Proc. Natl. Acad. Sci. USA Vol. 73, No. 11, pp. 4187-4189-, November 1976

Medical Sciences

f3-Endorphin: Cross tolerance to and cross physical dependence on morphine

(antinociceptive response/withdrawal jumping/enkephalin)

LIANG-FU TSENG*, HORACE H. LOH*, AND CHOH HAO Lit * Departments of Psychiatry and Pharmacology and t Hormone Research Laboratory, University of California, San Francisco, Calif. 94143

Contributed by Choh Hao Li, August 3,1976

ABSTRACT The effects of #-endorphin on the antinociceptive responses and abrupt withdrawal jumping in morphine-dependent mice were studied. Mice were rendered morphine dependent by implantation of a morphine pellet (75 mg base) for 3 days. The analgesic response to fi-endorphin decreased after morphine pellet implantation, as evidenced by an eight-fold increase in the median antinociceptive dose. A similar increase in the median antinociceptive dose of morphine was found. In small doses (0.09-0.17 1&g per mouse), fi-endorphin suppressed abrupt withdrawal jumping. Met-enkephalin, even in high doses (200 Ag per mouse), did not suppress abrupt withdrawal jumping. We have recently demonstrated that fl-endorphin, an untriakontapeptide isolated from camel pituitary gland (1), is a potent analgesic agent when administered either intracerebroventricularly (i.c.v.) (2) or intravenously (3). The analgesic responses to fl-endorphin were completely blocked or reversed by naloxone. Moreover, chronic infusion of fl-endorphin into the periaquaductal gray of rat brain for 3 days using miniosmotic pumps (4) resulted in the induction of physical dependence (2). f3-Endorphin also displaced competitively [3H]dihydromorphine or [3H]naloxone in binding assays and inhibited the naloxone-reversible contractions of guinea pig ileum induced by transmural stimulation (5, 6). These studies strongly suggest that the biological behavior of f3-endorphin is similar to that of opiate alkaloids. The characteristics of the pharmacological effects of opiate alkaloids are cross tolerance and cross physical dependence. To elucidate the mechanism of fl-endorphin action, it is necessary to establish whether or not cross tolerance and cross physical dependence exist between fl-endorphin and morphine. MATERIALS AND METHODS fl-Endorphin was synthesized as previously described (7). Methionine-enkephalin (Met-enkephalin) was purchased from Bachem, Inc. (Marina Del Rey, Calif.) and morphine sulfate from Mallinckrodt Chemical Works (St. Louis, Mo.). Naloxone HCI was a gift from Endo Laboratories (Garden City, N.Y.). Morphine pellets containing 75 mg of morphine base were formulated according to Gibson and Tingstad (8). Assessment of Tolerance. Male ICR mice (25-30 g, Simonsen Labs., Gilroy, Calif.) were rendered tolerant to morphine by subcutaneous implantation of one morphine pellet per mouse for 3 days. Placebo pellets were used for the control mice. The pellets were removed 72 hr after implantation. Five hours after pellet removal, mice were injected i.c.v. with fl-endorphin or morphine sulfate in a volume of 5 JAI per mouse according to the method described by Haley and McCormick (9). The antinociceptive or analgesic properties of 1f-endorphin and morphine were assessed by the tail-flick method (10) and the Abbreviations: i.c.v., intracerebroventricularly; AD50, median antinociceptive dose. 4187

hot-plate method (11). The analgesic responses to f3-endorphin and morphine are expressed as "percent analgesia" according to Harris and Pierson (12). With a two-fold increase in latency of reaction time as a quantal index of inhibition, the median antinociceptive dose (AD50) and 95%-confidence limits were calculated according to the method of Litchfield and Wilcoxon (13). At least six to eight animals were tested at each dose with three to five dose levels used for determining the AD50. Assessment of Physical Dependence. The incidence of abrupt withdrawal jumping was used as an index for the quantitative measurement of physical dependence (14). Male Swiss-Webster mice (Simonsen Labs., Gilroy, Calif.) weighing 25-30 g were rendered morphine dependent by pellet implantation in exactly the same manner that they were made tolerant (14). Morphine pellets were removed after 72 hr and 5-6 hr later, mice, in groups of four, were placed in a 2-liter glass beaker. Mice which jumped to the top of the beaker were then separated from the nonjumpers. The jumpers, four in each group, were then injected with various doses of ,B-endorphin, morphine, or saline, placed in the same 2-liter beakers, and the incidence of jumping was observed for 30 min. RESULTS AND DISCUSSION Cross tolerance between ,B-endorphin and morphine Placebo-implanted mice injected i.c.v. with 1 ,ug of ,B-endorphin showed 80-100% analgesia in both the hot-plate and tail-flick tests. This effect lasted about 60 min (Fig. la and b). The same dose of fl-endorphin produced much less inhibition in both the hot-plate and tail-flick test responses to nociceptive stimuli in morphine-pellet-implanted mice (Fig. la and b). A quantitative comparison of the analgesic potencies of flendorphin and morphine in placebo-implanted and morphine-pellet-implanted mice is shown in Fig. 2. In placebo pellet-implanted mice, the AD50 of ,B-endorphin was found to be 0.15 ,ug per mouse in both the hot-plate and tail-flick tests, while the AD50 of morphine was 0.61 and 0.47 ,ug per mouse in the hot-plate and tail-flick tests, respectively. When potency was compared on a molar basis, ,B-endorphin was 42 times more potent than morphine in the hot-plate test and 32 times more potent in the tail-flick test. These values are comparable to the data reported in our previous studies (2). The degree of cross tolerance to 3l-endorphin was measured by the increase of the AD50 of f,-endorphin after morphine pellet implantation. In morphine-pellet-implanted mice, the AD50 of ,B-endorphin was eight- to nine-fold higher than that in placebo-implanted mice. A similar increase in the AD50 of morphine was found. Suppression of abrupt withdrawal jumping ,B-Endorphin, in doses ranging from 0.043 to 0.17 ,ug per mouse, caused a dose-related inhibition of abrupt withdrawal jumping

Medical Sciences: Tseng et al.

