Methionine oxidation enhances opioid activity of an enkephalin analog

Life Sciences, Vol. 32, pp. 889~893 Printed in the U.S.A.

Pergamon Press

METHIONINE OXIDATION ENHANCES OPIOID ACTIVITY OF AN ENKEPHALIN ANALOG Judith A. Kiritsy-Roy #, S.K. Chan* and Edgar T. lwamoto #** Departments of Pharmacology # and Biochemistry*, and the Tobacco and Health Research Institute**, Albert B. Chandler College of Medicine, University of Kentucky, Lexington, Kentucky 40536, USA (Received in final form November 9, 1982) Summary 2 5 D-ala -met-sulfoxide -enkephalinamide, DALA(0),~was synthesized by oxidizing the 5-methionine residue of D-alaL-met 5enkephalinamide (DALA). Antinociception was assessed on the hotplate and catalepsy estimated using an immobility test in rats administered DALA, DALA(0) and morphine intraventricularly. By comparing areas under time-effect curves, DALA(0) was 30 times more antinociceptive and up to 40 times more cataleptogenic than DALA. For comparison, morphine induced one-tenth the antinociception and one-fortieth the immobility caused by DALA(0). These results demonstrate that the opiate activity of DALA is clearly enhanced by oxidation of its terminal methionine.

The C-terminal methionine (met) residue of met-enkephalin is unstable and can oxidize to met sulfo~ide [met(0)] during storage (1-3). The potency of met(0)-enkephalin reportedly differs from that of met-enkephalin in two biologic assays. While met-enkephalin is 5 times more potent than met(0)-enkephalin in inhibiting the electrically-evoked twitch of the guinea pig ileum (4), it is 8-fold less active in delaying extinction of a pole-jump avoidance response (5). The peptidase-resistant met-enkephalin analog, D-ala -met5-enke phalinamide (DALA), commonly used in pharmacologic studies of opioid peptides because of its prolonged duration of action (6), might also be subject to spontaneous oxidation at its met residue. We therefore compared the effects of DALA and D-ala2-met(0)5-enkephalinamide [DALA(O)] in two tests for opioid activity, analgesia and catalepsy. Methods DALA(0) was prepared by treating 3 mg of DALA (Beckman Bioproducts) with 4.5 ml of 0.i M hydrogen peroxide at room temperature for 30 min. The reaction mixture was diluted two-fold with ice cold distilled water and immediately lyophilized. The product was chromatographed on silica gel (Cat. 5775, E. Merck) in butanol:acetic acid:water (4:1:1) and its amino acid composition was determined on an automated analyzer (7) after hydrolysis in 3.75 N NaOH at II0°C for 18 h as previously described (8). Male, Sprague-Dawley rats weighing 240-325 g were prepared for intraventricular (i.v.t.) microinjections by surgically implanting stainless steel guide cannulae (25 gauge) aimed 1.5 nun above the right lateral ventricle. The coordinates for the inner injector cannulae (31 gauge) were 1 mm posterior, 2 mm lateral and 4.2 mm ventral from bregma (horizontal skull). Experiments were conducted at least one week after surgery. Following injection of the 0024-3205/83/080889-05503.00/0 Copyright (c) 1983 Pergamon Press Ltd.

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test solution or saline, catalepsy was assessed by measuring the latency (L, in sec) to ~ovement of a hindlimb placed on a 3.5 cm high cork (9). The maximum L was 180 sec. The cataleptic response was calculated for each animal at each time point as the percent of the maximum possible response (i00 X L/180). Analgesia was then assessed in the same animals by measuring the latency (L, in sec) to a hindpaw lick, a lifting of one hindpaw for at least 2 seconds, or an escape attempt after being placed upon a 52°C hot plate (i0). A baseline (B) nociceptive latency (sec) was averaged from 3 measurements taken at 30 minute intervals before the i.v.t, injection. The maximum L was 45 seconds and the analgesic response was calculated for each animal at each determination as the percent of the maximum possible change in response latency [I00 X (L-B)/(45-B]. Results Thin layer chromatography of the product obtained from reacting DALA with hydrogen peroxide revealed the presence of a single spot migrating with an Rf value of 0.38 compared to 0.59 for DALA. The amino acid composition of the product (met(0), 0.77; gly, 1.03; ala, 0.99; tyr, 0.96; phe, 1.02) was consistent with that expected for DALA(0). I00

