Introduction

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Opioid receptor agonists are classified by their activity at three opioid receptor subtypes known as µ (Wang et al., 1994),  (Mansson et al., 1994), and  (Knapp et al., 1994). Members of this class of compounds have been demonstrated to have antitussive effects. For example, compounds such as morphine and codeine, considered to be the most potent and effective antitussive drugs currently on the market, are categorized generally as agonists at the µ-opioid receptor (Eddy et al., 1969; Karlsson et al., 1990). In addition, -opioid-selective agonists have been shown to inhibit cough in laboratory animals (Kamei et al., 1990).

Although the involvement of -opioid receptors in cough has been described (Kamei et al., 1992; Dondio et al., 1997), some of the data are conflicting. Some reports indicate that -receptor antagonists produce an antitussive effect against capsaicin-induced cough in rats and mice (Kamei et al., 1993c, 1994b). Furthermore, evidence has been presented that -opioid receptor agonists can either reduce (Kamei et al., 1991) or enhance (Kamei et al., 1993d) the antitussive effect of µ-receptor agonists [e.g., D-Ala²-Me-Phe⁴-Gly-ol⁵-enkephalin (DAMGO), morphine]. The reason(s) for these discrepancies is not known but may be linked to the limited selectivity of the compounds that were investigated.

The goal of this study was to clarify the influence of -receptors in a cough model. Specifically, we investigated the ability of the novel -opioid receptor agonist, SB 227122 ([10R,4bS-(4b,9a)]-7-diisopropylaminocarbonyl-8,14-dimethyl-4- hydroxy-3-methoxy-4b,5,9,9a,10,11-hexahydro-(6H)-[2,3-h]-pyrrolo[10,4-b]iminoethenophenanthrene; Fig. 1), to inhibit citric acid-induced cough in the guinea pig. SB 227122 is a nonpeptide agonist with high affinity for the human -receptor (K_i = 6.9 nM from binding studies) and K_i > 2 µM versus µ- or -receptors (Petrillo et al., 1998). In addition, we attempted to confirm the mechanism of the antitussive activity of SB 227122 by using subtype-selective opioid receptor antagonists.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Structure of SB 227122 ([10R,4bS-(4b,9a)]-7-diisopropylaminocarbonyl-8,14-dimethyl-4-hydroxy-3-methoxy-4b,5,9,9a,10,11-hexahydro-(6H)-[2,3-h]-pyrrolo[10,4-b]iminoethenophenanthrene).

	Materials and Methods

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Cell Lines. Stable expression of human -opioid receptors (h-DORs) and µ-opioid receptors (h-MORs) in Chinese hamster ovary (CHO) cells and human -opioid receptors (h-KORs) in human embryonic kidney 293 (HEK-293) cells were prepared in-house. The h-DOR was cloned by screening primers based on the public sequence (GenBank accession no. U10504); reverse-transcription polymerase chain reaction (RT-PCR) was done using whole human brain poly(A)⁺ RNA as the template. The h-MOR was cloned by screening a whole brain human cDNA library with a coding probe from a truncated h-KOR cDNA. A truncated version of the h-KOR was cloned by screening a whole brain cDNA library using the ORL-1 receptor as the probe. The missing 5'-end was cloned by RT-PCR using whole brain poly(A)⁺ RNA as the template. The full-length cDNA for the three opioid receptors was inserted into Asp718 and HindIII sites of pCDN mammalian expression vector (Aiyar et al., 1994). CHO or HEK-293 cells were transfected by electroporation then plated for isolation of single clones. CHO-transfected cells were grown in suspension culture in serum-free medium (1017 S03; Proprietary-SmithKline Beecham Pharmaceuticals, King of Prussia, PA) in the presence of 0.05% pluronic acid (F68; GIBCO BRL, Paisley, Scotland), maintained at 37°C, and gassed with 5% CO₂. Selection for CHO transfectants was performed by growth in the absence of nucleotides. The maximum cell density for these cell lines was 4 × 10⁶ cells/ml. From binding studies it was determined that transfected CHO cell lines express h-DOR and h-MOR at a density of 11.5 and 6.9 pmol/mg of protein, respectively.

