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NEUROPHARMACOLOGY

Evidence for a Selective Role of the -Opioid Agonist [8R-(4bS*,8a,8a,12b)]7,10-Dimethyl-1-methoxy-11-(2-methylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline Hydrochloride (SB-235863) in Blocking Hyperalgesia Associated with Inflammatory and Neuropathic Pain Responses

Departments of Neurobiology Research (P.P., O.A., G.F., M.G., P.F.Z., G.P., O.P., M.S., M.A.S.) and Medicinal Chemistry (G.M.D.), GlaxoSmithKline Pharmaceuticals, Milano, Italy; and Neurobehavioral Research, Neurology Centre of Excellence for Drug Discovery, Essex, United Kingdom (S.B., T.O.S., N.U.)

Received June 11, 2003; accepted August 22, 2003.

	Abstract

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The specific involvement of the -opioid receptor in the control of nociception was explored by investigating the pharmacological activity in vivo of a selective, orally active, and centrally penetrant -opioid agonist. [8R-(4bS*,8a,8a,12b)]7,10-dimethyl-1-methoxy-11-(2-methylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochloride (SB-235863) is a new pyrrolomorphinan with high affinity (K_i = 4.81 ± 0.39 nM) for the -opioid receptor, full agonist activity, and binding selectivity versus the µ- and -opioid receptors of 189-fold and 52-fold, respectively. Perorally administered SB-236863 was inactive in the rat tail-flick and hot-plate tests of acute pain response, but potently reversed thermal hyperalgesia in rats resulting from a carrageenan-induced inflammatory response. This activity could be blocked by the -opioid antagonist naltrindole (3 mg/kg s.c.), but selective µ- and -opioid antagonists were ineffective. Naltrindole (1 µg i.c.v.) also blocked the activity of 10 mg/kg (p.o.) SB-235863, showing that the compound activates -opioid receptor sites in the central nervous system. SB-235863 was additionally effective at reversing chronic hyperalgesia in the Seltzer rat model of partial sciatic nerve ligation after peroral administration. These data show that the -opioid receptor plays a selective role in regulating evoked and lasting changes in nociceptive pain signaling. Classical side effects of µ- and -opioid receptor activation (slowing of gastrointestinal transit and motor incoordination, respectively) were not observed after administration of 70 mg/kg (p.o.) SB-235863, nor was evoked seizure activity affected. These results suggest a selective and limited role of -opioid receptors in the modulation of nociception.

Supporting evidence of a central site of action has come from in vivo studies using selective opioid peptides. For instance, the selective enkephalin analog DPDPE effectively reverses the mechanical and cold allodynia that develops in spinally injured rats, a model of chronic pain (Hao et al., 1998). Studies of [D-Ala²,Glu⁴]deltorphin in the rat paw formalin model, using both intrathecal and intracerebroventricular administration, have further suggested the existence of a descending inhibitory pain pathway that controls tonic nociceptive input at the spinal level (Kovelowski et al., 1999). However, the antinociceptive effects of DPDPE after spinal administration are blocked by a µ-opioid antagonist (He and Lee, 1998). Furthermore, the antinociceptive activity of both deltorphin-II and DPDPE is reduced in mice lacking the µ-opioid receptor (Matthes et al., 1998). These results suggest that the analgesic activity of such -preferring peptides may be directly mediated via interaction with the µ-opioid receptor, or via a synergistic action of - and µ-opioid receptors.

In vivo studies with peptides that selectively activate the -opioid receptor have also been shown to lack classical morphine-like side effects such as inhibition of gastrointestinal transit (Tavani et al., 1990), respiratory depression (Su et al., 1998), and motor incoordination (Franck et al., 1991). These results suggest that activation of the -opioid receptor may not lead to the broader set of actions that complicate clinical usage of the µ- or -opioid agonists.

Small molecule agonists representing several independent structural classes, and which have better central and systemic bioavailability than the peptide ligands, have become available for experimental use. These include the octahydroisoquinoline TAN-67 (Nagase et al., 1998) and the diarylpiperizine derivative SNC80 (Bilsky et al., 1995). Pharmacological studies of (–)TAN-67 have shown that intrathecal administration leads to increased mouse thermal tail-flick latencies (Tseng et al., 1997). This activity of (–)TAN-67 proved to be greater in streptozocin-induced diabetic mice (Kamei et al., 1997), and -opioid agonists may be useful in treating the painful neuropathies associated with this chronic illness. The small molecule agonist SNC80 has been shown to have a dose- and time-dependent activity in a warm-water tail withdrawal assay in rats (Bilsky et al., 1995). SNC80 was also active in the rat hot-plate test of analgesic activity if the compound was administered intracerebroventricularly. However, a notable proconvulsant activity of SNC80 was also observed (Bilsky et al., 1995; Hong et al., 1998), bringing into question whether this activity is associated with -opioid receptor activation or is an intrinsic feature of this compound class.

The nonpeptidic, selective -opioid agonist SB-235863 ([8R-(4bS*,8a,8a,12b)]7,10-dimethyl-1-methoxy-11-(2-methylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]-isoquinoline hydrochloride) was designed according to an extension of the message-address concept proposed by Portoghese (1989). In this instance, the aromatic moiety (address) of the morphinan backbone was replaced with a nonaromatic, lipophilic ester. We now describe the in vitro affinity and selectivity of this -opioid agonist, and demonstrate its in vivo antinociceptive properties after systemic oral administration. These results with a selective -opioid agonist clarify the role of -opioid receptor activation in controlling the thermal hyperalgesia, but not tactile allodynia, which develops in response to inflammation or tissue injury. Further examination of the possible activity of SB-235863 in tests of µ- and -opioid-mediated side effects, and seizure activity, suggests a lack of involvement of the -opioid receptor in these events.

	Materials and Methods

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Materials. The following drugs and chemicals used in the described studies were obtained from the following sources: [³H]DAMGO (50 Ci/mmol) and [³H]DADLE (55 Ci/mmol) were purchased from New England Nuclear (Bruxelles, Belgium); [³H]U69593 (60 Ci/mmol) was purchased from Amersham-Italia (Milan, Italy) and U69593 [GenBank] from RBI (Natick, MA); naloxone hydrochloride, morphine hydrochloride, and 7,8-dihydrocodeinone hydrochloride were purchased from Salars (Como, Italy); [(D-Pen²,D-Pen⁵]-enkephalin (DPDPE), [(D-Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO), and nor-binaltorphimine dihydrochloride (nor-BNI) were purchased from Sigma-Aldrich (Milan Italy); SNC80, naltrindole (NTI), and -funaltrexamine (-FNA) were purchased from Tocris Cookson Inc. (Bristol, UK); lambda carrageenan, forskolin, 3-isobutyl-1-methylxanthine (IBMX), and pluronic acid (F68) were purchased from Sigma-Aldrich; sodium pentobarbital was from Siegfried Chemic (Zofingen, Switzerland); BRL-52656 was synthesized as described by Brooks et al. (1997); SB-235863 was synthesized in-house.¹

Tissue culture plastics were purchased from NUNC (Milan, Italy). Cell culture medium reagents and FuGENE transfection reagent were purchased from Life Technologies (San Giuliano Milanese, Italy). Geneticin (G418 sulfate) and hygromycin B were purchased from Calbiochem Inalco (Milan, Italy). Culture plates and reagents used in the luciferase gene-reporter assay were from Packard Bioscience (Milan, Italy).

