and the last step was repeated two more times, saving the supernatants each time. The low-speed supernatants were pooled and centrifuged at high speed at 4°C. The pellets obtained were resuspended in buffer A containing 0.32 M sucrose and 5 mM EDTA. The membranes were stored in this buffer until use at –80°C at a concentration of 2 to 5 mg protein/ml (ca. 10 x 10⁶ cells/ml).

Measurement of Specific Radioligand Binding. The radioligands [³H]DAMGO, [³H][D-Ala²,D-Leu⁵]-enkephalin, [³H]U-69593, and [³H]nociceptin were used to label µ-, -, -, and ORL-1 receptors, respectively (Gillan and Kosterlitz, 1982; Petrillo et al., 1989; Wang et al., 1994; Dooley et al., 1997). µ- and -Opioid receptor binding studies were performed in 25 mM potassium phosphate buffer, pH 7.4, in 96-well polypropylene square plates in a final volume of 0.7 ml. -Opioid and ORL-1 receptor binding studies were performed in 25 mM potassium phosphate buffer, pH 7.4, containing 3 mM MgCl₂, in a final volume of 0.5 ml. The protease inhibitors bacitracin (100 µg/ml), leupeptin (4 µg/ml), and chymostatin (2 µg/ml) were added to the incubation buffer of the ORL-1 receptor assay to prevent radioligand degradation. Nonspecific binding was determined in the presence of 10 µM naloxone (µ-, -, and -opioid receptor assays) or 10 µM nociceptin (ORL-1 receptor assay). Incubation was carried out for 60 min at 25°C. The reaction was terminated by filtration using a Packard Filtermate harvester with GF/B Unifilter plates pretreated with buffer (µ- and -opioid receptor assays) or 0.3% polyethylenimine (-opioid and ORL-1 receptor assays). After filtration, Unifilter plates were dried, each well filled with 50 µl of Packard Microscint 30, and radioactivity was counted by a Packard TopCount NXT. Competition experiments were performed using a membrane concentration of 5 to 20 µg protein/ml and radioligand concentrations close to the experimental K_d of each radioligand, as obtained from saturation binding studies. IC₅₀ values were determined as described by Leatherbarrow (1990) using the nonlinear least-squares fitting program GraFit (Erithacus Software Limited, Horley, UK) and results transformed into K_i values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

In Vitro Assessment of Receptor and Enzyme Interactions. SB-612111 was tested at a concentration of 10 µM by CEREP (Le Bois L'Eveque, France) in a broad profile of radioligand binding assays specific for different classes of receptors, ion channels, and enzymes. SB-612111 was initially dissolved in dimethyl sulfoxide to yield a concentration of 10 mM. Further dilutions of the compound for testing were in water. Assays were performed in duplicate and results expressed as the percentage of inhibition of specific radioligand binding or specific enzyme activity. Affinity values (K_i) were further established for SB-612111 in those receptor assays for which a significant interaction was detected.

Measurement of Receptor-Mediated Cellular Signaling. Receptor signaling studies were performed using a cAMP responsive CRE-luciferase-gene reporter assay, as described previously by Garnier et al. (2003). Receptor expressing cell lines were generated by stably expressing plasmid containing cDNA for the human ORL-1 receptor (pcDNA 3.1/Hygro/ORL1) or µ-opioid receptor (pcDNA 3.1/Hygro/MOR) in a HEK293-Luc cell line derived from HEK293 cells and stably expressing a cAMP-responsive luciferase gene reporter construct. Recombinant clones expressing the hORL-1 receptor (HL-ORL-1) or human µ-opioid receptor (HL-MOR) were obtained by selection using zeocin (400 µg/ml) and hygromycin (300 µg/ml), respectively. HL-ORL-1 receptor and HL-MOR clones that expressed high levels of mRNA for each receptor were chosen for use in the signaling assays. These cell lines were grown in Eagle's minimal essential medium culture medium containing 2 mM L-glutamine, 1% (w/v) non-essential amino acids, 0.4 mg/ml active geneticin, and 0.4 mg/ml zeocin (HL-ORL) or 0.3 mg/ml hygromycin (HL-MOR), supplemented with 10% (v/v) FBS (Invitrogen). Cells were incubated at 37°C (5% CO₂) on Falcon plastic culture dishes precoated with poly-D-lysine (Sigma).