Cells Membrane Preparation. Membranes were prepared by hypotonic lysis according to previously described methods with minor modification (Scheideler and Zukin, 1990). Briefly, cells were harvested in PBS (1 × 10⁶ cells/ml) and collected by centrifugation (800g for 5 min). The pellets were resuspended in the same volume of ice-cold 10 mM potassium phosphate buffer, pH 7.2 (buffer A), and centrifuged at 40,000g for 10 min. The cells were hypo-osmotically lysed by resuspension in the same volume of buffer A for 20 min in ice, centrifuged at 800g (5 min), and the supernatant saved. The resulting pellets were resuspended in buffer A, and the last step was repeated two additional times, saving the supernatants each time. Supernatants derived from the three low-speed centrifugations were pooled and centrifuged at the high speed. Pellets were resuspended in buffer A containing 0.32 M sucrose and 5 mM EDTA (buffer B) to wash and concentrate the membranes. The final pellets were resuspended in buffer B at a final concentration of 1 to 2 mg of protein/ml (ca. 40 × 10⁶ cells/ml) and stored at 80°C. Protein was determined with the protein assay kit from Sigma Chemical Co. (Milan, Italy).

Radioligand Binding Assays. [³H]D-Ala²-D-Leu⁵-enkephalin ([³H]DADLE; K_d = 1.2 nM), [³H]DAMGO (K_d = 1.2 nM), and [³H]U69593 (K_d = 1.6 nM) were used to label -, µ-, and -binding sites, respectively. Binding experiments were performed in triplicate at a final protein concentration of 10 µg/ml in buffer A and a radiolabeled ligand concentration of 0.4 to 0.5 nM. The nonspecific binding was determined in the presence of 10 µM naloxone, and was less than 1% of the radioligand added under these assay conditions. Samples (final volume of 2 ml) were incubated for 60 min at 37, 30, and 25°C for -, µ-, and -binding assays, respectively. The reaction was terminated by rapid filtration through Whatman GF/B filters and two washes with cold assay buffer A (4 ml) using a M48 Brandel Cell Harvester (Biomedical Research and Development Laboratories, Inc., Gaithersburg, MD). Filters used for [³H]U69593 binding were presoaked in buffer A containing 0.05% polyethylenimine. Radioactivity on the filters was measured by liquid scintillation counting with a Camberra Packard 2500TR beta counter (Milan, Italy).

cAMP Accumulation. Whole CHO cells, expressing the human -receptor, were incubated with vehicle or test compounds in 200 mM Krebs-Ringer, buffered with HEPES, containing 125 mM NaCl, 5 mM KCl, 0.4 mM KH₂PO₄, 1.2 mM MgSO₄, 1.2 mM CaCl₂, 25 mM NaHCO₃, 12 mM glucose, and 1 mM isobutylmethylxanthine. Cells were treated with 10 mM forskolin to stimulate cAMP synthesis, and cAMP content was determined after 10 min using a double antibody ¹²⁵I-cAMP radioimmunoassay (RPA 509; Amersham, Milan, Italy).

Cough Model. All animal studies conform to Animal Care and Use Committee protocols filed at SmithKline Beecham Pharmaceuticals (King of Prussia, PA).

Drugs. [³H]DADLE [specific activity (S.A.) 55.3 µCi/nmol] and [³H]DAMGO (S.A. 54.5 µCi/nmol) were obtained from New England Nuclear (Brusselles, Belgium). [³H]U-69593 (55 µCi/nmol) was supplied by Amersham (Milan, Italy). Codeine and hydrocodone were purchased from Mallinckrodt, Inc. (St. Louis, MO) and Salars (Como, Italy), respectively. BRL 52974 [4-(pyrrolidin-1-yl)methyl-5-5-(3,4-dichlorophenyl)acetyl- 4,5,6,7-tetrahydroimidazo[4,5,-c]pyridine]; SB 227122 ([10R,4bS-(4b,9a)]-7-diisopropylaminocarbonyl-8,14-dimethyl-4-hydroxy-3-me-thoxy-4b,5,9,9a,10,11-hexahydro-(6H)-[2,3-h]-pyrrolo [10,4-b]iminoethenophenanthrene); -FNA [(E)-4-[[(5,6)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6yl]amino]-4-oxo-2-butenoic acid methyl ester hydrochloride]; Nor-BNI dihydrochloride tetrahydrate; naltrindole hydrochloride (8R-(4bS*,8,8a,14b)]-7-(cyclopropyl-methyl)-5,6,7,8,14,14b-hexahydro-4,8-methanobenzofuro[2,3-a]pyrido[4,3-b]carbazole-1,8a(9H)-diol hydrochloride); and SB 244525([8R-(4bS*,8,8a,12b)]-7-allyl-11-isobutylcarbonyl-1-methoxy-10-methyl-5,6,7,8,12,12b-hexahydro-(9H)-4,8-methano-benzofuro[3,2-e]pyrrolo[2,3-g]isoquin-olin-8a-ol hydrochloride) were synthesized by the Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals. Naloxone (4,5-epoxy-3,14-dihydroxy-17-(2-propenyl)morphinan-6-one hydrochloride); dextromethorphan (d-3-methoxy-N-methylmorphinan); and rimcazole (cis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]-9H-carbazole dihydrochloride) were purchased from Sigma (St. Louis, MO). Citric acid monohydrate was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). All chemicals were dissolved in 0.9% saline for i.m., s.c., i.p., and aerosol drug administration except for dextromethorphan, which was solubilized in 4% acetic acid, Nor-BNI, which was dissolved in 0.1 N HCl, and SB 227122 and BRL 52974, which were dissolved in 0.1 N CH₃SO₃H for parenteral administration.