Cell Cultures and Transfection. Cell cultures used in preparing membranes for specific radioligand binding assays and in cellular signaling assays were routinely maintained at 37°C in a humidified atmosphere containing 5% CO₂. Chinese hamster ovary (CHO) cells [obtained from European Type Culture Collection (ETCC), 317] were grown in suspension in 1017S03 culture medium (GlaxoSmithKline, Harlow, UK) containing 10% (v/v) fetal bovine serum and 0.05% (v/v) pluronic acid (F68). Human embryonic kidney (HEK293) cells (obtained from ETCC, 85120602) were grown in monolayer in Eagle's minimal essential medium supplemented with 10% (v/v) fetal bovine serum and 2 mM L-glutamine. The human (hDOR) and µ (hMOR) receptors were expressed in CHO cells, and the human -opioid receptor (hKOR) in HEK-293 cells, using a pCDN vector. Subclones stably expressing receptors 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 further selected for additional characterization.

HEK293-Luc cells derived from HEK293 cells by stably expressing a cAMP-responsive MRE/CRE luciferase gene reporter construct (as described by Fitzgerald et al., 1999) were stably transfected using the FuGENE transfection reagent with an expression plasmid containing the pcDNA3.1/Hygro/hDOR or pcDNA3.1/Hygro/hMOR cDNAs and a gene conferring resistance to hygromycin B. Selection of hDOR- or hMOR-expressing clones was achieved by growing the cells in the presence of 0.4 mg/ml hygromycin B. HEK293 cells stably expressing hKOR were transiently transfected using FuGENE reagent together with 40 µg of the cAMP-responsive MRE/CRE luciferase reporter construct. HEK293 cells transfected with the CRE luciferase reporter construct and expression vectors for either hDOR or hMOR were cultured in the presence of 0.5 mg/ml geneticin (G-418), 1% (w/w) non-essential amino acids, and 0.4 mg/ml hygromycin.

Measurement of Specific Opioid Receptor Binding. Radioreceptor binding assays used to establish drug affinities at the human opioid receptors were performed using membranes from cells expressing the receptors, prepared by lysis in hypotonic phosphate buffer via a modification of the method described by Scheideler and Zukin (1990). Briefly, cells were harvested in phosphate-buffered saline (approximately 30 x 10⁶ cells/30 ml tube) and collected by centrifugation (800g, 5 min). Each pellet was then washed by resuspension (30 ml/pellet) in 10 mM potassium phosphate buffer, pH 7.2 (buffer A) and centrifuged at 40,000g for 10 min. The pellets obtained were resuspended in the same volume of buffer A, incubated on ice for 20 min, and centrifuged at 800g for 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 (40,000g, 10 min). The pellets obtained were resuspended in buffer A containing 0.32 M sucrose and 5 mM EDTA (buffer B) and stored at –80°C. Protein concentrations were determined with the protein assay kit from Sigma Aldrich.

[³H]DAMGO, [³H]DADLE, and [³H]U69593 were used to selectively label -, µ-, and -opioid receptors expressed at high levels in the cell membrane preparations, as described by Petrillo et al. (1989) and Wang et al. (1994); - and µ-opioid binding experiments were performed in 50 mM Tris buffer, pH 7.4, in 5-ml polypropylene tubes (final volume of 2 ml). -Opioid binding was measured in 25 mM potassium phosphate buffer containing 3 mM MgCl₂, pH 7.4, in a final volume of 1 ml. Nonspecific binding was determined in the presence of 10 µM naloxone. Incubation was carried out for 60 min at 25°C (-opioid assay), 30°C (µ-opioid assay), or 37°C (-opioid assay). The reaction was terminated by filtration using cold assay buffer (3 x 4 ml) on an M48 Brandell Cell Harvester (Biomedical Research and Development Laboratories Inc., Gaithersburg, MD). The radioactivity present on the disks was determined by liquid scintillation counting using a Packard Bioscience 2500TR beta counter (Milan, Italy). Experiments were performed using a membrane concentration of 5 to 20 µg protein/ml and radioligand concentrations close to the experimental K_D values obtained from saturation studies. The data obtained from competition binding experiments were analyzed with nonlinear fitting analysis using the software GraFit (Erithacus Software Limited, Horley, Surrey, UK). The K_i values were determined from IC₅₀ determinations using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). The K_D value for specific [³H]DAMGO binding to hMOR was 1.2 ± 0.2 nM. The K_D value for specific [³H]DADLE binding to hDOR was 1.2 ± 0.1 nM. The K_D value for specific [³H]U69593 binding to hKOR was 1.1 ± 0.3 nM.

Measurements of specific opioid binding, using membranes prepared from whole brain minus cerebellum of male CD rats, were performed as described by Brooks et al. (1997). -Opioid receptors were specifically labeled with 0.7 to 1.0 nM [³H]DADLE. DAMGO (40 nM) was included in the incubations to suppress binding to µ-opioid receptors. µ-Opioid receptors were specifically labeled with 0.7 to 1.0 nM [³H]DAMGO. -Opioid receptors were specifically labeled with 0.7 to 1.0 nM [³H]U69593. Nonspecific binding was assessed in each assay in the presence of 10 µM naloxone. K_D values for specific binding of [³H]DADLE, [³H]DAMGO, and [³H]U69593 to the -, µ-, and -opioid receptors were 1.3 ± 0.2 nM, 1.2 ± 0.4 nM, and 4.0 ± 0.8 nM, respectively.

In Vitro Assessment of Receptor and Enzyme Interactions. SB-235863 was tested at a concentration of 10 µM by CEREP (Le Bois L'Eveque, France) in a broad profile of 48 radioligand binding assays specific for G-protein-coupled receptors, nuclear hormone receptors, and voltage- and ligand-gated ion channels. The compound was further tested for activity in nine enzyme assays with specificity for ATPase, elastase, protein kinase C, tyrosine kinase, and phosphodiesterase subtypes. SB-235863 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.