The luciferase-gene reporter assay was performed in 96-well format. Briefly, cells were plated in white Packard CulturPlates (Millipore S.p.A., Milan, Italy) at a density of 10⁴ cells/well in phenol red-free medium. Twenty-four hours after seeding, cells were preincubated for 30 min in the presence of 0.5 mM phosphodiesterase inhibitor 3-isobutyl-1-methylxantine, as a means of inhibiting cAMP breakdown by endogenous phosphodiesterase activity. Cells were further incubated for 4 h in the presence of forskolin and test drugs at various concentrations, in a final volume of 100 µl. All compounds were freshly dissolved as 1 mM solutions in ethanol/H₂O (v/v), further diluted in culture medium, and added to cell cultures as 10-fold concentrated solutions. Reactions were stopped by the addition of 100 µl of reconstituted Luclite reagent. Plates were dark-adapted for 10 min and luciferase expression (luminescent count per second) measured using a 12–channel Packard TopCount scintillation counter. The amount of luciferase expressed in each well, which is proportional to the luminescent count per second measured, was expressed as the percentage of response measured in forskolin-treated control cells. Dose-response curves were fitted by nonlinear fit using GRAFIT version 4.09 to determine EC₅₀ values.

In Vivo Pharmacological Assays
Animals. Male CD-1 mice weighing 25 to 35g and male CD rats weighing 220 to 280 g were obtained from Charles River Italica (Calco, Italy). Mice and rats were housed in groups of 10 and two, respectively, in Plexiglas cages with food and water available ad libitum. Animals were maintained at 22 ± 0.5°C with an alternating 12-h light/dark cycle and were used only once in all experiments.

Hot-Plate Test. Antinociception was assessed utilizing the hot-plate apparatus (Ugo Basile, Comerio, Italy), maintained at a constant temperature of 55 ± 0.1°C, as described by Eddy and Leimbach (1953). Briefly, each mouse was placed on the hot-plate and a reaction time measured starting from the placement of the mouse on the hot-plate and continuing until the initiation of licking or rapid movement of the hindpaw. Control latencies were approximately 4 to 9 s. A test cut-off time of 25 s was chosen to avoid possible tissue damage resulting from the test.

Thermal Hyperalgesia. Thermal hyperalgesia was assessed using the rat plantar test (Ugo Basile), as described by Hargreaves et al. (1988). Briefly, hyperalgesia was induced by an intraplantar injection of 0.1 ml of a 2% (w/v) suspension of lambda carrageenan into the right hindpaw of the animal. Thermal hyperalgesia was evaluated 3 h after the carrageenan injection.

Induction of Morphine Tolerance. Mice were treated once a day for 4 days with a high dose of morphine hydrochloride (50 mg/kg s.c.), as described by Zarrindast et al. (1996). On day 5, 24 h after the last administration of morphine, mice were challenged with a lower dose of morphine (9 mg/kg s.c.), and the degree of developed tolerance was measured by the hot-plate test.

Drug Administration. Nociceptin, lambda carrageenan, and morphine hydrochloride were dissolved in 0.9% (w/v) NaCl. SB-612111A was dissolved in 10% (w/v) encapsin containing 5% (w/v) glucose. Intracerebroventricular administration was performed as described by Porreca et al. (1984). Mice were lightly anesthetized with ether and an incision made in the scalp. The injection was made at a point 2 mm caudal and 2 mm lateral from bregma, using a 10-µl Hamilton syringe. Compounds were injected at a depth of 3 mm in a volume of 5 µl.

Statistical Analysis. Results are presented as mean value ± S.E.M. The statistical significance between groups was established by analysis of variance followed by Duncan's multicomparison test. P > 0.05 was considered as indicative of significance.