Statistics. Data were analyzed using StatView statistical analysis software (Abacus Concepts, Inc., Berkeley, CA). ANOVA was performed for each experiment, followed by a Fisher's protected least-squares difference (PLSD) test. P values are provided under Results, with a value less than .05 regarded as significant. Results are presented as means or means ± S.E.

	Results

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In Vitro Studies

Receptor Binding. The binding affinity of agonists and antagonists used in cough studies were determined in assays using the h-DOR, h-MOR, and h-KOR cell lines (Table 1). The -receptor agonist SB 227122 demonstrated a 294- and >725-fold greater affinity for the -receptor than the µ- or -receptor, respectively. The -receptor antagonist SB 244525 also proved to be highly selective for the -receptor with a µ/- and /-ratio from binding studies (K_i values) of 294 and 170, respectively. The selectivities of the other opioid receptor agonists (codeine, hydrocodone, and BRL 52974) and antagonists (-FNA, Nor-BNI, and naloxone) as well as the poor affinities of -receptor agonist (dextrome-thorphan) and antagonist (rimcazole) for the opioid receptors were also confirmed (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Agonist and antagonist binding to human -, -, and µ-opioid receptors

Functional Experiments. The functional in vitro activities of SB 227122 and SB 244525 were assessed by determining their effects on cAMP levels in CHO cells expressing the human cloned -opioid receptor. SB 227122 (0.1-1000 nM) displayed full agonist activity for decreasing cAMP (IC₅₀ = 12 nM); this effect was completely antagonized by naltrindole (100 nM). SB 244525 alone, at concentrations up to 1 µM, lacked any significant effect on cAMP levels. However, SB 244525 (0.1-1000 nM) potently antagonized the inhibitory effects of SB 227122 or ()TAN-67, a nonpeptide -opioid receptor agonist, on cAMP levels (data not shown).

In Vivo Studies

Antitussive Activity of Receptor Subtype-Selective Agonists. The antitussive activities of four subtype-selective opioid receptor agonists, codeine, hydrocodone, BRL 52974, and SB 227122, were determined in the guinea pig citric acid-induced cough model. Cough incidence in vehicle-treated animals ranged from 10 to 18 coughs, averaging 15 ± 1 coughs in the 13-min monitoring period (n = 8). All four opioid agonists produced dose-related inhibition of the cough response resulting in at least 60% maximum inhibition (Fig. 2). The ED₅₀ values of the µ-receptor agonists, codeine and hydrocodone, were determined to be 5.2 mg/kg (n = 5) and 5.1 mg/kg (n = 5), respectively, when administered i.p. 30 min before citric acid challenge. The -receptor agonist BRL 52974 and the -receptor agonist SB 227122 had ED₅₀ values of 5.3 mg/kg (s.c.; 30-min pretreatment) and 7.3 mg/kg (i.p.; 20-min pretreatment), respectively (n = 4-8; Fig. 2).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Inhibition of citric acid-induced cough in guinea pigs after the administration of selective µ- (codeine and hydrocodone), - (BRL 52974), and - (SB 227122) opioid receptor agonists. Codeine and hydrocodone were administered i.p. 30 min before cough challenge, whereas SB 227122 and BRL 52974 were given i.p. 20 min and s.c. 30 min before treatment, respectively. The results are expressed as percentage of inhibition of the citric acid-induced cough response as compared with vehicle-treated animals; n = 4-8. Standard error bars are omitted for clarity.

