The Abilities of Specific x -Opioid Agonists, U-50,488H and U-62,066E, to Cause Antitussive Tolerance Were Lower than That of Morphine

Junzo Kamei, Hiroaki Tanihara and Yutaka Kasuya

Department of Pharmacology, School of Pharmacy, Hoshi University, 4-41, Ebara 2-chome, Shinagawa-ku, Tokyo 142, Japan

Received April 23, 1991 Accepted June 10, 1991

ABSTRACT-We examined whether the chronic administration of selective x-opioid agonists could produce antitussive tolerance, in a comparison with the ,u-opioid mor phine. A certain degree of tolerance to the antitussive effects of morphine appeared in rats treated chronically with this drug. However, chronic administration of U 50,488H and U-62,066E, highly selective agonists for the x-opioid receptor, does not result in the development of tolerance to their respective antitussive effects. These results suggest that the ability of x-opioid agonists to cause tolerance to their respec tive antitussive effects was lower than that of a ,u-opioid agonist.

There is evidence that specific x-opioid of U-50,488H. To ascertain the specificity of agonists, such as U-50,488H and U-62,066E, the involvement of serotonin receptors, the not only have potent analgesic effects (1) but binding of [3H]serotonin to receptors on the also potent antitussive effects (2). One of the brainstem membranes of rats chronically potential disadvantages to the analgesic use of treated with U-62,066E or morphine were also U-50,488H and U-62,066E is that these drugs examined. induce tolerance (1, 3). Thus, it is possible Male Sprague-Dawley rats (Tokyo Animal that chronic administration of x-opioid ago Laboratory, Inc., Tokyo, Japan), weighing nists causes the development of tolerance to about 250g, were used. Rats were injected their respective antitussive actions. Therefore, with U-50,488H (10 mg/kg, i.p.), U-62,066E we examined whether the chronic administra (10 mg/kg, i.p.), morphine (1 mg/kg, i.p.) or tion of selective x-opioid agonists, U-50,488H vehicle (1 ml/kg) twice a day (10 : 00 and 17 : and U-62,066E, could produce antitussive 00), for four days. On day five, tolerance to tolerance, in a comparison with the u-opioid the antitussive action of these drugs was deter morphine. mined by the previously described method (6). Previously, we have reported that tolerance In brief, animals were exposed to a nebulized to the antitussive effects of opiates is thought solution of capsaicin (30 ,umol) under identical to result from the development of serotonin conditions, using a body plethysmograph. The receptors subsensitivity (4). Furthermore, Ho number of coughs produced per 5-min period and Takemori (5) suggested that the reduction of exposure to capsaicin was counted. The rats of the affinity of serotonin receptors in the were exposed to capsaicin 30 min before the spinal cord may be involved in the develop injection of drugs to determine the frequency ment of tolerance to the antinociceptive action of control coughs; then they were exposed to capsaicin 15 min after i.p. injection of drugs or (10 mg/kg, i.p.) also significantly increased vehicle. The number of coughs produced 15 the percent inhibition of the number of coughs min after the injection of drugs was compared in rats treated chronically with vehicle (vehi with the number produced by vehicle-injected cle, 4.8 ± 2.9%; U-50,488H, 73.2 ± 5.0%; U control rats. The antitussive effect was ex 62,066E, 80.8 ± 5.7%). A certain degree of pressed in terms of the percentage reduction tolerance to the antitussive effects of mor in number of coughs relative to the number of phine appeared in rats treated chronically with control coughs. morphine. Thus, the antitussive response to The serotonin binding assay was performed morphine (1.0 mg/kg, i.p.) in chronically using the brainstem (medulla oblongata and morphine-treated rats was only half that pons) in 50 mM Tris buffer containing 10 ,uM observed in rats treated chronically with vehi pargyline and 4 mM CaC12 (pH 7.4), according cle (Table 1). As can be seen from Table 1, to the method used by Bennet and Snyder (7). however, the percent inhibition of the number Samples of membrane preparations (60,ug of coughs in chronically U-50,488H or U protein/tube) were put in test tubes. 62,066E-treated rats in response to a challenge [3H]Serotonin was added at concentrations dose of morphine was similar to that observed over the range of 1.5 to 50 nM, and 10 ,uM in chronically vehicle-treated rats. In chroni unlabelled serotonin was used to determine cally U-50,488H-treated rats, the antitussive nonspecific binding. Incubations were per action induced by U-50,488H was similar to formed in a volume of 1 ml at 22°C for 30 that observed in chronically vehicle-treated min. The incubation was terminated by adding rats. There was no significant difference in the of 5 ml of ice-cold Tris buffer and rapid filtra U-50,488H-induced antitussive effect between tion through Whatman GF/C filters. The fil chronically vehicle-treated and chronically ters were washed twice with 5 ml Tris buffer morphine or U-62,066E-treated rats, respec and transferred to scintillation vials and then tively. The antitussive effects of U-62,066E in counted in a Packard Tri-Carb liquid scintilla rats chronically treated with U-62,066E were tion spectrometer in 5 ml aqueous scintillant similar to those in chronically vehicle-treated (Echonoflow, New England Nuclear). rats. Furthermore, when U-62,066E was given U-50,488H and U-62,066E were generously to chronically morphine or U-50,488H-treated supplied by Upjohn Company, Kalamazoo, rats, the antitussive effects of U-62,066E were MI, U.S.A. Morphine hydrochloride was also not significantly different from the anti purchased from Sankyo Co., Ltd., Tokyo, tussive effect of U-62,066E in chronically Japan. 5-Hydroxy(G-3H)tryptamine creatinine vehicle-treated rats. sulfate was obtained from Amersham Inter Table 2 shows the results of binding of national (Amersham, Buckinghamshire, U.K.). [3H]serotonin to brainstem membranes from All antitussive drugs were dissolved in 0.9% rats chronically treated with morphine or U saline immediately before use. 62,066E. There were no significant changes in Data are expressed as the mean ± S.E.M. both the B„,a,, and apparent Kd values in for a given number of rats. The statistical sig brainstem tissues of rats chronically treated nificance of differences was assessed by with either morphine or U-62,066E as com Student's t-test after an analysis of variance pared to the vehicle-treated animals. (significance level was set at P = 0.05). The present study clearly demonstrates that Morphine injection (1 mg/kg, i.p.) into chronic administration of U-50,488H and vehicle-treated rats had a typical antitussive U-62,066E, highly selective agonists for the effect. The percent inhibition of the number x -opioid receptor, does not result in the de of coughs, 15 min after the administration of velopment of tolerance to their respective morphine, was 84.4 ± 4.3%. Administration antitussive effects. There is, furthermore, no of U-50,488H (10 mg/kg, i.p.) and U-62,066E cross-tolerance between U-50,488H and U Table 1. Development of tolerance to and cross-tolerance between morphine, U-50,488H and U-62,066E with respect to antitussive activity