	Results

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Selective Binding Site Interaction of SB-612111 with hORL-1. The affinity and selectivity in vitro of SB-612111 (structure shown in Fig. 1), and of the corresponding enantiomer SB-612112, with human opioid receptors was evaluated in radioreceptor binding assays using membranes prepared from clonal cell lines expressing the human ORL-1 receptor, hMOR, hDOR, and hKOR opioid receptors. SB-612111 and SB-612112 both competitively displaced binding of the ORL-1 receptor radioligand [³H]nociceptin to hORL-1, yielding K_i values in these assays of 0.33 ± 0.03 and 1.3 ± 0.23 nM, respectively (Table 1). Competition studies in assays that measure specific [³H]DAMGO, [³H]DPDPE, and [³H]U-69593 binding to hMOR, hDOR, and hKOR receptors showed that SB-612111 is highly selective versus µ-(174-fold), -(6391-fold), and (486-fold)-opioid receptors. By comparison, SB-612112 had lower affinity for the hORL-1 receptor and was substantially less selective toward the µ-opioid receptor (27-fold). The - and -opioid receptor selectivities were, however, similar for both ligands. The data for SB-612111 compare favorably with results obtained in these radioreceptor assays for JTC-801, and the (+)-enantiomer of J-113397, ligands previously described as ORL-1 receptor antagonists. In this study, the observed µ-opioid/ORL-1 receptor selectivities of JTC-801 and (+)-J-113397 were only 3- and 15-fold, respectively, and the -opioid/ORL-1 selectivity of (+)-J-113397 was 29-fold. Furthermore, significant separation in the ORL-1 receptor affinity values was observed for the J-113397 enantiomers (79-fold). As expected, the µ-opioid (DAMGO, morphine), -opioid (DPDPE), and -opioid (BRL 52626, U-69593) ligands and the opioid antagonist naloxone did not compete for [³H]nociceptin binding at concentrations of up to 1 µM, but effectively displaced radioligand binding to the receptors for which they are known to have high affinity, demonstrating the selectively of the assays used.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Chemical structure of SB-612111.

To assess the specificity by which SB-612111 regulates ORL-1 receptor activity, a broad profiling of its interaction in vitro with receptors from outside of the opioid receptor family, as well as ion channels and enzymes, was undertaken. SB-612111 weakly inhibited activity in several assays (predicted K_i values of <500 nM). These interactions were then investigated in more detail. Affinity (K_i) values were established for competitive displacement of [³H]prazosine binding to the _1A-adrenergic receptor (K_i = 196 nM), [³H]RX 821002 binding to the _2A-adrenergic (K_i = 187 nM) and _2C-adrenergic (K_i = 395 nM) receptors, ¹²⁵I-cyanopindolol binding [in the presence of 1 µM (–)-propranolol] to the ₃-adrenergic receptor (K_i = 86 nM) and ¹²⁵I-aminopotentidine binding to the H₂-histimine receptor (K_i = 444 nM).

Measurement of hORL-1 Receptor Antagonism by SB-612111 in Vitro. The functional ORL-1 receptor antagonist activity of SB-612111 was evaluated by measuring its ability to antagonize inhibition by nociceptin of forskolin-induced luciferase expression in HEK293 cells stably expressing both hORL-1 and a CRE-luciferase gene reporter construct. Nociceptin inhibited forskolin-induced luciferase expression in a concentration-dependent manner (EC₅₀ = 0.009 ± 0.001 nM). SB-612111 produced a dose-dependent rightward shift of this nociceptin response (Fig. 2a). Moreover, SB-612111 was able to effectively reverse the inhibitory effect of 0.1 nM nociceptin on luciferase expression in a dose-dependent manner (K_b = 5 nM), as shown in Fig. 2b. Importantly, SB-612111 did not show any agonist or antagonist activity in HEK293 cells lines stably expressing both hMOR and a CRE-luciferase gene reporter construct, when tested at concentrations of up to 1 µM (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 2. In vitro antagonist activity of SB-612111. Activity in the CRE-luciferase gene reporter assay. a, cells were treated with increasing concentrations of nociceptin in the absence () or in the presence of 10 nM (), 30 nM () or 100 nM () of SB-612111. b, cells were treated with increasing concentrations of nociceptin () or SB-612111 (), or, varying concentrations of SB-612111 in the presence of 0.01 nM nociceptin (). Results are expressed as percentage of forskolin-induced luciferase expression and represent the mean ± S.E.M. from four independent experiments.