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Time After Injection (rain.) FIG. i Time course of the cataleptic effect of i.v.t, administered A, DALA, B, DALA(0), and C, morphine sulfate with ( 0 ) or without ( O ) a simultaneous injection of naloxone (N, 1 mg/kg, s.c.). Each point is the mean of 6 rats and the doses are shown beside the curves in nmol/rat. The mean cataleptic response of 6 control rats at 30, 60, 90 and 120 min after i.v.t, saline microinjection was 0.4 (0.i), 0.6 (0.3), 0.3 (0) and 4.1 (3.2) percent (S.E.M.) respectively. *p
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At doses greater than 20 nmol, DALA produced a shor~lasting, dose-related (r=0.674; p<0.01) cataleptic response that peaked at 30 min and disappeared by 60 min after i.v.t, administration (Fig. IA). In contrast, rats developed strong cataleptic immobility after DALA(O) (Fig. IB). The response to the oxidized peptide was also dose-related (r=0.883; p<0.01) (1.25 to 5 nmol) and l a s ~ ed at least 1 h after the 2.5 and 5 nmol doses. Morphine sulfate, used as a standard for comparison, induced mild catalepsy after i.v.t, administration but the effect was not dose-related (Fig. IC) (r=0.219; p>0.05). Naloxone blocked the cataleptic response to 80 nmol of DALA, 5 nmol of DALA(0) and 80 nmol of morphine (Fig. IA-C). Rats treated with saline never exhibit a cataleptic response. After i.v.t, injection, DALA induced a dose-dependent (r=0.598; p<0.Ol) antinociceptive response at doses of 20 to 80 nmol (Fig. 2A). The effect peaked at 30 min and was considerably diminished 60 min after injection. DALA(0) on the other hand, produced dose-dependent analgesia (r=0.952; p
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Time After hjection FIG. 2 Time course of the analgesic response to i.v.t, microinjection of A, DALA, B, DALA(O) and C, morphine sulfate with ( O ) or without ( O ) a simultaneous injection of naloxone (i mg/kg, s.c.). Each point is the mean of 6 rats and the doses are given beside the curves in nmol/rat. The mean analgesic response of 6 control rats at 33, 63, 93 and 123 min. after i.v.t, saline microinjection was 1.3 (1.2), -1.5 (1.9), 1.2 (4.2) and -0.5 (1.8) percent (S.E.M.) respectively. *p<0.05 **p
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(1.25-5 nmol) of DALA(0) by an order of magnitude. Naloxone (i mg/kg s.c.) completely blocked the effects of 80 nmol of DALA and 5 nmol of DALA(0) and reduced the antinociceptive response to 80 nmol of morphine (Fig. 2, A-C). Microinjection of saline did not elicit an antinociceptive effect. Discussion These results clearly demonstrate a pronounced enhancement of opioid activity after oxidation of the terminal met residue of the enkephalin analog, DALA, to met(0). Table 1 summarizes the results presented in Figures 1 and 2. As little as 2.5 nmol of DALA(0) elicited greater a n t i n o c i c e p t i o n and catalepsy than 80 nmol of DALA. In comparison with morphine, DALA was a p p r o x i m a t e l y onethird to one-half as potent and DALA(0) was over 15-fold more potent in the hot plate test for analgesia. As reported previously (ii), morphine elicited unpredictable catalepsy after i.v.t, injection that apparently was not doserelated.

TABLE Summary of the A n t i n o c i c e p t i v e DALA and DALA(0). Dose i.v.t. (nmol/rat) 1.25 2.5 5 i0 20 40 80

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HOTPLATE ANALGESIA* MOR DALA DALA(0) .29 .73 .97 .30 .ii .44 .09 .56 .27 1.00 .34