Table 2. 3H-Serotonin binding sites in the brain mg/kg, i.p.) and U-62,066E (10 mg/kg, i.p.) stem of rats chronically treated U-62,066E or each reduced the number of coughs by about morphine 80%, respectively. Moreover, chronic adminis tration of an analgesic dose of U-50,488H (25 mg/kg, i.p.), on the same treatment schedule as that in the present study, resulted in the de velopment of tolerance with respect to the analgesic effect of the drug (8). Thus, the abil ity of these x-opioid agonists to cause toler ance to their respective antitussive effects was lower than that of a u-opioid agonist. In the present study, no changes in affinity and density of serotonin receptors were observed in the brainstem of rats chronically treated with U-62,066E as compared to the vehicle-treated animals. Although we did not, 62,066E. In contrast, tolerance to the anti in this study, assess the effects of U-50,488H tussive effect of morphine, a u-opioid receptor on the functions of serotonin receptors be agonist, developed in rats that were chronical cause of the small quantity of sample, there is ly treated with morphine. However, in rats reason to believe that also no changes in affin made tolerant to morphine, there was no ity and density of serotonin receptors might cross-tolerance to U-50,488H or U-62,066E. have occurred in the brainstem of rats chroni The results of previous study (2) under similar cally treated with U-50,488H. Indeed, U conditions indicated that administration of 50,488H has the same antitussive potency as morphine (1 mg/kg, i.p.), U-50,488H (10 U-62,066E (2). In addition, the antitussive effects of U-50,488H and U-62,066E are medi ever, greater changes in receptor affinity of ated by the same modes of interaction at the the x-opioid receptor were found in the spinal serotonin receptors (2). Ho and Takemori (5) cord than in the other brain regions (5). reported that antinociceptive tolerance to U Several lines of evidence indicate that the 50,488H was accompanied by a decrease in spinal cord is the more important antinocicep the affinity of [3H]serotonin to serotonin re tive locus for the action of U-50,488H (9-11). ceptors in the spinal cord, but not in the supra By contrast, the supraspinal brain regions, spinal regions. Most opioid and nonopioid particularly the brainstem, are the most im antitussive drugs have their site of action at portant antitussive locus for x-opioid agonists, the supraspinal level. Furthermore, U-50,488H as mentioned above. Taken together, both and U-62,066E have their site of action at the these studies and the present results, it can be supraspinal level (2). It seems likely, there speculated that the subtype of receptors for fore, that the differential ability of x-opioid the x-opioid agonist responsible for the regula receptor agonists to cause tolerance to their tion of antitussive effects in the brainstem may own antinociceptive effects and to their own be different from that responsible for the anti antitussive effects may be due to differences nociceptive effects in the spinal cord. between their modes of action on the seroto ninergic systems at the supraspinal level and at Acknowledgments the spinal level. Furthermore, it is possible We would like to express our thanks to Y. Ohhashi, that the mechanisms involved in the develop H. Igarashi and F. Kamimura for their capable techni ment of tolerance to the analgesic and antitus cal assistance and thank Upjohn for the generous sup sive effects may be different. plies of drugs. 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