In Vivo Antagonist Activity of SB-612111. The ability of SB-612111 to antagonize nociceptin-induced antinociception was evaluated in the mouse hot-plate test. Intracerebroventricular nociceptin administration (0.6–10 nmol/mouse) led to a significant reduction in paw withdrawal latencies at the higher doses, as shown in Fig. 3a. Paw withdrawal latencies of nociceptin-treated animals were 2.15 ± 0.35 s versus saline values of 7.06 ± 0.62 s. Intravenous (i.v.) administration of SB-612111 (0.1–3 mg/kg) antagonized the effect elicited by i.c.v. administration of nociceptin (5 nmol) in this test in a dose-dependent manner [ED₅₀ = 0.62 mg/kg, 95% confidence limit (CL)_. = 0.22–1.89], as shown in Fig. 3b. However, the administration of 1.3 mg/kg i.v. SB-612111 alone did not cause any significant effect on paw withdrawal latencies (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Activity in the mouse hot-plate test. a, effect of nociception. Groups of mice were administered with saline alone or with varying doses of nociceptin. Paw withdrawal latencies (seconds) were measured 10 min after administration. Each value shown represents the mean ± S.E.M. for nine mice. *, P < 0.05 compared with the saline-injected control. Paw withdrawal latencies for mice treated with 5 µl/mouse i.c.v. saline were 7.12 ± 0.40 s. b) SB-612111 antagonism of the nociceptin activity. Groups of mice were administered varying doses of SB-612111 and 20 min later received an i.c.v. injection of nociceptin. Paw withdrawal latencies (seconds) were measured 10 min after nociceptin administration. Each value represents the mean ± S.E.M. for eight mice. *, P < 0.05 compared with the nociceptin-treated group. Paw withdrawal latencies for mice treated with 5 µl/mouse i.c.v. saline were 7.06 ± 0.62 and 2.15 ± 0.35 s in mice treated with 5 nmol i.c.v. nociceptin.

The ability of SB-612111 to reverse the nociceptin antagonism of morphine analgesia was also evaluated in the hot-plate test in mice (Fig. 4). Nociceptin (5 nmol/mouse i.c.v.) inhibited morphine (5 mg/kg s.c.)-induced analgesia in the mouse hot-plate test, measured as a reduction in paw withdrawal latencies: morphine administration (5 mg/kg s.c.) produced a withdrawal latency of 21.84 ± 1.07s, whereas the latency after morphine administration (5 mg/kg s.c.) together with nociceptin (5 nmol i.c.v.) was shortened to 5.55 ± 0.68 s. Administration of SB-612111 i.v. concomitantly with 5 mg/kg s.c. morphine, and 20 min before the administration of 5 nmol/mouse i.c.v. nociceptin, led to a dose-dependent inhibition of the antagonism by nociceptin of the analgesia induced by morphine (ED₅₀ = 0.69 mg/kg i.v., 95% CL = 0.34–1.21), as shown in Fig. 4. Together, these results demonstrate that SB-612111 can act in vivo to antagonize acute nociceptive activity mediated by the ORL-1 receptor.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Antagonism of the antimorphine effect of nociceptin by SB-612111. Groups of mice were treated with 5 mg/kg s.c. morphine and increasing doses (i.v.) of SB-612111. After 20 min, animals were administered i.c.v. nociception, and paw withdrawal latencies (seconds) were measured 10 min thereafter. Each value represents the mean ± S.E.M. for eight mice. *, P < 0.05 compared with the morphine + nociception-treated group. Paw withdrawal latencies were 21.84 ± 1.07 s after the administration of 5 mg/kg s.c. morphine, and 5.55 ± 0.68 s after the administration of 5 mg/kg s.c. morphine and 5 nmol/mouse i.c.v. nociceptin.