Effect of Naloxone on Opioid Antitussive Activity. Naloxone, a nonselective opioid receptor antagonist, was administered (3 mg/kg, i.m.) after vehicle; codeine or the -receptor agonist SB 227122 were administered 10 min before cough challenge. Higher doses of naloxone produced central nervous system effects, such as ataxia and sedation, as well as antitussive activity (data not shown). Naloxone (3 mg/kg, i.m.) alone was without influence on the incidence of cough after citric acid challenge: control = 17 ± 2.5 coughs (n = 4); +naloxone = 17 ± 1.2 coughs (n = 4). Guinea pigs treated with codeine (10 mg/kg, i.p.) coughed an average of 8.6 ± 1.4 times (n = 5), which represented 49% inhibition of the cough reflex (P < .05, ANOVA). In the group of codeine-treated animals that received naloxone (3 mg/kg, i.m.), the incidence of cough was 14 ± 2.8 (P < .05 compared with the group receiving codeine alone, ANOVA; n = 4; Fig. 3a), demonstrating an attenuation of the antitussive effect of codeine by naloxone.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   The effect of the opioid antagonist naloxone (3 mg/kg, i.m.; 10-min pretreatment) on the antitussive activity of codeine (10 mg/kg, i.p.; 30-min pretreatment; panel a) or SB 227122 (5 or 10 mg/kg, i.p.; 20-min pretreatment; panel b). The results are expressed as the total number of coughs induced by a 1-min 0.4 M citric acid aerosol during the 13-min observation period; n = 4-5. *P < .05, ANOVA, Fisher's PLSD, as compared with vehicle-treated animals; and P < .05, ANOVA, Fisher's PLSD, as compared with opioid-agonist-treated animals.

Effect of Selective Opioid Receptor Antagonists on the Antitussive Activity of Opioid Agonists. To test the activity of a selective µ-receptor antagonist, -FNA (20 mg/kg, s.c.) or vehicle was administered 24 h before the selective µ-receptor agonist hydrocodone (12 mg/kg, i.p.; 30-min pretreatment) (Kamei et al., 1993b). -FNA alone had no effect on citric acid-induced cough occurrences (control = 16.3 ± 0.9 coughs, n = 8; +-FNA = 15 ± 1.9 coughs, n = 4). However, -FNA substantially reduced the ability of hydrocodone (12 mg/kg, i.p.) to inhibit the citric acid-induced cough reflex (hydrocodone = 4.8 ± 1.4 coughs, n = 8; hydrocodone + -FNA = 12.8 ± 1.8 coughs, n = 8; P < .05, ANOVA; Fig. 4a). The cough incidence in animals treated with both hydrocodone and -FNA was not significantly different from vehicle-treated animals (P > .05, ANOVA; Fig. 4a).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   a, the effects of selective opioid µ-antagonist (-FNA; 20 mg/kg, s.c.; 24-h pretreatment) on the antitussive effects of hydrocodone (12 mg/kg, i.p.; 30-min pretreatment), a selective µ-receptor agonist; b, the effects of Nor-BNI, a selective -antagonist (20 mg/kg, s.c.; 4-h pretreatment), on the antitussive effects of BRL 52974, a selective -agonist (10 mg/kg, s.c.; 30-min pretreatment); c, the effects of a combination of -FNA (µ) and Nor-BNI () on the antitussive effects of SB 227122 (20 mg/kg, i.p.; 20-min pretreatment); d, the effects of selective -opioid receptor antagonist SB 244525 (10 mg/kg, i.p.; 25-min pretreatment) on the antitussive effects of SB 227122 (20 mg/kg, i.p.; 20-min pretreatment). The results are expressed as the total number of coughs induced by a 1-min 0.4 M citric acid aerosol during the 13-min observation period; n = 4-5. *P < .05, ANOVA, Fisher's PLSD, as compared with vehicle-treated animals; and P < .05, ANOVA, Fisher's PLSD, as compared with opioid agonist-treated animals.

