European Journal of Pharmacology, 186 (1990) 137-141 137 Elsevier

EJP 20693

Short communication Selective modulation of anfinocicepfion, but not development of tolerance, by 8 receptor agonists

Qi Jiang, Henry I. Mosberg 1 and Frank Porreca Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724. U.S.A. and I College of Pharmacy. University of Michigan, Ann Arbor, Mi 48109, U.S.A.

Received 2 July 1990, accepted 10 July 1990

Co-administration of 8 agonists at doses which do not produce measurable antinociception were demon- strafed to produce an increase in the antinociceptive potency of morphine in the mouse tail-flick test. In contrast, co-administration of equi-antinociceptive combinations of a 8 agonist plus morphine for three days resulted in the development of less tolerance to morphine antinociceptive actions. The data indicate that while acute antinociceptive effects of opioid It agonists are modulated by 8 agonists, the development of antinociceptive tolerance is not.

Antinociception; Morphine; 8- agonists; Tolerance

1. Introduction Ocomplexed (¢~cx) receptors to designate their ex- istence within the/t-~ receptor complex (Rothman Previous reports from our laboratory (Heyman et al., 1989) while those /t and 8 receptors not et al., 1989a,b; Jiang et al., in press; Porreca et al., interacting were termed the ~non-complexed (~tncx) 1990) and others have shown that 8 opioid agonists and 8,on-complcx~ (8,cx)receptors. can modulate the potency and efficacy of opioid p The possibility that occupation of the opioid agonists such as morphine. The modulatory action Sex site could increase the potency (Heyman et al., (i.e. increases or decreases in potency and/or ef- 1989a,b; Porreca et al., 1990) and efficacy (Jiang ficacy), but not the direct antinociceptive effect of et al., in press) of opioid ~t agonists was of inter- morphine, was blocked by pretreatment with the est, for example, in using /~ agonists of lower selective 8 , ICI 174,864 (Cotton efficacy to produce equi-effective pain relief clini- et al., 1984), and this compound given alone did cally. Such a possibility was particularly interest- not produce antinociception nor modulation of ing in that the route of administration of the morphine effects. These studies, and others (see modulating 8 agonist seemed to be umimportant. Rothman et al., 1989 for review) have supported In this regard, i.c.v, administration of the highly the hypothesis that some opioid/t and 8 receptors selective 8 agonist, [D-Pen2,D-PenS] may exist in a functionally associated state. In this (DPDPE) (Mosberg et al., 1983) or i.p. adminis- hypothesis, opioid receptors thought to function- tration of [LeuS]enkephalin produced an increase ally interact have been termed ~complexed (~cx) and in the antinociceptive potency of morphine given by the same route. In spite of these findings, however, it remained unclear whether all of the pharmacological actions Correspondence to: F. Porreca, Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ of morphine would be similarly subjected to mod- 85724, U.S.A. ulation by O opioid agonists, or whether selective