CRE-Luciferase Gene Reporter Assay. Potential drug agonist or antagonist activity was tested using the luciferase signaling assay, as previously described by Fitzgerald et al. (1999). Cells were plated in white polystyrene 96-well format CulturPlates at a density of 1 x 10⁴ cells/well. One day after seeding, cells were incubated for 30 min in the presence of 0.5 mM phosphodiesterase inhibitor IBMX. Cells were further treated with 300 nM forskolin (FSK) in the presence or absence of drugs at the indicated concentrations in a final volume of 100 µl. After 4 h, incubation was terminated by the addition of an equal volume of LucLite reagent. Luciferase activity measured in counts per second (cps) was measured using a TopCount microplate scintillation and luminescence counter (Packard Bioscience). Results were expressed as percentage of response measured in control (forskolin-treated) cells. Dose-response curves were fit by nonlinear fit using GraFit version 4.09 to determine EC₅₀ values.

Measurement of Intracellular cAMP. The ability of SB-235863 to alter -opioid receptor-mediated signal transduction was confirmed by direct measurement of the intracellular accumulations of cAMP in cultured HEK293-Luc cells stably expressing hDOR or hMOR. Briefly, cells (5 x 10⁴ cells/well in white 96-well CulturPlate) were treated with 10 µM FSK and test compounds in the presence of 0.5 mM IBMX. After 15 min of incubation at 37°C, medium was replaced with 50 µl of reconstituted lysis reagent, and 150 µl of immunoreagent mix containing [¹²⁵I]cAMP (0.0143 µCi/well) was added. The cAMP ¹²⁵I-Scintillation Proximity Assay (SPA) Direct Screening Assay System (Amersham Biosciences Inc., Cologno Monseze, Italy) was used to measure second-messenger synthesis, and cAMP quantitated using a microtiter plate -scintillation counter (Packard Bioscience). The amount of cAMP present was extrapolated from a standard curve consisting of 6.25 to 25.6 pmol of cAMP per well. Data were expressed as the percentage of FSK-stimulated cAMP accumulation from independent experiments, each performed in triplicate.

Animals. Male Sprague-Dawley rats (Charles River Laboratories, Calco, Italy) and Wistar (Harlan Olac) rats weighing 200 to 300 g were used in the studies of nociception and opioid side effect activity. Animals were housed in a temperature-controlled room (22 ± 0.5°C) with a relative humidity between 40 and 60%, and maintained on a 12-h light/dark cycle. Animals were fasted for 16 to 18 h before acute, oral drug treatments. Water was available ad libitum. In experiments involving chronic drug administration animals were allowed food and water ad libitum. All experiments were performed in accordance with National Institutes of Health guidelines for the care and use of animals.

Intracerebroventricular cannula placement in rats was performed under pentobarbital anesthesia (65 mg/kg i.p.). An incision was made slightly to the cranial midline, the skin was retracted, and underlying tissue removed. A polyethylene cannula (PE-10), cut to a length of approximately 5 cm, was implanted into the right lateral cerebral ventricle (coordinates: L 1.5 mm, P 0.8 mm, V 4.5 mm). Rats were allowed to recover in individual cages for 3 days before further experimentation. Placement of the cannula was verified post mortem with Evans blue dye.

Rat Tail-Flick and Hot-Plate Tests. The antinociceptive activity of SB-235863 was assessed using a radiant heat tail-flick test. The tail was positioned under a hot light beam and the time required for the rat to remove its tail from the thermal stimulus recorded, as described by Suh and Tseng (1988). The lamp intensity was adjusted such that each animal in the control group flicked its tail within 4 to 7 s. Rats were submitted to a noxious thermal stimulus at 30, 60, 90, and 120 min after compound or vehicle administration and tail-flick threshold determined. To avoid tissue damage, a cutoff limit to the test of 15 s light exposure was observed. The antinociceptive effect afforded by the test compound was expressed as the statistical increase in the tail-flick latencies of treated animals in comparison to the vehicle-treated group.

The effect of SB-235863 on nociceptive thresholds was assessed in the rat hot-plate test by evaluating the reaction time of rats placed on a Socrel hot plate (Ugo Basile, Comerio, Italy) maintained at 52 ± 0.5°C, according to the method of Tung and Yaksh (1982). Before the administration of test compounds the baseline hot-plate latency of each rat was determined. After treatment, a maximal possible latency of 60 s was allowed, after which rats not responding were removed from the hot-plate surface to avoid tissue damage.

Measurement of Carrageenan-Induced Hyperalgesia in Rats. The ability of SB-235863 to alter thermal hyperalgesia resulting from inflammation was tested in rats after intraplantar injection of 2% (w/w) lambda carrageenan (0.1 ml dissolved in sterile saline) or saline (control animals). The hindpaw withdrawal latency to noxious radiant heat application was measured in unrestrained animals using the plantar test apparatus (Ugo Basile, Comerio, Italy), as described by Hargreaves et al. (1988). Rats were placed in a clear plastic chamber, a radiant heat source focused on the plantar surface of the hindpaw, and the paw withdrawal time automatically determined. The intensity of the heat source was adjusted so that baseline latency was 9 to 12 s. A test cutoff time of 22 s was observed to avoid tissue damage. Carrageenan-induced hyperalgesia resulted in significantly shorter paw withdrawal latencies compared to saline-treated paws. The percentage of reduction of hyperalgesia was expressed as follows: 100 x [(latency carrageenan + drug) – (latency carrageenan + vehicle)]/[(latency saline + vehicle) – (latency carrageenan + vehicle)]. Paw edema was assessed using an electronic rat paw plethysmometer (Ugo Basile).

The subcutaneous doses of antagonist and times of pretreatment that were used in studies in the carrageenan model aimed at confirming the -opioid receptor-selective activity of SB-235863 were as follows: -FNA (10 mg/kg, 24 h); NTI (3 mg/kg, 20 min); nor-BNI (20 mg/kg, 4 h). The antagonists -FNA, NTI, and nor-BNI were initially tested in this model to establish doses effective in reversing the activity of an opioid receptor agonist, using information from a previous study in rats by Negus et al. (1993), which suggested approximate effective antagonist doses and administration times. Agonist doses, selected from dose-response studies in the rat carrageenan test after peroral administration, and pretreatment times used were as follows: SB-235863 (10 mg/kg, 90 min); morphine (1 mg/kg, 60 min); BRL-52656 (10 mg/kg, 45 min).