Effect of Selective -Antagonist Rimcazole on Dextromethorphan or SB 227122. The -receptor antagonist rimcazole (3 mg/kg, i.p.) alone had no effect on citric acid-induced cough: control = 16 ± 1.0 coughs, n = 4; +rimcazole = 16.3 ± 1.1, n = 4; P > .05, ANOVA (Table 2). The -receptor agonist dextromethorphan inhibited citric acid-induced cough at doses of 10 and 30 mg/kg, i.p. (12 ± 2.4 coughs, n = 5 and 6 ± 1.8 coughs, n = 7, respectively; P < .05, ANOVA at the 30 mg/kg dose only). The antitussive activity of dextromethorphan (30 mg/kg, i.p.) was significantly inhibited by rimcazole (3 mg/kg, i.p.): cough incidence = 6 ± 1.8 coughs (n = 7) in dextromethorphan-treated animals versus 15 ± 2.2 coughs (n = 5) in the animals treated with rimcazole and dextromethorphan (P < .05, ANOVA). In contrast, rimcazole was without effect on SB 227122 (20 mg/kg, i.p.)-induced inhibition of coughs: +SB 227122 = 4.6 ± 1.2 coughs, n = 8; +SB 227122 and rimcazole = 6.2 ± 3.0 coughs, n = 4; P > .05, ANOVA; Table 2).

	Discussion

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The major findings of this study, using the citric acid-induced cough model in guinea pigs, are: 1) the four subtype-selective opioid receptor agonists, codeine (µ), hydrocodone (µ), BRL 52974 (), and SB 227122 (), demonstrated dose-dependent antitussive activity; 2) the opioid antagonist naloxone inhibited the antitussive activity of codeine (µ-selective receptor agonist) or SB 227122 (-selective receptor agonist); and 3) the antitussive activity of the -opioid receptor agonist SB 227122 was not affected by treatment with a combination of µ-receptor- and -receptor-selective antagonists, or rimcazole, a -receptor-selective antagonist but was inhibited by SB 244525, a -receptor-selective antagonist (Petrillo et al., 1998). Collectively, these data indicate that the antitussive activity of the -receptor agonist SB 227122 is due to stimulation of this specific opioid receptor.

Selective agonists for each of three opioid receptors, i.e., codeine and hydrocodone for the µ-receptor, BRL 52974 for the -receptor, and SB 227122 for the -receptor (Giardina et al., 1995), demonstrated dose-dependent antitussive activity with a range of ED₅₀ values from 5.1 to 7.3 mg/kg (i.p.) across the group of agonists. In addition to similar ED₅₀ values, all four opioid agonists produced similar degrees of inhibition (60%) at the maximum dose tested.

SB 227122 has been identified previously as a selective agonist at the -opioid receptor with a K_i of 6.9 nM in a binding assay using human -receptor; selectivity is indicated by the µ/- and µ/-ratio from binding studies of 294 and >725, respectively (Petrillo et al., 1998; Table 1). This study represents the initial comprehensive description of the binding and functional activity profiles of SB 227122 and SB 244525. The results clearly indicate that SB 227122 is a potent and selective -opioid receptor agonist, whereas SB 244525 is a potent and selective -opioid receptor antagonist.

To determine whether the antitussive activity of SB 227122 was attributable to activity at an opioid receptor, it was tested in the presence of naloxone, a nonselective opioid receptor antagonist. Codeine was used as a standard opioid receptor agonist, whose antitussive activity has been shown previously to be inhibited by naloxone (Karlsson et al., 1990). Naloxone has a K_i of 33.8, 2.4, and 2.5 nM for the -, µ-, and -human opioid receptors, respectively (Table 1). The inhibition by naloxone of the antitussive properties of both codeine and SB 227122 strongly suggests that the latter exerts its antitussive activity via activation of opioid receptors.

Receptor subtype-selective ligands were used to further explore the mechanism of action of SB 227122. -FNA has demonstrated µ-receptor-selective antagonist activity when administered 24 h before challenge with a µ-receptor agonist (Hayes et al., 1985). -FNA alone had no effect on citric acid-induced cough, but it substantially reduced the antitussive activity of hydrocodone, a µ-receptor selective agonist. Hydrocodone has an affinity (K_i) for the human µ-receptor of 36.4 nM, which is 28 and 20 times greater than its affinity for the -opioid and -opioid receptors, respectively (Maguire et al., 1993). BRL 52974 has a K_i of 0.48 nM against h-KOR and K_i values of greater than 18,000 nM for both the µ- and -opioid receptors (Table 1). Nor-BNI has been identified as a -opioid-selective receptor antagonist and this was confirmed in our study, in which Nor-BNI inhibited the antitussive effects of BRL 52974, the -receptor selective agonist (Portoghese et al., 1992). Pretreatment with a combination of -FNA (µ) and Nor-BNI (), at doses that inhibited their corresponding selective agonists, had no effect on the antitussive activity of SB 227122. This finding suggests that the antitussive effect of the compound in this model is independent of activity at the µ- or -opioid receptors.