0014-2999/90/$03.50 © 1990 Elsevier Science Pubfishers B.V. (Biomedical Division) 138 modulation of specific effects could be achieved. 1989a,b; Jiang et al., in press), while morphine The present study has addressed whether DPDPE and [LeuS]enkephalin were given i.po and testing or [LeuS]enkephalin would increase the rate of took place after 20 min, a time shown to result in development of tolerance to morphine antinoci- a maximal response for the combination (Porreca ceptive actions following chronic administration et al., 1990). of equi-antinociceptive combinations. We now re- A cutoff time of 15 s was employed; if the port that although these compounds increase mouse failed to respond within this time, the tail morphine antinociceptive potency, the rate of de- was removed from the water and that animal was velopment of antinociceptive tolerance depends assigned a maximum score. Mice not responding only on the amount of morphine administered. within 5 s in the initial control trial were eliminated from the experiment. Antinociception at each time point was calculated according to the following 2. Materials and methods formula: ~ antinociception ffi 100 y (test latency- control latency)/(15-control latency). 2.1. Animals 2.4. Induction of antinociceptive tolerance Male, ICR mice (20-30 g) were used for all experiments. Animals were kept in groups of five in a temperature-controlled room with a 12 h In the tolerance experiments, mice were in- light-dark cycle (lights on 7 : 00 a.m. to 7: 00 p.m.). jected twice daily (8:00 a.m. and 5:p.m.) for 3 Food and water were available ad libitum until days, alid testing took place on the morning of the time of the experiment. day 4. For the DPDPE/morphine experiments, animals received i.c.v, saline, morphine alone (1.2 2.2. Injection techniques or 6 nmol), DPDPE alone (1.55 nmol) or morphine plus DPDPE (1.2 and 1.55 nmol), respectively. I.c.v. administrations were made directly into This combination of i.c.v, morphine plus DPDPE the lateral ventricle as previously described (Hey- was shown to produce an equivalent degree of man et al., 1989a,b). The mouse was tightly antinociception as i.c.v, morphine given at the 6 anesthetized with ether, an incision was made in nmol dose. After each i.c.v, injection, the scalp the scalp, and the injection was made 2 mm lateral incision was closed using wound clips. No signs of and 2 mm caudal to bregma at a depth of 3 mm distress were noted in these animals over the 3 using a 10/~l Hamilton microliter syringe with a days (six injections) period. In the [LeuS]enkepha- 26-gauge needle. I.c.v. injections were made at a lin/morphine experiments, mice received i.p. volume of 5/H. saline, morphine alone (9 or 24 ~tmol/kg), [LeuS]enkephalin (18/~mol/kg) or morphine plus 2.3. Taii flick assay [LeuS]enkephalin (9 and 18 /tmol/kg), respec- tively. The combination of i.p. morphine plus The thermal nociceptive stimulus was 55°C [LeuS]enkephalin was shown to produce an equiv- warm water with the latency to tail-flick or alent degree of antinociception as i.p. morphine withdrawal taken as the endpoint (Heyman et al., given at the 24/~mol/kg dose. 1989a, b). After the determination of control latencies, the mice received graded i.c.v, doses of 2.5. Chemicals vehicle, morphine, or concurrent combination of morphine plus DPDPE or [LeuS]enkephafin. Morphine sulfate was purchased from Mal- Morphine and DPDPE were given by the i.c.v. linckrodt Chemical Co. (St. Louis, MO) while route and testing took place after 10 min, a time [LeuS]enkephalin was purchased from Sigma previously shown to result in a maximal response Chemical Company (St. Louis, MO). DPDPE was for both compounds (Heyman et al., 1987; synthesized as previously described (Mosberg et 139 al., 1983). All compounds were dissolved in dis- 100 tilled water just before using. QO 80 2.6. Statistics ,H 70 I10 o') SO Regression lines, As0 values (i.e. the dose pro- td 40 ducing a 50~ antinociception response) and 95~; < confidence limits (C.L.) were determined with each Z