Reversal of Morphine Tolerance by SB-612111. The ability of SB-612111 to alter pain response in the mouse hot-plate test was further evaluated in mice exhibiting a reduced response (tolerance) to morphine after chronic administration of the drug. Mice were made morphine-tolerant after 4 days of administering 50 mg/kg s.c. morphine once a day. One day after the last morphine dosage animals were challenged with a lower dose of morphine (9 mg/kg s.c.), and analgesic activity was measured on the hot-plate apparatus at 15, 30, 45, and 60 min after drug administration. The peak analgesic activity afforded by the acute morphine administration occurred after 30 min in both drug naive and chronically treated animals (data not shown). As expected, a reduction in the antinociceptive effect of morphine was observed after chronic morphine administration, which is consistent with the development of opiate tolerance (Fig. 5). In drug-naive mice, the administration of 3 mg/kg i.v. SB-612111 60 min before performing the hot-plate test failed to elicit any significant antinociceptive effect. However, the administration of SB-612111 to animals chronically treated with morphine produced a marked enhancement in the analgesic effect of an acute dose (9 mg/kg s.c.) of morphine. Together, these results demonstrate that an ORL-1 receptor antagonist can act to resensitize animals to the analgesic effect of morphine after chronic opiate exposure. It should be further noted that although the potential analgesic activity of SB-612111 per se in morphine-tolerant mice was not tested in this study, Ueda et al. (2000) reported earlier that the ORL-1 antagonist J-113397 does not alter response in tail pinch or tail-flick analgesia tests using morphine-tolerant mice.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Sensitization of response after SB-61211 treatment of morphine-insensitive mice. SB-612111 (3 mg/kg i.v.) and morphine (9 mg/kg s.c.) were administered 60 and 30 min before performing the hot-plate test, respectively. Experiments with tolerant animals were performed 24 h after the final high-dose (50 mg/kg s.c., once a day) of morphine was administered and 5 days after initiating the chronic opiate dosing. Each value shown represents the mean ± S.E.M. for 10 mice. *, P < 0.05 compared with the vehicle + saline-treated group. #, P < 0.05 compared with the vehicle + morphine-treated group. $, P < 0.05 compared with vehicle + morphine (chronic)-treated group.

Antihyperalgesic Activity of SB-612111. To test a suspected role of the ORL-1 receptor in persistent pain responses, we used the carrageenan plantar model of inflammatory pain to evaluate the potential antihyperalgesic effect of SB-612111. After injection of carrageenan into the rat hindpaw, a painful inflammatory response develops that can be measured by a shortening in ipsilateral paw withdrawal latencies to a noxious stimulus. Paw withdrawal latencies in the carrageenan-treated animals were 4.00 ± 0.50 versus 11.05 ± 0.52 s in saline-treated animals. In this model, SB-612111 caused a significant inhibition of the carrageenan-induced reduction in paw withdrawal latencies at doses of 3 and 5 mg/kg i.v., whereas paw withdrawal latencies in the contralateral, untreated paw were uneffected (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Antihyperalgesic activity of SB-612111 in the rat carrageenan plantar test. Paw withdrawal latencies are shown for the ipsilateral (filled columns) and contralateral paw (open columns). Each value represents the mean ± S.E.M. for eight rats. *, P < 0.05 versus carrageenan. Paw withdrawal latencies for saline-treated rats were 11.05 ± 0.52 (ipsilateral) and 10.97 ± 0.79 s (contralateral), whereas for carrageenan-treated rats these values were 4.00 ± 0.50 s (ipsilateral) and 11.22 ± 0.51 s (contralateral). *, P < 0.05 compared with vehicle + carrageenan-treated group.

	Discussion

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The availability of the high-affinity and selective antagonist SB-612111 has allowed us to probe the role of the ORL-1 receptor in regulating nociception. SB-612111 is a member of a novel class of nonpeptide antagonists that is structurally unrelated to other nonpeptide ORL-1 receptor ligands that have been described, such as J-113397 (Kawamoto et al., 1999) and JTC-801 (Shinkai et al., 2000).

