Analgesic effects of dynorphin-A and morphine in mice

Peptides, Vol. 6, pp. 75-78, 1985. ''° Ankho InternationalInc. Printed in the U.S.A.

0196o9781/85$3.00 + .00

Analgesic Effects of Dynorphin-A And Morphine in Mice T. N A K A Z A W A , M. IKEDA, T. K A N E K O A N D K. Y A M A T S U Department o f Pharmacology, Tsukuba Research Laboratories, Eisai Co., Ltd., 1-3 Tokodai 5-chome Toyosato-machi, Tsukuba-gun, lbaraki 300-26, Japan R e c e i v e d 19 July 1984 NAKAZAWA, T., M. IKEDA, T. KANEKO AND K. YAMATSU. Analgesic
Morphine

Analgesia

Analgesic Tests

THE opioid peptide dynorphin-A (DYN) is widely distributed throughout the central nervous system, but its physiological role remains unknown. Conflicting reports have appeared regarding the analgesic effect of this peptide [2, 5, 7, 9, 10, 1I, 16]. Recently, evidence has been accumulating that DYN is active against pressure and chemical noxious stimuli [5, 7, 1 1] and that it is active predominantly at the spinal level [9, 10, Ill. The purpose of this study was to investigate whether or not DYN is analgesic, and if it is, to compare its action with that of morphine (MOR) in three different analgesic tests (i.e., acetic acid writhing, tail pinch and tail flick tests) after administration by two different routes (i.e., intracerebroventricular (ICY) and intrathecal (IT) injections) in mice.

Analgesic activities were measured by the acetic acid writhing, tail pinch and tail flick tests. In the acetic acid writhing test, mice were injected intraperitoneally with 0.7%, acetic acid 5 min after ICV injection of drugs and then the writhing syndrome was observed for 15 min. Results are expressed as percentage of inhibition of writhing. In the tail pinch test, a clip, exerting a pressure of 300 g, was placed at the base of the tail including the anal mucosa, and the latencies to biting of the clip were measured I131. The tail flick test was performed by the method of D'Amour and Smith [ 11. The tail flick latencies were measured. The light intensity was adjusted to give control readings of about 3 sec. In both the tail pinch and tail flick tests, animals were screened for tail clip or radiant heat nociception before experiments and those mice not biting within 3 sec or not flicking within 4 sec were eliminated from the experiments. Control latency to biting or flicking was 1.09_+0.49 or 2.95+_0.62 sec Imean+S.D.), respectively. To avoid tissue damage, the cutoff was arbitrarily selected as 8 sec in these tests. In both tests, a latency of more than 6 sec was used as the criterion for analgesia. Results are expressed as percentage of mice which showed latencies of more than 6 sec. The ED~, values and their 95% confidence limits were determined by the method of Litchfield and Wilcoxon I81.

METHOD

h!iections and Dru~,s Male ddY strain mice (20-27 g body weight, usually studied in groups of 10) were injected ICV or IT. The ICV injection procedure followed that of Haley and McCormick 141. Dye injection showed that the vehicle filled the ventricular system, including the lateral ventricles as well as the third and occasionally the fourth ventricles. The IT injection was performed by a modification of the method of Hylden and Wilcox [61. Under local anesthesia, a small incision was made in the skin to aid visualization of the vertebral column. The drug was injected IT via the spaces between TI3 and L1 using a bent 30 gauge needle. Drugs were dissolved in 0.9% saline and the solutions were injected ICV or IT in a volume of 5/~1. The following drugs were used: morphine HC1 (Shionogi Co., Ltd., Osaka, Japan); dynorphin-A and dynorphin-A-( 113) (Protein Research Foundation, Osaka, Japan); and naloxone HC1 (Endo Lab., Garden City, N J, U.S.A.).

RESULTS

Analgesic Profile of lCV DYN and MOR in Various Tests The analgesic effects of DYN and MOR following ICV injection in the tail pinch test are shown in Fig. 1. Injection of 0.9% saline did not cause any significant increase of the biting latency. Both DYN and MOR produced significant analgesic effects in a dose-dependent manner (DYN 1.25, 2.5, 5

75

76

NAKAZAWA, IKEDA, KANEKO AND YAMATSU

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FIG. 1. Analgesic effects of dynorphin-A (DYN) and morphine (MOR) fifllowing intracerebroventricular (ICV) injections and their antagonism by naloxone in the mouse tail pinch test. Each dose was given to 7-10 mice. Naloxone 0.5 mg/kg was injected subcutaneously 10 rain before ICV injection of DYN or MOR. *p<0.05, **p<0.01 vs. DYN 10 nmol or MOR I nmol alone treated group at a given time (X'-'test).