3. Results resulted in a slight tolerance to the antinociceptive actions of morphine as shown by the 1.85-fold I.c.v. morphine produced a dose-related anti- rightward displacement of the dose-response line; nociceptive effect in the mouse tail-flick test with the As0 for morphine in animals pretreated with an As0 value of 0.92 (0.65-1.3) nmol. When morphine at 1.2 nmol was calculated to be 2.13 morphine and DPDPE were co-administered i.c.v., (1.44-3.15) nmol (fig. 1). Pretreatment with i.c.v. the morphine dose-response line was displaced to morphine at 6 nmol produced a locater degree of the left; the As0 of this combination was calcu- tolerance resulting in approximately a 12.84-fold lated as 0.21 (0.12-0.34) indicating an approxi- rightward displacement of the i.c.v, morphine mately 4.4-fold increase in potency. Similarly, i.p. dose-response line. Additionally, the maximum morphine produced a dose-related antinociceptive antinociceptive effect of i.c.v, morphine in animals repsonse with an As0 value of 15.3 (12.99-18.02) pretreated with this higher dose of morphine was ~tmol/kg and when co-administered with i.p. shown to be approximately 60~;; the calculated [LeuS]enkephalin the dose-response line for the As0 value for these pretreated mice was 14.77 combination was to the left of that for morphine (9.21-23.68) nmol, indicating severe antinocicep- alone, with an As0 of 4.25 (3,41-5.31) ~tmol/kg, a tive tolerance (fig. 1). In contrast to the degree of shift of approximately 3.6-fold. From these pairs tolerance seen with the higher dose of morphine, of curves, equi.antinociceptive combinations were administration of the lower morphine dose plus calculated and given twice daily in the tolerance DPDPE, a combination which produced an equi- experiments. antinociceptive effect, resulted in a much lower Construction of the i.c.v, morphine dose-re- degree of tolerance. I.c.v. co-administration of sponse line in animals pretreated twice daily with morphine (1.2 nmol) plus DPDPE (1.55 nmol) for saline showed no change from non-injected 3 days produced a rightward displacement of the animals with an As0 of 1.15 (0.87-2.09) nmol, and i.c.v, morphine dose-response line of only 1.8S-fold similarly, pretreatment i.c.v, with the modulating (fig. 1). The morphine A so value in mice pre- dose of DPDPE (1.55 nmol) did not affect the treated i.c.v, with the combination was 2.13 (1.55- i.c.v, dose-response line for morphine resulting in 2.93) nmol. an As0 of 1.27 (0.86-1.86) nmol (fig. 1). Pretreat- Construction of the i.p. morptfine dose-re- merit with i.c.v, morphine at 1.2 nmol for 3 days sponse line in a_~mals pretreated twice daily with too i & 3 days produced a rightward displacement of the 9o i.p. morphine dose-response line of only 2.1-fold (fig. 2). The i.p. morphine As0 value in mice pretreated i.p. with this combination was 32.48 - & (26.71-39.51)/tmol/kg. m so o 4o 4. Discussion N lo Previous studies had reported that the anti- o 8 IO 3o 1oo 3oo nociceptive potency of morphine could be en- DOSE MORPHINE Ozrnol/kg, i.p.) hanced by compounds acting at the 8 receptor, such as [LeuS]enkephalm (Vaught and Takemori, Fig. 2. Dose-response lines for i.p. morphine in mice pretreated with saline (circles), [LeuS]enkephalin (18 pmol/kg, inverted 1979) suggesting that the endogenous triangles), morphine (9 /~mol/kg, squares), morphine (24 might have an indirect, modulatory role in anti- /~mol/kg. triangles) and morphine (9 ~mol/kg) plus (LeuS]en- nociception. The present study, and our previous kephalin (18/xmol/kg)(diamonds). work (Heyman et al., 1989a,b; Jiang et al., in press; Porreca et al., 1990) continue to support this hypothesis. Though the enhancement by 8 agonists of desirable/~ effects such as antinocicep- saline showed no change from non-injected tion was attractive, the possibility remained that animals with an Aso of 15.49 (13.41-19.03) non-desirable effects such as the development of /~r,ol/kg, and similarly, pretreatment i.p. with the antinociceptive tolerance would also be simulta- modulating dose of [LeuS]enkephalin (18 neously enhanced. In the study of Vaught and /~mol/kg) did not affect the i.p. dose-response line Takemori (1979) mice were pretreated with an for morphine resulting in an Aso of 14.91 (13.01- acute s.c. injection of morphine alone, or a combi- 18.71) /tmol/kg (fig. 2). Pretreatment with i.p. nation of s.c. morphine plus i.p. [LeuS]enkepha!in morphine at 9/tmol/'kg for 3 days resulted in a and the degree of antinociceptive tolerance to slight tolerance to the antinociceptive actions of morphine established after a 3 h period. In t~s morphine as shown by the 2.29-fold rightward experiment, the degree of tolerance was increased displacement of the dose-response line; the Aso by co-administration of morphine plus the 8 for morphine in animals pretreated with i.p. agonist. However, it should be noted that this morphine at 9/xmol/kg was calculated to be 35.52 experimental protocol emphasized the phenome- (30.65-41.16)/tmol/kg) (fig. 2). Pretreatmeti: with non of acute tolerance. The present studies have Lp. morphine at 24/tmol/kg produced a greater re-evaluated this issue by administration of ~t-8 degree of tolerance resulting in approximately a combinations over a 3 days period. 