Measurement of Hyperalgesia in Rats after Partial Sciatic Nerve Ligation. The ability of SB-235863 to alter the thermal hyperalgesia that accompanies nerve injury was tested in rats as described by Seltzer et al. (1990). After general anesthesia induced by 3% isoflurane, partial denervation of the left hindpaw was achieved by exposure of the left sciatic nerve at high thigh level, and a 7-0 mersilk suture inserted such that the dorsal third to half of the nerve thickness was trapped in the ligature. Sham animals were also prepared in which insertion of the suture and ligation was not performed. Thermal hyperalgesia was assessed using the plantar method as described for the carrageenan test, with the exception that both hindpaws were tested. Withdrawal latencies assessed 1 day before surgery served as the baseline latencies for repeated measures. The rats that exhibited an altered paw posture on the side ipsilateral to the nerve injury, and an ipsilateral latency/contralateral latency ratio (percentage of hyperalgesia) of 80% or less, were considered to be hyperalgesic and selected for inclusion in the study. The percentage of rats from each surgical group that met this criterion was >90%. Saline or SB-235863 (30 mg/kg/day) was administered subcutaneously from day 15 to day 21 postsurgery, and hyperalgesia assessed 30 min after drug administration on days 15, 17, and 21 after surgery. In a separate experiment SB-235863 (100 mg/kg/day) or 1% (w/v) methyl cellulose was given orally for 14 days, commencing on day 15 postsurgery. Testing took place 60 min after drug administration on days 15, 17, 21, 24, and 28 after surgery.

Rat Rotarod Test. Potential motor incoordination/ataxia caused by SB-235863 was evaluated utilizing an accelerating Rotarod (Ugo Basile). Rats were trained on the Rotarod during six 5-min sessions (each separated by 1 h), on two consecutive days before drug administration and tested on day 3, as described by Sutters et al. (1990). On day 1, during the first three sessions of training, the Rotarod accelerated from 2 to 20 rpm in a period of 5 min, whereas for the remaining training sessions of day 1 and for the second day of training, the belt-gear ratio was set so that the rotor would accelerate from 3 to 30 rpm in the 5-min period. These latter conditions were also adopted for experiments. Rats were placed on the Rotarod and the animal's performance measured as time on the Rotarod. The degree of the motor incoordination/ataxia afforded by the compound was determined using the following formula: percentage of activity = (1 – T/C) x 100, where T and C refer to mean performance time on the Rotarod of the drug-treated and control groups, respectively.

Rat Gastrointestinal Motility. Potential alterations in colonic motility caused by SB-235863 were assessed as described by Tavani et al. (1990). Animals received, by gavage, 2 ml of a mixture of 12.5% (w/w) gum arabic and 12.5% (w/w) vegetable charcoal in water. Five minutes after the charcoal meal administration rats were sacrificed, their small intestine removed, and its length measured from the pyloric sphincter to the ileocecal junction, also recording the transit distance of the charcoal meal. The percentage of the gastrointestinal transit was calculated as (LC/LT) x 100. LC refers to the distance of charcoal meal transit and LT is the total length of the small intestine.

Rat Maximal Electroshock Seizure Threshold and Metrazol Infusion Tests. Assessment of potential proconvulsant or anticonvulsant effects of SB-235863 was made as previously described by Upton et al. (1997). Results of the maximal electroshock seizure test were expressed as CC₅₀ values, i.e., the threshold current required for the induction of tonic hindlimb extension seizures. Statistical analysis of the results was based upon a comparison to corresponding vehicle-treated controls. Results of the metrazol infusion test were expressed as the dose of metrazol (mg/kg i.v.) required to produce myoclonus forelimb clonus. Statistical analysis of the results was based upon a comparison to corresponding vehicle-treated controls using the Kruskal-Wallis test, followed by the Mann-Whitney U test.

	Results

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Selective Binding Site Interaction of SB-235863 with hDOR. Selective in vitro interaction of SB-235863 (structure shown in Fig. 1) with the human opioid receptors was evaluated in radioreceptor binding assays using membranes prepared from clonal cell lines expressing the human (hDOR), µ (hMOR), and (hKOR) opioid receptors (Fig. 2). SB-235863 competitively displaced binding of the preferential -opioid radioligand [³H]DADLE to hDOR (K_i value of 4.81 ± 0.39 nM, n_H = 1.04); this value was similar to that determined for the selective peptide DPDPE (Fig. 2A, Table 1). Morphine weakly displaced the radioligand binding, whereas the µ-opioid peptide DAMGO competed for [³H]DADLE binding only at concentrations in excess of 1 µM (Table 1). SB-235863 was relatively ineffective at inhibiting the binding of [³H]DAMGO and [³H]U69593 to the hMOR and hKOR receptors, respectively (Fig. 2, B and C). Relative receptor selectivities were µ/ = 189 and / = 52.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Chemical structure of SB-235863.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Competitive displacement of specific radioligand binding from hDOR, hMOR, and hKOR. A, competition binding was measured for DPDPE (), TAN-67 (), SNC80 (), NTI (), and SB-238563 () using membranes from hDOR/CHO cells and the radioligand [3H]DADLE. B, competition binding was measured for DAMGO (), morphine (), -FNA () and SB-235863 () using membranes from hMOR/CHO cells and the radioligand [3H]DAMGO. C, competition binding was measured for BRL-52656 (), U69593 [GenBank] (), nor-BNI (), and SB-235863 () using membranes from hKOR/HEK cells and the radioligand [3H]U69593. Representative experiments are shown. These were performed and data analyzed as described under Materials and Methods.

A high-affinity and selective interaction of SB-235863 with opioid receptor sites in rat brain membrane was also observed. The K_i value for SB-235863 displacement of [³H]DADLE binding to -opioid receptor sites was 14.7 ± 4.30 nM. K_i values for the displacement of specific [³H]DAMGO and [³H]U69593 binding to µ- and -opioid receptor sites were 3568 ± 575 nM and 1014 ± 72 nM, respectively, confirming the high selectivity observed for SB-235863 at the human -opioid receptor.

To assess the specificity by which SB-235863 regulates -opioid receptor activity, a broad profiling of its interaction in in vitro assays for receptors from outside the opioid receptor family, as well as ion channels and enzymes, was undertaken. At a test concentration of 1 µM SB-23586 yielded a >25% inhibition of activity in only 3 of the 55 assays in which it was tested. The specific binding of [³H]pirenzepine to M₁(h) muscarinic receptors expressed in CHO cell membranes was inhibited by 26%. The specific binding of [³H](+)-PN200-100 to rat cerebral cortex membranes, a measure of interaction with the dihydropyridine site of L-type calcium channels, was inhibited by 38%. The specific binding of [³H]batrachotoxinin to rat cerebral cortex membranes, a measure of interaction with site 2 of the sodium channel, was inhibited by 37%.