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Dose (nmol / mouse) FIG. 2. Effects (dose-response curves) of DYN (A), DYN-(1-13) (11) and MOR (O) in the acetic acid (AcOH) writhing (left), tail pinch (middle) and tail flick tests (right) at the peak time after ICV i[!jection into mice. The peak effects of DYN, DYN-(I-13) and MOR were observed 15, 15 and 5 rain after ICV injection, respectively, in all the tests. Each dose was given to 6-13 mice.

and 10 nmol/mouse, M O R 0.1,0.3 and 1 n m o l / m o u s e , X2 test; p < 0 . 0 5 ) . The peak effects o f D Y N and M O R were o b s e r v e d 15 and 5 min after ICV injection, respectively. ICV injection of higher doses of D Y N caused transient "barrel-rolling.'" H o w e v e r , mice which exhibited such m o t o r dysfunction did not always show analgesia. Sedation and diuresis were also o b s e r v e d after ICV injection o f D Y N , Such effects were not o b s e r v e d with M O R . Analgesic effects (dose-response curves) at the peak time following ICV injection of D Y N , D Y N - ( I - 1 3 ) and M O R in the three different tests are shown in Fig. 2. The peak times of D Y N , D Y N - ( I - 1 3 ) and M O R were 15, 15 and 5 min after ICV injection, respectively. ED50 values calculated from the d o s e - r e s p o n s e curves are summarized in Table 1. D Y N s h o w e d a potent analgesic effect in the acetic acid writhing

and tail pinch tests, but a weak effect in the tail flick test. In the tail flick test, the analgesic activity of D Y N could be d e t e c t e d only at high doses which caused "barrel-rolling.'" In contrast, MOR produced a potent analgesia in all the tests. DYN-(1-13) showed the same analgesic profile as D Y N , but it was 3 to 8 times less potent than D Y N . In the tail pinch test (Fig. 1), pretreatment with naloxone 0.5 mg/kg SC totally reversed M O R analgesia (;(2 test: p < 0 . 0 1 at 5, 15 and 30 rain), but only partially reversed D Y N analgesia (X2 test: not significant at 15 rain, p < 0 . 0 5 at 5, 30, 60 and 90 rain). In the acetic acid writhing test, the antagonistic effect of various doses (0. I, 0.3, 1 and 3 mg/kg) of naloxone on D Y N - and M O R - i n d u c e d analgesia was investigated. N a l o x o n e ADs, values (with 95% confidence limits) measured in the presence of a fixed dose of DYN (8 nmol) or

ANALGESIC E F F E C T OF DYNORPHIN-A

77 TABLE 1

S U M M A R Y O F T H E ED~ V A L U E S O F DYN, DYN-(I-13) A N D MOR IN T H E ACETIC ACID W R I T H I N G , T A I L P I N C H A N D T A I L F L I C K TESTS AT T H E P E A K TIME A F T E R ICV I N J E C T I O N INTO MICE

EDa,, (nmol/mouse) tail pinch

tail flick

3.8 (2. I-6.8) 15.2 (8.4-27.4) 0.19 (0.06-0.56)

15.8 ( 11.5-21.8) 51.3 (27.5-95.9) 0.14 (0,08-0.24)

AcOH writhing DYN

1.0 (0,65-1.56) 8.2 (5.0-13.4) 0.46 (0.18--1.14)

DYN-(I-13) MOR

ED:,, values are nmol doses required to produce analgesia in 5(VA,of mice. Values in parentheses are 95% confidence limits.

Morphine

Dynorphin 100 -

100'

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FIG. 3, Analgesic effects of DYN and MOR following intralhecal (IT) injections in the mouse tail pinch test. Each dose was given to 7-10 mice.

MOR (3 nmol) were more than 3 or 0.12 (0.04-0.38) mg/kg, respectively. Thus DYN analgesia was relatively resistant to naloxone blockade. A nal~,esic Activity of i T D YN and M O R in the Tail Pinch Test

The analgesic effects of DYN and MOR following IT injection in the tail pinch test are shown in Fig. 3. DYN and MOR produced a dose-related analgesia following IT as well as ICV injection (DYN I, 3 and 10 nmol/mouse, MOR 0.1, 0.3 and 1 nmol/mouse, X2 test; p<0.05). IT injection of higher doses of DYN caused long lasting paralysis of the hindlimbs. The peak analgesic effects of DYN and MOR were observed 15 and 5 rain after IT injection, respectively. The ED:,o values (with 95% confidence limits) at the peak times of DYN and MOR were 0.68 (0.36-1.31) and 0.18 (0.06-0.54) nmol/mouse, respectively. The ratios of ED5o

values (1CV/IT) of DYN and MOR were 5.6 and 1.1, respectively. Thus DYN was more potent by IT injection, while MOR was equipotent by both injection routes. DISCUSSION

Although it has been reported that DYN has a very potent opioid activity in vitro 13], most experiments have failed to demonstrate its analgesic activity [2,16]. In these studies, its analgesic activity was exclusively assayed by the use of radiant heat noxia. In the present study, the analgesic activity of this peptide could be detected in the acetic acid writhing and tail pinch tests, but only a weak effect could be detected in the tail flick test in the condition which analgesic effect of MOR was equipotent in all the tests. This result is consistent with the report that DYN was effective against pressure noxious stimuli but less effective against heat noxi-