8.96-fold rightward displacement of the i.p. Our results clearly demonstrate that using a morphine dose-response line. The calculated As0 chronic tolerance paradigm, that co-administra- value for these mice pretreated with i.p. morphine tion of/~-8 combinations result in a smaller degree at 24 /xmol/kg was 138.77 (114.47-168.22) of tolerance to morphine than administration of ~tmol/kg (fig. 2). In contrast to the degree of an equi-antinociceptive (higher) dose of morphine tolerance seen with i.p. pretreatment with this alone. This result was demonstrated regardless of higher dose of morphine, administration of the whether both compounds were given by the i.c.v. lower morphine dose plus [LeuS]enkephalin, a or the i.p. routes. From the results of these experi- combination which produced an equi-antinocicep- ments it would appear that the degree of anti- five effect, resulted in a much lower degree of nociceptive tolerance depends only on the amount tolerance. I.p. co-administration of morphine (9 of morphine administered. These results are in ~tmol/kg) plus [LeuS]enkephalin (18/tmol/kg) for agreement with the observation of a lack of cross- 141 tolerance between morphine and DPDPE (Porreca Heyman, J.S., Q. Jiang, R.B. Rothman, H.I. Mosberg and F. et al., 1987; Kov~cs et al., 1988). Porreca, 1989a, Modulation of p-mediated an~ception The failure of the 8 agonists to modulate the by B agonists in the mouse: pharmacological characteriza- tion with antagonists, European J. Pharmacol. 169, 43. development of antinociceptive tolerance while Heyman, J.S., S.A. Mulvaney, H.I. Mosberg and F. Porreca, enhancing the direct antinociceptive actions of the 1987, Opioid B receptor involvement in supraspinal and /~ agonists is of interest when interpreted in light spinal antinociception in mice, Brain Res. 420, 100. of the hypothesis of functionally complexed/t and Heyman, J.S., J.L. Vaught, H.I. Mosberg, R.C. Haaseth and F. 8 receptors. In this hypothesis, it agonists such as Porreca, 1989b, Modulation of p-mediated antinociception by 8 agonists in the mouse: selective potentiation of morphine can produce antinociceptive effects by morphine and by [D-Pen2,D-PenS]enkephalin, acting at either ltncx or/Lcx sites. In such a scenario, European J. Pharmacol. 165, 1. it would seem possible that while the modulation Jiang, Q., H.i. Mosberg and F. Porreca, Modulation of the of direct antinociceptive effects occurs via the 8c~ potency and efficacy of p-mediated antinociception by B receptor, and the development of tolerance is more agonists in the mouse, J. Pharmacol. Exp. Ther. (in press). Kov~cs, G.L., N. Nyolczas, M. Krivam and K. Guyla, 1988, closely related to the /tncx site. In any case, it and tolerance-inducing effects of the highly selec- would appear that the direct mediation of anti- tive 8 opioid agonist [D-Pen2,D-PenS]enkephalin in mice, nociceptive actions and the development of anti- European J. Pharmacol. 150, 347. nociceptive tolerance can be separated, at least in Mosberg, H.I., R. Hurst, V.J. Hruby, K. Gee, H.I. Yamamura, terms of the modulatory actions of 8 agonists. The J.J. Galligan and T.F. Burks, 1983, Bis-penicillamine en- kephalins show pronounced B opioid receptor selectivity, present results continue to support the concept of Proc. Natl. Acad. Sci. U.S.A. 80, 5871. functionally coupled/~ and 8 receptors and offer Porreca, F., J.S. Heyman, H.I. Mosberg and J.L. Vaught, 1987, the possibility that such combinations may be of Role of mu and delta receptors in the supraspinal and clinical use. spinal analgesic effects of [D-Pen2,D-PenS]enkephalin in the mouse, J. Pharmacol. Exp. Ther. 241,393. Porreca, F., Q. Jiang and R.J. Tallarida, 1990, Modulation of morphine antinociception by peripheral [LeuS]enkephalin: Acknowledgements a synergistic interaction, European J. Pharmacol. 179, 463. Rothman, R.B., J.B. Long, V. Bykov, A.E. Jacobson, K.C. Rice Supported by USPHS Grants DA 04285 and DA 06284 and J.W. Holaday, 1989, Beta-FNA binds irreversibly to from the National Institute on Drug Abuse. the receptor complex: in vivo and in vitro evidence, J. Pharmacol. Exp. Ther. 247, 405. Tallarida, R.J. and R.B. Murray, 1986, Manual of Pharmaco- logic Calculation, 2nd edition (Springer-Verlag, New York). References Vaught, J.L. and A.E. Takemori, 1979, Differential effects of leucine enkephalin and methionine enkephalin on Cotton, R., M.G. Giles, L. 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