Measurement of Specific hDOR Activation by SB-235863. The opioid agonist activity of SB-235863 was studied in vitro by evaluating its ability to inhibit forskolin-stimulated luciferase expression in cells expressing hDOR, hMOR, and hKOR, and a CRE-luciferase gene reporter construct (Fig. 3). SB-235863 inhibited signal transduction in a concentration-dependent manner in cells expressing hDOR (Fig. 3A). The measured EC₅₀ value of 16.9 ± 3.7 nM (Table 2) was severalfold higher than the K_i value determined in competitive radioligand binding experiments (Table 1). SB-235863 proved to be approximately 15- and 100-fold less potent than the octahydroisoquinoline agonist TAN-67 and the enkephalin analog DPDPE, respectively (Table 2), in this assay. SB-235863 reversed the forskolin-mediated signaling to the same extent as these -opioid agonists, demonstrating that it is similarly a full -opioid agonist. The selective -opioid receptor antagonist NTI completely reversed the SB-235863-mediated inhibition of luciferase expression in cells expressing hDOR (Table 2), demonstrating that its agonist activity is mediated via a specific -opioid receptor interaction. SB-235863 did not significantly inhibit forskolin-stimulated luciferase expression in hMOR cells at concentrations of up to 1 µM, indicating only weak activity at this receptor at the highest concentrations tested (Fig. 3B), whereas DAMGO exhibited a potent activity in these cells that was reversed by the selective µ-opioid antagonist -FNA. Subsequent direct measurement of cellular cAMP allowed us to estimate an EC₅₀ value of 2208 ± 659 nM at hMOR. The EC₅₀ value measured for hDOR in this assay (15.99 ± 2.70 nM) was the same as measured in the gene reporter assay, showing that the results of these two assays are directly comparable. Thus, SB-235863 is 21-fold less potent than morphine and 130-fold less potent than the enkephalin analog DAMGO in activating hMOR signaling in vitro. In experiments performed using cells expressing hKOR, SB-235863 concentrations required to inhibit forskolin-stimulated luciferase expression (EC₅₀ = 795 ± 135 nM) were four orders of magnitude higher than those at which the -opioid agonist BRL-52656 produces an equivalent activity (Fig. 3C, Table 2). The inhibitory effects of both agonists were fully antagonized by coaddition of the selective -opioid antagonist nor-BNI, as expected for an hKOR-mediated activity. The relative potencies of 138 for µ/ and 47 for / compare well to the relative affinities measured for SB-235863 at these receptor sites.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Opioid inhibition of forskolin-stimulated luciferase expression. A, concentration-dependent activation of hDOR by DPDPE (), TAN-67 (), and SB-235863 (). Open symbols represent drugs tested in the presence of 10 nM NTI. B, activation of hMOR by DAMGO (), morphine (), and SB-235863 (). Open symbols represent drugs tested in the presence of 100 nM -FNA. C, activation of hKOR by BRL-52656 () and SB-235863 (). Open symbols represent drugs tested in the presence of 100 nM nor-BNI. Assays were performed as described under Materials and Methods. Data are expressed as the percentage of forskolin-stimulated luciferase expression. Data points represent the mean ± S.E.M. from three to four independent experiments, each performed in triplicate.

Antinociceptive Activity of SB-235863. A 10 mg/kg (p.o.) dose of morphine produced maximal protection in the rat hot-plate test 30 min after administration, the test latency measured in vehicle-treated animals (2.47 ± 3.3 s) increasing to 17.60 ± 6.0 s (P < 0.05) after morphine treatment. SB-235863 has a high central nervous system penetrance,² yet a 30 mg/kg (p.o.) dose of SB-235863 failed to change response in this test 30 or 60 min after administration, and high doses of SB-238653 (100, 300 mg/kg p.o.) failed to induce any significant effects in the rat tail-flick test of acute nociceptive signaling when response was evaluated at times ranging from 30 to 120 min after administration (data not shown). Morphine (30 mg/kg p.o.) slowed the rat reaction time at all time points in this test, reaching a maximum protective effect of 63.9 ± 14.6% (P < 0.05) 90 min after drug administration. These results suggested to us that -opioid receptor activation by SB-235863 does not modify acute nociceptive signaling.

Further experiments were aimed at examining the activity of SB-235863 in inflammatory (subchronic) and neuropathic (chronic) pain conditions in which changes in an evoked pain response are measured. After the injection of carrageenan into the rat hindpaw, a painful inflammatory response develops that can be measured by a shortening in paw withdrawal latencies to a noxious thermal stimulus. SB-235863 (5 mg/kg p.o.) reversed the shortened paw withdrawal latencies in this model in a time-dependent manner (data not shown). Significant changes in withdrawal latencies were seen after 60 min (peak threshold increase) and lasted for 3 h. Morphine, the selective -opioid agonist BRL-52656, and SB-235863 each produced a dose-dependent reduction of hyperalgesia after oral administration (Fig. 4). ED₅₀ values in this test for morphine and BRL-52656 were 1.9 mg/kg (95% CL 1.0–3.3) and 0.09 mg/kg (95% CL 0.04–0.15), respectively. Furthermore, morphine and BRL-52656 reached peak levels of activity (100% reversal of hyperalgesia) at doses of 30 mg/kg and 1 mg/kg, respectively. At these maximal doses both compounds induced sedation in 50% of the treated rats. SB-235863 was severalfold less active than morphine (ED₅₀ = 7.5 mg/kg, 95% CL 4.4–13.9), yielding a 77.2 ± 7% reversal of hyperalgesia at the highest dose tested (100 mg/kg). No sedation was observed at any dose of SB-235863 tested. The selective -opioid antagonist NTI (3 mg/kg s.c.) significantly antagonized the effect of SB-235863 (5 mg/kg p.o.), confirming the involvement of the -opioid receptor in mediating its antihyperalgesic activity (Fig. 5A). The selective µ-opioid antagonist -FNA, administered at a dose of 10 mg/kg (s.c.), was unable to antagonize the antihyperalgesic effect of a 5 mg/kg (p.o.) dose of SB-235863, but completely reversed the activity of an equipotent dose (1 mg/kg p.o.) of morphine (Fig. 5B). Similarly, the -opioid antagonist nor-BNI, administered at a dose of 20 mg/kg (s.c.) was effective in antagonizing an equipotent dose (0.16 mg/kg p.o.) of the -opioid agonist BRL-52656, but failed to antagonize the effect of 5 mg/kg (p.o.) of SB-235863 (Fig. 5C). Taken together, these results demonstrate a selective action of SB-235863 in reversing hyperalgesia via interaction with the -opioid receptor.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Opioid agonist reversal of thermal hyperalgesic activity. The antihyperalgesic activity of SB-235863 (), morphine () and BRL-52656 () was determined in the rat carrageenan plantar test. Paw withdrawal latencies to a thermal stimulus were measured 3 h after carrageenan injection. SB-235863, morphine, and BRL-52656 were given orally 90, 60, and 45 min before the test. Each value represents the mean ± S.E.M. for 6 to 12 animals. Details of the experimentation are described under Materials and Methods.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Opiate antagonism of the antihyperalgesic activity of SB-235863. The antihyperalgesic activity of SB-235863 was determined in the rat carrageenan plantar test after administration of the following receptor-selective antagonists: A, the -opiate antagonist NTI (3 mg/kg s.c.); B, the µ-opiate antagonist -FNA (10 mg/kg s.c.); C, the -opiate antagonist nor-BNI (20 mg/kg s.c.). Paw withdrawal latencies were measured by the plantar test 3 h after carrageenan injection. SB-235863 (5 mg/kg p.o.), morphine (1 mg/kg p.o.) and BRL-52656 (10 mg/kg p.o.) were given 90 min, 60 min, and 45 min before the plantar test, respectively. NTI, -FNA, and nor-BNI were given 20 min, 24 h, and 4 h, respectively, before the test. Groups of 6 to 12 animals were used. , P < 0.05 versus the respective groups treated with antagonist alone, as determined using Duncan's multiple range test. Further details of the experimental procedures can be found under Materials and Methods.