Effects

of Morphine,

CATALEPSY (CORK TEST)* MOR DALA DALA(0) .26 1.18 2.10 .14 .04 1.04 .07 .28 .54 !.00 .78

*Results are expressed as pharmacologic intensity relative to the area under time-effect curve for 80 nmol of morphine (N=6 for all values). Oxidation of m e t - e n k e p h a l i n has been reported to occur spontaneously (I10%) during storage of solutions of the peptide (1-3). It is also likely that DALA would be subject to oxidation at its 5-met residue under similar conditions. Our results indicate that spontaneous oxidation of even a small fraction of a D A L A test solution would result in a serious over-estimation of the analgesic or cataleptic potency of DALA. For example, assuming that the analgesic effects of DALA and DALA(0) are simply additive, it can be estimated that a 3% impurity of DALA(0) in experimental samples of DALA would impart equipotence between D A L A and morphine in the hotplate test (1.25 nmol of DALA(0) in a 40 nmol D A L A sample being equipotent with about 40 nmol of morphine i.v.t.; see Table i). Whether DALA and DALA(0) differ in potency in other measures of opioid activity remains to be determined. In any case, the purity of DALA solutions should be examined before biological activity is measured. The m e c h a n i s m for the enhancement of DALA-induced analgesia and catalepsy by met oxidation is unclear. One possibility is that sulfoxide derivative possesses higher affinity for the opiate receptor m e d i a t i n g analgesia and c a t a l e p s y than the reduced peptide. However, structure-activity studies (12) have shown that opiate receptor binding affinity decreases with decreasing l i p o p h i l i c i = y of the 5 residue of enkephalin and since met(0) is less lipophilic than met, this observation probably does not explain the enhanced potency of DALA(0). A second possibility is that oxidation stabilizes the peptide against proteolytic enzymes of the brain. Recent evidence suggests

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that the enkephalins are catabolized in vivo by a dipeptidyl carboxypeptidase (13,14). An alteration in the electronic character of the C-terminal residue in the peptide, as would result from met oxidation, might confer protection against a carboxy terminal-directed exopeptidase. Indeed, the prolonged duration of action of DALA(0) compared to DALA indicates that this explanation is feasible. Alternatively, a change in the permeability of the peptide from the lateral ventricle to the active sites responsible for the opioid analgesia and catalepsy may explain the enhanced potency of DALA(0). Acknowledgements This study was supported in part by a Biomedical Research Support Grant RR05374 and by a University of Kentucky Tobacco and Health Research Grant from the Kentucky Tobacco Research Board to E.T.I. Portions of this work were done in partial fulfillment of the requirements for the Ph.D. degree (J.A.K.-R.). J.A.K.-R. is a recipient of an Advanced Predoctoral Fellowship from the Pharmaceutical Manufacturers Association Foundation, 1980-1982. An abstract of this work appears in the Pharmacologist 24:229, 1982. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii.

12.

13. 14.

V. CLEMENT-JONES, P.J. LOWRY, L.H. REES and G.M. BESSER, Nature 283, 295297 (1980). V. CLEMENT-JONES, P.J. LOWRY, L.H. REES and G.M. BESSER, J. Endocr. 86, 231-243 (1980). J.A. KING and R.P. MILLAR, Peptides i, 211-216 (1980). N. LING and R. GUILLEMIN, Proc. natn. Acad. Sci. U.S.A. 73, 3308-3310 (1976). D. de WIED, B. BOHUS, J.M. van REE, and I. URBAN, J. Pharmac. exp. Ther., 204, 570-580 (]978). C.B. PERT, A. PERT, J.-K. CHANG and B.T.W. FONG, Science 194, 330-332 (1976). T.H. LIAO, G.W. ROBINSON and J. SALINIKOW, Analyt. Chem. 45, 2286-2288 (1973). N.P. NEUMANN, Meth. Enzym. ii, 487-490 (1967). R. FOG, Acta neurol, scand. 48 (Suppl. 50), 7-66 (1972). J.P. O'CALLAGHAN and S.G. HOLTZMAN, J. Pharmac. exp. Ther. 192, 497-505 (1975). A. HERZ and J. BLAsIG, J., in Endorphins in Mental Health Research, E. Usdin, W.E. Bunney, and N.S. Kline, Eds., pp. 269-278, Oxford University, New York (1979). C.R. BEDDELL, R.B. CLARK, G.W. HARDY, L.A. LOWE, F.B. UBATUBA, J.R. VANE, S. WILKINSON, K.-J. CHANG, P. CUATRECASAS and R.J. MILLER, Proc. R. Soc. B198, 249-265 (1977). A. GUYON, B.P. ROQUES, F. GUYON, A. FOUCAULT, R. PERDRISOT, J.-P. SWERTS and J.-C. SCHWARTZ, Life Sci. 25, 1605-1612 (1979). Z. VOGEL and M. ALTSTEIN, Prog. biochem. Pharmac. 16, 49-59 (1980).