78

NAKAZAWA, IKEDA, KANEKO AND YAMATSU

ous stimuli in rats [5,11]. It has been d e m o n s t r a t e d that such an analgesic profile is typical of kappa r e c e p t o r agonists [14,151. It has been reported that D Y N is active predominantly at the spinal level [9, 10, I l]. Our present results in mice are consistent with these reports, although our previous result in rats indicated that D Y N was more active after 3rd ventricular or N R G C - N R P G injection than after IT injection [71. T h e r e may be another site of analgesic action of D Y N as well as the spinal cord. Higher doses of D Y N caused a transient " b a r r e l - r o l l i n g " and long lasting paralysis of the hindlimbs after ICV and IT injection, respectively. It was reported that such motor dysfunction was also caused by des-Tyrl-DYN, which failed to bind to opioid receptors, in rats [10,16]. It was d e m o n s t r a t e d

that bestatin, an amino-peptidase inhibitor, selectively potentiated the analgesic action of IT D Y N but not the motor dysfunction in rat tail pinch test [12]. Our preliminary data also showed that the analgesic effect of DYN potentiated by bestatin could be d e t e c t e d without motor dysfunction in the mouse tail pinch test. Thus, the analgesic effect of DYN could be discriminated from the motor dysfunction effect. D Y N induced analgesia was different from that of MOR in the following respects: (1) it was effective against pressure and chemical noxious stimuli but less effective against heat noxious stimuli; (2) it was active predominantly at the spinal level; (3) it was relatively resistant to naloxone. These resuits suggest that the analgesic m e c h a n i s m of D Y N is at least partly different from that of M O R , which is mediated by mu opioid receptor.

REFERENCES

1. D'Amour, F. E. and D. L. Smith. A method for determining loss of pain sensation. J Pharmacol Exp Ther 72: 74-79, 1941. 2. Friedman, H. J., M. F. Jen. J. K. Chang, N. M. Lee and H. H. Loh. Dynorphin: A possible modulatory peptide on morphine or /3-endorphin analgesia in mouse. Eur ,I Pharmacol 69: 357-36(I, 1981. 3. Goldstein+ A., S. Tachibana, L. 1. Lowney, M. Hunkapiller and L. Hood. Dynorphin-(I-13), an extraordinary potent opioid peptide. Proc Natl Acad Sci USA 76: 6666-6670, 1979. 4. Haley, T. J. and W. G. McCormick. Pharmacological effects produced by intracerebral ir~iection of drugs in the conscious mouse. B r J Pharmacol 12: 12-15, 1957. 5. Hayes, A. G., M. Skingle and M. B. Tyers. Antinociceptive profile of dynorphin in rats, Ltilb Sci 33: 657-660, 1983. 6. Hylden, J. L. K. and G. L. Wilcox. lntrathecal morphine in mice: a new technique. Eur J Pharmacol 67: 313-316, 1980. 7. Kaneko, T., T. Nakazawa, M. lkeda, K. Yamatsu, T. lwama, T. Wada, M. Satoh and H. Takagi. Sites of analgesic action of dynorphin. Lift, Sci 33: 661-664, 1983. 8. Litchfield, J. T. and F. Wilcoxon. A simple method for evaluating dose-effect experiments, J Pharmacol k:rp Ther 96:99-113. 1949. 9. Piercey, M. F., K. Varner and L. A. Schroeder. Analgesic activity of intraspinally administrated dynorphin and ethylketocyclazocine. E u r J Pharmacol 80: 283-284, 1982.

10. Przewj'ocki, R., G. T. Shearman and A. Herz. Mixed opioid/nonopioid effects of dynorphin and dynorphin relayed peptides after their intrathecal injection in rats. NeuropeptMe.~ 3: 233-240, 1983. I I. Przewlocki, R., 1+. Stala, M. Greczek, G. T. Shearman. B. Przewj'ocka and A. Herz. Analgesic effects of p.-, a- and K-opiate agonists and, in particular, dynorphin at the spired level. L~lb Sci 33: 649-652, 1983. 12. Satoh, M., M. Yasui, K. Fujibayashi and H. Takagi. Bestatin potentiates analgesic effect of intrathecally administered dynorphin in rats. IRCS Med Sci 11: 965-966, 1983. 13. Takagi, H.. T. lnukai and M. Nakama. A modification of Haffner's method for testing analgesia. Jim .I Pharmacol 16: 287294, 1966. 14. Tyers, M. B. A classification of opiate receptors thal mediate antinociceptive in animals. B r J Pharmacol 69: 503-512, 1980. 15. Upton, N., R. D. E. Swell and P. S. J. Spencer. Differentiation of potent /x- and K-opiate agonists using heat and pressure antinociceptive profiles and combined potency analysis. /!ur ,I Pharma~'ol 78:421-429, 1982. [6. Walker, J. M., H, C. Moises, D. H. Coy and H. Akil. Nonopiate effects of dynorphin and des-Tyr-dynorphin. A'cicm'e 218: 1136-1138, 1982.