Both SB-235863 (100 mg/kg p.o.) and lamotrigine (100 mg/kg p.o.) failed to modulate tactile allodynia response in the rat carrageenan test. A von Frey filament force of 20 ± 3.1g was required to elicit a stimulus response versus a filament strength of only 3.7 ± 0.5g with carrageenan-treated rats. By contrast, morphine (10 mg/kg p.o.) caused a strong reversal of this carrageenan-induced sensitivity to mechanical stimulus to a value of 11 ± 0.4g (P > 0.01), 3 h after injecting carrageenan into the rat hindpaw.

To establish whether the antihyperalgesic activity of SB-235863 measured in the rat carrageenan plantar test involves -opioid receptor sites in the central and/or peripheral nervous system we tested whether central administration of the -opioid antagonist NTI could antagonize the activity of SB-235863 after its systemic administration. After administering a dose of 10 mg/kg (p.o.) of SB-235863 we showed that its antihyperalgesic activity can be blocked by 1 µg of NTI coadministered via intracerebroventricular injection (Table 3). This result demonstrates that central -opioid receptors can participate in this antinociceptive response.

We further tested the ability of SB-235863 to modify activity in the partial sciatic nerve Seltzer ligation model of established neuropathic pain, in which a strong thermal hyperalgesia is present 14 days postsurgery. This hyperalgesic response could be shown by a decrease in the paw withdrawal latency to thermal stimulus on the side ipsilateral to the nerve ligation (Fig. 6A). By contrast, no hyperalgesia was detected on the contralateral side on day 14 (Fig. 6B). Starting from day 15 postsurgery, rats were treated once a day with SB-235863 (10–100 mg/kg p.o.) and thermal paw withdrawal latencies tested on days 15, 17, 21, 24, and 28. SB-235863 administration produced a dose-dependent reversal of the hyperalgesia at days 15, 17, 21, and 28 after surgery. The reversal of hyperalgesia produced by 10, 30, and 100 mg/kg (p.o.) doses of SB-235863 ranged from 50 to 100% at day 15, 70 to 104% at day 17, 61 to 91% at day 21, and 51 to 105% at day 28. All three doses of SB-235863 tested produced a strong reversal of hyperalgesia (84–91%) at day 24. At the maximum dose used (100 mg/kg p.o.) SB-235863 increased the response latency of the ipsilateral foot to presurgery levels at all days during the dosing period at which response was tested (days 15, 17, 21, 24, and 28), compared to rats administered vehicle (Fig. 6A). Furthermore, SB-235863 at all doses tested had no significant effects on the thermal paw withdrawal response of the contralateral foot, apart from a transitory effect observed for the highest dose tested (100 mg/kg) on day 21 (Fig. 6B). SB-235863 (100 mg/kg p.o.) failed to modulate tactile allodynia response in the rat sciatic nerve ligation model. By contrast, lamotrigine (100 mg/kg p.o.) was able to partially reverse the tactile allodynia that develops 3 days after sciatic nerve ligation. A 45% return to the mean response level of sham-operated animals was measured 14 days after surgery (P > 0.01).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Reversal by SB-235863 of thermal hyperalgesia resulting from sciatic nerve ligation. From days 14 to 21 postsurgery rats were perorally administered vehicle () or 100 mg/kg SB-235863 (). A, latency on response(s) of the foot ipsilateral to partial nerve ligation. B, latency on response of the foot contralateral to partial nerve ligation. The results are expressed as mean ± S.E.M. of 11 to 15 rats. Comparisons were made between drug and vehicle groups at each time point tested using analysis of variance with repeated measures, followed by post hoc Dunnett's test (, P < 0.05 compared to vehicle). Further details of the experimental procedures can be found under Materials and Methods.

Effect of SB-235863 on Gastrointestinal Transit and Motor Coordination. The ability of SB-235863 to induce opioid-like side effects was tested using the gastrointestinal transit and motor incoordination/ataxia tests, respectively, of µ- and -opioid side effect potential. At a dose of 4 mg/kg (p.o.) morphine caused a 50% reduction (P > 0.05) in the transit of a charcoal meal in rats during a 5-min observation period. In contrast to this potent activity of morphine, a dose of SB-235863 (70 mg/kg p.o), which was maximally effective in reversing hyperalgesia, was unable to produce a slowing of gastrointestinal transit, in comparison to vehicle-treated animals (Table 4). In the rat accelerated Rotarod test of motor incoordination the -opioid selective agonist BRL-52656 (1 mg/kg p.o.) significantly reduced performance time (Table 5). By contrast, at all doses tested (7–70 mg/kg p.o.), SB-235863 did not alter the performance of trained rats in comparison to vehicle-treated animals.