Profiling of SB-612111 in competition radioligand binding experiments indicated some preference of the (–)-enantiomer (SB-612111) for the ORL-1 receptor and a higher selectivity versus the µ-opioid receptor, and all further studies were carried out using SB-612111. SB-612111 proved in these tests to have both higher affinity for the ORL-1 receptor and greater selectivity versus the µ-opioid receptor than the other ORL-1 antagonists tested (JTC-801 and J-113397). Interestingly, the resolved enantiomers of J-113397 differed considerably in their affinity for the ORL-1 receptor, with the (+)-enantiomer having 79-fold higher affinity than that measured for the (–)-enantiomer. Extended profiling of SB-612111 in assays specific for a variety of receptors, ion channels, and enzymes revealed several weak interactions that were explored in more detail. However, the highest measured affinity interaction of SB-612111, to the ₃-adrenergic receptor, was >260-fold weaker than its interaction with the ORL-1 receptor. Cellular signaling assays further established that SB-612111 is an ORL-1 receptor antagonist with low potential for either activation or antagonism of the µ-opioid receptor. In summary, these data suggest that SB-612111 would allow us to examine ORL-1 receptor function in vivo.

We established that intracerebroventricular injection of nociceptin dose dependently shortens hot-plate latencies in mice. These results are in agreement with behavioral studies demonstrating that i.c.v. injection of low doses of nociceptin produces a hyperalgesic response in mice, as measured by the hot-plate or tail-flick test (Meunier et al., 1995, Reinscheid et al., 1995). Intravenous injection of SB-612111 dose dependently inhibited acute pain signaling induced by i.c.v. nociceptin in the mouse hot-plate assay without altering baseline hot-plate latencies. These results demonstrated that SB-612111 acts as an ORL-1 receptor antagonist in vivo as well as in vitro. The lack of antinociceptive or hyperalgesic effects of SB-612111 per se in the mouse hot-plate test suggests that ORL-1 receptor activation does not directly modulate acute nociceptive signaling. Earlier results by Yamada et al. (2002) demonstrating a potent effect of JT-801 alone in this test may be explained by a possible interaction with µ-opioid receptors, because our in vitro data (summarized in Table 1) revealed a low µ-opioid/ORL-1 receptor selectivity for this compound. Our data also agree with studies showing that J-113397 can inhibit nociceptin-induced hyperalgesia in the mouse tail-flick assay without altering baseline tail-flick latencies (Ozaki et al., 2000) and that ORL-1 receptor knockout mice display the same nociceptive threshold as control mice in acute pain models (Nishi et al., 1997). These earlier findings have suggested, and our data agree with, the hypothesis that the nociceptin/ORL-1 system is activated in evoked pain states. SB-612111 likely has a central site of action in these studies, as evidenced by its ability to antagonize the action of centrally administered nociceptin. However, its action at peripheral ORL-1 receptors to regulate thermal nociception cannot be ruled out, as previously demonstrated by Ko et al. (2002).

In the present study, we additionally demonstrated that SB-612111 behaves as an ORL-1 antagonist in vivo by dose dependently reversing an inhibition of morphine analgesia promoted by central i.c.v. administration of nociceptin. The ability of SB-612111 to counter the effects of centrally administered nociceptin on morphine responsiveness is consistent with a mechanism in which both µ-opioid and ORL-1 receptor activation influences the release of neurotransmitters such as GABA in descending pain pathways, as described by Vaughan et al. (2001). The similar ability of the µ-opioid and ORL-1 receptors to regulate GABA release, yet opposing effects on nociceptive signaling, has been suggested to result from differential on- and off-cell expression and activity of these receptors in the rostral ventromedial medulla. An alternative view, which builds upon the observation of µ-opioid and ORL-1 receptor coexpression on the same cells, uses evidence of receptor cross talk and heterologous receptor desensitization to explain the opposing actions of µ-opioid and ORL-1 receptor activation. For instance, Mandyam et al. (2002) have shown that ORL-1 receptor activation can cause desensitization of µ-opioid receptors expressed on the same cell by stimulating translocation of protein kinase C isozymes to the cell membrane. Thus, an ORL-1 antagonist would be expected to prevent desensitization of the µ-opioid receptor resulting from nociceptin-mediated ORL-1 receptor activation, and thereby potentiate the action of morphine.