4188

Proc. Natl. Acad. Sci. USA 73 (1976)

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5 10 20 30 60 120 Time after -endorphin injection, I g i.c.v. (min) Time after /8-endorphin injection,I1,g i.c.v. ( min FIG. 1. Antinociceptive effects following i.c.v. injection of fi-endorphin in placebo-pellet-implanted mice and morphine-pellet-implanted mice. The antinociceptive effects were measured by the (a) hot-plate and (5) tail-flick tests. Mice, 10 per group, were implanted with either one morphine pellet or one placebo pellet for 3 days. The pellets were removed after 72 hr. Five hours later, mice were injected with fl-endorphin (1 ,g per mouse, i.c.v.) at 0 time. The vertical bars indicate the SEM. in morphine-dependent mice (Fig. 3). When the potency of molar compared with that of morphine f3-endorphin on a

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This work was supported by National Institute of Drug Abuse Grants DA-01314 and DA-00564 and by National Institutes of Health Grant GM-2907. H.H.L. is a recipient of a National Institute of Mental Health Research Scientist Career Development Award, K2-DA-70554. The authors thank Miss Shelley Petluck for her technical assistance and Miss Barbara Halperin for her editorial and secretarial assistance in the preparation of this manuscript.

T i

i-ENDORPHIN

basis, f3-endorphin was about 10 times more potent than morphine in the suppression of abrupt withdrawal jumping. Metenkephalin, even in a high dose of 200 Ag per mouse, did not suppress abrupt withdrawal jumping (Fig. 3). We have previously demonstrated that fl-endorphin possessed at least two classical pharmacologic properties of morphine, namely antinociceptive or analgesic activity and the ability to cause physical dependence (2). In the present studies, we demonstrated a cross tolerance between fl-endorphin and morphine and the ability of fB-endorphin to suppress abrupt withdrawal jumping. Collectively, our results suggest that f3endorphin elicits its pharmacological actions in a manner similar to that of opiate alkaloids. In contrast to fl-endorphin, Met-enkephalin, even in high doses, was found to have little effect on the suppression of abrupt withdrawal jumping in morphine-dependent mice. Met-enkephalin also was found to have a very weak effect on the production of analgesic response (2, 15). These findings provide further support for a more significant in vivo role for f3-endorphin than enkephalin as the true endogenous opiate ligand.

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FIG. 2. Median antinociceptive doses (AD50) of fl-endorphin and morphine sulfate in placebo-pellet-implanted and morphinepellet-implanted mice estimated by the hot-plate (left) and the tailflick test (right). After control latency was obtained prior to drug injection, groups of 8 to 10 mice were injected i.c.v. with various doses of ,-endorphin or morphine sulfate and the antinociceptive effect was determined 10 min later. The AD50 values (vertical bars) and 95% confidence limits (vertical lines) are expressed in gg per mouse.

1. Li, C. H. & Chung, D. (1976) Proc. Natl. Acad. Sci. USA 73, 1145-1148. 2. Loh, H. H., Tseng, L. F., Wei, E. & Li, C. H. (1976).Proc. Nati. Acad. Sci. USA 73,2895-2898. 3. Tseng, L. F., Loh, H. H. & Li, C. H. (1976) Nature, in press. 4. Wei, E. & Loh, H. H. (1976) Science, in press. 5. Bradbury, A. F., Smyth, D. G., Snell, C. R., Birdsell, N. M. M. & Hulme, E. C. (1976) Nature 260,793-795. 6. Cox, B. M., Goldstein, A. & Li, C. H. (1976) Proc. Natl. Acad. Sci. USA 73, 1821-1823. 7. Li, C. H., Lemaire, S., Yamashiro, D. & Doneen, B. A. (1976)

Medical Sciences: Tseng et al.

Proc. Natl. Acad. Sci. USA 73(1976)

4189

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FIG. 3. Effects of fl-endorphin, morphine, and methionine-enkephalin on the suppression of abrupt withdrawal jumping in morphine-dependent mice. Mice were rendered dependent on morphine by implantation of a single morphine pellet (75 mg base). Three days after pellet implantation, pellets were removed. Various doses of j-endorphin, morphine sulfate (5 Al), and methionine-enkephalin (10 Il) were injected i.c.v. Bars indicate the percent of animals jumping. The ratios within the bars denote the number of mice that jumped per number of mice treated. Biochem. Biophys. Res. Commun. 71,19-25. Gibson, R. D. & Tingstad, J. E. (1970) J. Pharm. Sci. 59, 426427. 9. Haley, T. J. & McCormick, W. G. (1957) Br. J. Pharmacol. 12, 12-15. 10. D'Amour, F. E. & Smith, D. L. (1941) J. Pharmacol. Exp. Ther. 72,74-79. 11. Antier, S. (1974) Eur. J. Pharmacol. 27, 1-4. 8.

12. Harris, L. S. & Pierson, A. K. (1964) J. Pharmacol. Exp. Ther. 143, 141-148. 13. Litchfield, J. T. & Wilcoxon, F. (1949) J. Pharmacol. Exp. Ther. 96,99-113. 14. Way, E. L., Loh, H. H. & Shen, F. H. (1969) J. Pharmacol. Exp. Ther. 167, 1-8. 15. Belluzzi, J. D., Grant, N., Garsky, V., Sarantakis, D., Wise, C. D. & Stein, L. (1976) Nature 260, 625-626.