Evaluation of a Potential Anticonvulsant Activity of SB-235863. The rat maximal electroshock seizure threshold (MEST) test was used to assess whether SB-235863 can induce proconvulsant activity. In the MEST test picrotoxin (2 mg/kg i.p.) lowered the threshold current (CC₅₀) for the induction of tonic hindlimb extension seizures from 49.2 ± 1.0 mA (vehicle) to 40.0 ± 3.0 mA, a reduction of 19% (P > 0.01). Using a range of i.p. doses of SB-235863 that produce increasing antihyperalgesic activity,³ we observed an increase in seizure threshold that was not dose-related. An i.p. dose of SB-235863 of 2 mg/kg elevated the measured CC₅₀ value to 65 ± 4.9 mA (P > 0.01), a 32% increase. SB-235863, administered at i.p. doses of 7.5 and 30 mg/kg, resulted in CC₅₀ test values of 60.8 ± 1.9 (a 24% increase, P > 0.001) and 63 ± 4.7 (a 29% increase, P > 0.01), respectively. This effect was consistent with a modest anticonvulsant activity.

In the rat metrazol infusion test a 30 mg/kg (i.p.) dose of FG7412 reduced the i.v. dose of metrazol required to induce myclonic and forelimb tonic seizures from 23.6 mg/kg to 18.9 mg/kg (P < 0.01), as expected. SB-235863 (2, 7.5, and 30 mg/kg i.p.) did not significantly change the doses of metrazol required to induce myoclonic and forelimb tonic seizures (data not shown).

By comparison, the -agonist SNC80 produced anticonvulsant activity against maximal seizures in both the MEST and metrazol assays. The CC₅₀ value in the MEST test was increased from 56.7 ± 1.6 mA to 80 ± 8.6 mA (p < 0.05) after the administration of 3 mg/kg (i.p.) SNC80 30 min before testing. This same 3 mg/kg (i.p.) dose of SNC80 increased the dose of metrazol required to induce forelimb tonus from 36.9 mg/kg (i.v.) to 52.4 mg/kg (P < 0.05), whereas a dose of 10 mg/kg (i.p.) changed this value to 64.7 mg/kg (P < 0.01). SNC80 (10 mg/kg i.p.) significantly reduced the dose of metrazol required to elicit focal seizures (myoclonus), from 23.8 mg/kg (i.v.) to 18.4 mg/kg (P < 0.01), demonstrating a propensity for the compound to exacerbate absence seizures. Taken together, these data show a differential ability of SB-235863 and SNC80 to elicit a proconvulsant activity.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In vitro competition radioligand binding experiments and cellular signal transduction measurements establish SB-235863 as a morphinan analog with high affinity for the -opioid receptor, selectivity versus other opioid receptors, and full agonist activity. Determinations of opioid receptor affinities in rat brain membranes confirmed that the high measured affinity and selectivity can be observed for both the human and rodent receptors. Extended profiling of the possible interaction of SB-235863 with a wide panel of receptors, ion channels, and enzymes did not reveal any other additional significant interactions at ligand concentrations that are predicted to lead to full occupancy of the -opioid receptor.

SB-235863 has a similar affinity for the -opioid receptor as other agonists, including the peptide enkephalin derivative DPDPE and the octahydroisoquinoline TAN-67, and comparable selectivity toward the other opioid receptors. However, measurements of signal transduction activity showed a higher degree of selectivity of SB-235863 toward the µ- and -opioid receptors than is reflected by its affinity for these sites. These data suggest that although SB-235863 is a full agonist at these other receptors, it is relatively ineffective at initiating a signaling response at ligand concentrations which lead to full activation of the -opioid receptor.

SB-235863 was ineffective at modulating an acute nociceptive signaling response in both the rat tail-flick and rat hot-plate assays, tests of acute spinally mediated and central nociceptive signaling, respectively. By contrast, morphine was effective as an analgesic at low doses in both conditions. The lack of activity of SB-235863 in these tests cannot be explained by low oral exposure, as the compound has high central penetrance. Importantly, an antinociceptive activity of the compound is evident within 1 h postadministration in the rat carrageenan model. We had used this model to examine the activity of SB-235863 toward an evoked pain response, as earlier studies of opioid effects upon nociceptive signaling had shown an effect of nonselective opioid agonists in conditions of evoked pain, such as the hyperalgesia produced in response to inflammation. SB-235863 dose-dependently inhibited the thermal hyperalgesia caused by carrageenan-induced inflammation without affecting the paw edema that carrageenan injection also produces. The effect was shown to be specifically mediated via the -opioid receptor, as a selective -opioid antagonist blocked its effect, whereas selective µ- and -opioid receptor antagonists were without effect.

The antihyperalgesic response of SB-235863 in conditions of inflammatory pain suggested a further role of the -opioid receptor in neuropathic pain conditions in which thermal hyperalgesia is prominent. SB-235863 effectively reversed well established hyperalgesia in a rat surgical model of sciatic nerve ligation in a dose-dependent manner. Importantly, the ability of SB-235863 to alter paw withdrawal latencies in this model was not generalized to the nonoperated side of the animal. Furthermore, there was no reduction in the antihyperalgesic activity during an extended period of repeat dosing, an interesting result which suggests that the tolerance observed with the sustained usage of µ-opiates is unlikely to develop.

These results are consistent with reports of the ability of SNC80 to block capsaicin-induced thermal hypersensitivity in rhesus monkeys, measured as changes in warm-water tail withdrawal latencies (Brandt et al., 2001), and thermal hypersensitivity caused by a Freund's adjuvant-induced inflammatory response (Fraser et al., 2000). The current study extends these results by further describing activity after repeat dosing in a rat surgical model of neuropathic pain. The contribution of thermal hyperalgesia to the symptomatology evident in neuropathic pain conditions has been suggested by several studies and points to the significance of these results with SB-235863. A clinical study of postherpetic neuralgia patients has shown that the presence of thermal hyperalgesia is correlated with the severity of pain response (Rowbotham and Fields, 1996). In a large clinical study of diabetic polyneuropathy patients, Dyck et al. (2000) concluded that thermal hyperalgesia is notably present in patients with symptoms of mild diabetic polyneuropathy, and may be an early diagnostic indicator.