SB 244525 is a selective -receptor antagonist, which has a K_i of 4.8 nM for the human -receptor, a K_i of 1223 nM for the human µ-receptor, and a K_i of 810 nM for the human -receptor, and is without activity in the cAMP functional assay, suggesting that it has no -agonist activity (Petrillo et al., 1998). SB 244525 significantly inhibited the antitussive effect of SB 227122. This finding provides further evidence that the inhibition of cough associated with SB 227122 is mediated through the -receptor. The antagonist SB 244525 alone did not inhibit cough, which correlates with its lack of functional agonist activity at the -receptor. Moreover, SB 244525 (10 mg/kg) did not inhibit the antitussive effects of either the µ-opioid agonist hydrocodone (12 mg/kg) or the -opioid agonist BRL 52974 (10 mg/kg) (data not shown). It has previously been reported that a purported -receptor antagonist, naltrindole, demonstrated dose-dependent antitussive activity in mice and rats (Kamei et al., 1993c). This action was attributed to both direct -receptor antagonism and activation of -opioid receptors (Kamei et al., 1993c). In this study, we show significant binding of naltrindole to the µ- and -receptors (Table 1), albeit at least 20-fold less potent than to -receptors; these data are consistent with the results of a previous study (Rogers et al., 1990). Antitussive activity of naltrindole was confirmed in our laboratory (ED₅₀ = 8.2 mg/kg, i.p.), and was found to be inhibited by pretreatment with the combination or µ- (-FNA) and - (Nor-BNI) receptor antagonists (data not shown). This suggests that the antitussive influence of naltrindole is not due to -receptor antagonism per se but may be mediated via activation of µ- and -receptors.

In addition to opioid receptor-related antitussive activity of SB 227122, we investigated the possible involvement of the -receptors. Dextromethorphan is a commonly used nonnarcotic antitussive, which has a binding K_i of 15 nM at the -receptor (Chen et al., 1991). We confirmed dose-related antitussive activity of dextromethorphan in this guinea pig model of cough (Callaway et al., 1991; Braga et al., 1994) and its reduction by treatment with rimcazole, a -receptor antagonist (Kamei et al., 1993a). Rimcazole, at a dose which significantly inhibited the antitussive activity of dextromethorphan, did not affect the activity of SB 227122. Thus, although structurally both SB 227122 and dextromethorphan are classified as morphinans (although with opposite optical sign rotation), we have excluded the participation of the -receptor in the antitussive activity of SB 227122.

The selectivities of the compounds in this study were determined in human cloned receptors, whereas the antitussive studies were conducted in guinea pigs. It is possible that species differences exist in the affinities of compounds for guinea pig and human opioid receptors. To the best of our knowledge, the guinea pig - and µ-opioid receptors have not been cloned. However, it has been determined that homology among the human, rat, and mouse -opioid receptors is 93%, with 100% homology within the transmembrane regions (Quock et al., 1999). In addition, no binding discrepancy was detected in a direct comparison of human and mouse -opioid receptors (Simonin et al., 1994). In binding studies with several opioid ligands in guinea pig brain tissue and human cloned receptor, we have determined that the correlation coefficients for the affinities of the compounds for the guinea pig and human receptors were 0.9861, 0.9926, and 0.9274 for -, µ-, and -receptors, respectively (data not shown). Collectively, the data would suggest that no significant species differences are likely to exist in the affinities of SB 227122 and SB 244525 for human and guinea pig opioid receptors.

µ-receptor agonists have been recognized for some time to have significant side effects including respiratory depression, constipation, and physical dependence (Zenz and Willweber-Strumpf, 1993). Undesirable side effects with -agonists include diuresis, dysphoria, and sedation (Dionne et al., 1991; Coltro and Clarke, 1995). There is some evidence that the -opioid agonists may be less likely to produce some of the untoward effects associated with either the µ- or -agonists, suggesting that members of the former class of compounds may have a greater therapeutic index as potential antitussives (Rapaka and Porreca, 1991). We have evaluated several other -opioid agonists in the same structural class as SB 227122 and demonstrated antitussive activity of these compounds in the guinea pig model with ED₅₀ values ranging from 5 to 15 mg/kg, i.p. (data not shown).

In summary, we have demonstrated that the selective -opioid receptor agonist SB 227122 inhibits, in a dose-dependent manner, citric acid-induced cough in the guinea pig. The antitussive activity of SB 227122 is due to selective activation of the -subtype opioid receptor. Members of this class of compounds may have potential as novel antitussive drugs.