We further extended the morphine analgesia studies to include tests in mice treated chronically with morphine, and now show that a single administration of SB-612111 reverses established tolerance to the analgesic effects of morphine in the mouse hot-plate test. These data support the hypothesis, proposed by Nishi et al. (1997), that changes in neuronal plasticity observed in conditions of morphine tolerance may derive from increased activity of an antiopioid nociceptin system. Ueda et al. (2000) had previously reported the retarded acquisition of morphine tolerance in ORL-1 receptor knockout mice, and its reversal in mice after a single subcutaneous or intrathecal administration of J-113397. Thus, it will be of further interest to establish a linkage of the ORL-1 receptor to signaling pathway elements that can regulate µ-opioid receptor function. One such key signaling relationship may be with RGS4, a member of the regulator of G protein signaling (RGS) protein family. Recently, we reported a relationship between morphine tolerance and the expression of RGS4 and further established that this signal-regulating protein can influence µ-opioid receptor function (Garnier et al., 2003).

Previous work by Andoh et al. (1997) showed that peripheral inflammation induces nociceptin expression in primary sensory neurons and that the ORL-1 receptor may be associated with the production of nociceptive hypersensitivity associated with an inflammatory response. Our results, showing that SB-612111 had antihyperalgesic activity in the subchronic, rat carrageenan inflammatory pain model, are in line with this hypothesis. However, these data contrast with previous work by Yamamoto et al. (1997), showing that intrathecal injection of nociceptin reduces allodynia and thermal hyperalgesia induced by carrageenan injection into the rat paw. These discrepancies may be explained by the dose dependence of proversus antinociceptive activities of nociceptin, as recently described by Muratani et al. (2002) using the mouse formalin model of acute pain response. Our data showing that SB-612111 can reverse carrageenan-induced hyperalgesia are consistent with a role of nociceptin in the negative regulation of excitatory neurotransmission. The recent description of a role of the glutamate pathway in this hyperalgesia model (Inoue et al., 2003) is consistent with this view. However, our results do not suggest a pain-inhibitory action of nociceptin via its effects on substance P-ergic fibers in the spinal cord, as was also proposed in this report.

In this study, we have described the pharmacological characteristics of SB-612111, a member of a new class of selective, nonpeptidic ORL-1 receptor antagonists. We have further used this ligand to probe the role of the ORL-1 receptor in nociceptive signaling. The data demonstrate that SB-612111 can effectively antagonize the pronociceptive action of nociceptin in an acute pain model and potentiate the action of morphine in animals that are morphine-tolerant. In addition, SB-612111 proved to be effective alone in blocking hyperalgesia in an inflammatory pain model. Together, the results suggest that this novel class of selective ORL-1 antagonist may have therapeutic utility in the treatment of evoked pain responses such as those occurring in response to inflammation and in the prolongation of opiate analgesic therapy.

	Footnotes

 
DOI: 10.1124/jpet.103.055848.

ABBREVIATIONS: ORL, opiate receptor-like; DAMGO, D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin; DPDPE, [D-Pen²,D-Pen⁵]-enkephalin; hMOR, human µ-opioid receptor; hDOR, human -opioid receptor; hKOR, human -opioid receptor; CHO, Chinese hamster ovary; FBS, fetal bovine serum; HEK, human embryonic kidney; RGS, regulator of G protein signaling; SB-612111, (–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol; UFP-101, [N-Phe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂; JTC-801, N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide monohydrochloride; J-113397, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazole-2-one; U-69593, [(+)-(5,7,8b)-(+)-N-methyl-N[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide]; BRL 52656, S(–)-2-(1-pyrrolidinylmethyl)-1-(4-trifluoromethylphenyl)acetyl piperidine hydrochloride; RX 821002, 2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)4,5-dihydro-1H-imidazole.

Address correspondence to: Dr. Mark A. Scheideler, P.O. Box 16, 10325 Kensington Pkwy., Kensington, MD 20895. E-mail: mark.scheideler{at}att.net

	References

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