Whereas SB-235863 was effective toward thermal hyperalgesia, no effect on tactile allodynia response was seen in either the carrageenan model or the sciatic nerve ligation model, suggesting a selective role of the -opioid receptor in modifying abnormal pain responses. Gracely et al. (1992) had earlier proposed that the tactile allodynia response requires altered central processing, and differs from thermal pain responses in its ability to be blocked by local anesthetics. Further work by Pertova and Wei (2003) has suggested that descending excitatory pain pathways involving N-methyl-D-aspartate receptors can selectively modify the antiallodynic efficacy of morphine. Conversely, the mechanism of thermal hyperalgesia appears to involve a peripheral afferent signaling component. Rowbotham and Fields (1996) had observed the notable presence of thermal hyperalgesia in postherpetic neuralgia patients presenting with multiple sensory modalities, and suggested that primary afferent signaling was maintained in these cases and significantly contributed to pain signaling. In support of this observation, Zhou et al. (1998) had further shown that hyperalgesia elicited by injecting Freund's adjuvant into the hindpaw of a rat can be blocked by peripheral (intraplantar) administration of DPDPE. On the basis of this evidence we explored whether there could be a selective action of -opioid agonists at peripheral receptors that does not require participation of a central sensitization response. However, in this study our demonstration that centrally administered NTI can block the antihyperalgesic activity of systemically available SB-235863, while not excluding a participation of peripheral receptors, shows that central -opioid receptors can clearly modulate the activity of a -opioid agonist. Recent work by Cahill et al. (2003) showing up-regulation of the -opioid receptor in the spinal cord after the induction of inflammatory pain by intraplantar injection of Freund's adjuvant, has pointed to a possible role for the -opioid receptor in central pain signal processing. Furthermore, Kalra et al. (2001) have shown that injection of naltrindole into the rostral ventral medulla selectively blocks antihyperalgesia produced by high-frequency transcutaneous electrical nerve stimulation, and proposed -opioid activation of a descending inhibitory pathway.

Previous work by Danzebrink et al. (1995) has suggested a role of spinal -opioid receptors in regulating colonic motility. However, our results showing the lack of activity of SB-235863 in a gastrointestinal motility assay, together with our demonstration of the compound's ability to regulate central -opioid receptors, would suggest that the previously reported visceral activities of -opioid ligands may not be mediated via the -opioid receptor.

From previous studies with the -opioid agonist SNC80 showing a dose-dependent proconvulsant activity (Bilsky et al., 1995; Hong et al., 1998) it has been suggested that this is a feature of -opioid receptor activation. We have similarly observed this activity of SNC80 in vivo, but were unable to show a similar effect of SB-23863 in these same tests. The data suggest that this activity may be a feature of the diarylpiperidine structural class, and not of -opioid agonists per se.

As peripheral -opioid agonists have also been shown to have an antihyperalgesic property (Zhou et al., 1998), it was important to demonstrate that activities associated with -opioid receptor activation, such as the motor incoordination effects demonstrated previously for -opioid agonists, are not elicited by SB-235863. The antinociceptive activity of SB-235863 was not blocked by a selective -opioid agonist, showing that SB-235863 was unlikely to activate the -opioid receptor at effective doses in vivo. Importantly, SB-235863 was inactive in a test of motor incoordination that otherwise revealed strong -opioid agonist effects.

In conclusion, in this paper we have presented the in vitro and in vivo activity profile of SB-235863, a high-affinity and selective -opioid agonist. These results demonstrate a clear in vivo efficacy of this -opioid agonist in subchronic and chronic models of pain, but not in models of acute nociception. SB-235863 effectively and selectively reverses thermal hyperalgesia, yet is unable to elicit several of the side effects typically associated with opioid usage. The data obtained using SB-235863 shed new insight on the properties of the -opioid receptor, and describe a new class of compound with potential therapeutic utility in treating pain disorders mediated by the -opioid receptor.

	Acknowledgements

 
The expert technical assistance of Loredana Antonini, Maurizio Bassi, Concetta Belpasso, Mauro Diguigni, Calogero DiLiberto, Luigi Guibado, Elisa Tancorre, Sergio Oneta, and Armando Voluntieri is gratefully acknowledged.

	Footnotes

 
DOI: 10.1124/jpet.103.055590.

ABBREVIATIONS: DPDPE, [D-Pen²,D-Pen⁵]-enkephalin; TAN-67, (4aS*,12aR*)-4a-(3-hydrophenyl)-2-methyl-1,2,3,4,4a,5,12,12a-octahydropyrido(3,4b) acridine; SB-235863, [8R-(4bS*,8a,8a,12b)]7,10-dimethyl-1-methoxy-11-(2-ethylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochloride; DAMGO, [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin; DADLE, [D-Ala²,D-Leu⁵]-enkephalin; nor-BNI, nor-binaltorphimine dihydrochloride; NTI, naltrindole; -FNA, -funaltrexamine; IBMX, 3-isobutyl-1-methylxanthine; CHO, Chinese hamster ovary; HEK, human embryonic kidney; hMOR, human µ-opioid receptor; hDOR, human -opioid receptor; hKOR, human -opioid receptor; MRE, multiple response element; CRE, cAMP response element; FSK, forskolin; MEST, maximal electroshock seizure threshold; SNC80, (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; U69593 [GenBank] , 5,7,8-(–)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-phenyl-benzeneacetamide; BRL-52656, (2S)-1-[(4-trifluoromethylphenyl)acetyl]-2-[(1-pyrrolidinyl)methyl]piperidine.

¹ The synthesis of SB-235863 was performed as follows. In brief, 7.8-dihydrocodeinone hydrochloride (25 g, 74.4 mol) and iso-butyl-2-phenylhydrazono-3-oxobutyrate (58.5 g, 223.2 mol) were dissolved in a mixture of glacial acetic acid (150 ml) and sodium acetate (18.3 g, 223.2 mol) and warmed to 60°C. Zinc dust was added (29.2 g, 446.2 mol) under a nitrogen atmosphere, the mixture refluxed for 2 h, and then cooled to room temperature. The resulting salts were eliminated by decantation, washed with acetic acid, adjusted to pH 8.0 with NH₄OH, and extracted several times with CH₂CL₂. The organic phase was dried, evaporated to dryness in vacuo, and the resulting residue purified by flash chromatography using silica gel (230–400 mesh) and AcOEt/MeOH/NH₄OH (90:10:0.5) as eluant. The product was taken up in EtOH and the solution adjusted to an acidic pH with HCl/Et₂O. The resulting precipitate was filtered, washed, and dried, yielding 12 g of SB-235863. The melting point (278–279°C), of –512.5 (C = 0.1, MeOH), NMR, and IR spectra were consistent with the proposed structure.

² Intravenous infusion of SB-235863 (1.9 µmol/kg/h) for 8 h led to a rat brain/blood ratio at a steady state of 2.1 ± 0.7:1 and a brain concentration of approximately 1.5 µM.

³ SB-235863 doses of 3, 10, and 30 mg/kg (i.p.) inhibited hyperalgesia response in the rat carrageenan plantar test by 15.9 ± 8.2%, 41.7 ± 13.0%*, and 74.4 ± 12.1%* (*P > 0.001), respectively.

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

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