[Met]Enkephalin-Arg6-Phe7 (Opioid Peptide/Adrenal/Brain) CHARLES E

Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp. 5512-5514, September 1980 Neurobiology

Analgesic activity of the naturally occurring heptapeptide [Met]enkephalin-Arg6-Phe7 (opioid peptide/adrenal/brain) CHARLES E. INTURRISI*, JASON G. UMANS*, DOUGLAS WOLFF*, ALVIN S. STERNt, RANDOLPH V. LEWISt, STANLEY STEINt, AND SIDNEY UDENFRIENDt *Department of Pharmacology, Cornell University Medical College, New York, New York 10021; and tRoche Institute of Molecular Biology, Nutley, New Jersey 07110 Contributed by Sidney Udenfriend, May 20,1980 ABSTRACT [Met]Enkephalin-Arge-Phe7 is an opiate-like (Garden City, NY). Prostaglandin E2 was a gift from Upjohn. peptide normally found in We adrenal gland and brain that has [Met]Enkephalin-Arg6-Phe7 was synthesized by Peninsula analgesic (antinociceptive) activity when administered directly into the cerebral ventricles of mice. On a molar basis, [Met- Laboratories and purified by us by high-performance liquid enkephalin-Arg6-Phe7, with a median effective dose (EDso) of chromatography (7). 38.5 nmol/mouse, is 8 times more potent than [Metlenkephalin. Drug Administration. Intracerebralventricular (i.c.v.) in- As with [Metlenkephalin, analgesic activity is blocked by nal- jections were made by a modification of Pedigo et al. (9) of the oxone and intravenous administration does not produce char- method of Haley and McCormick (10). Intravenous (i.v.) in- acteristic opiate effects in tests for analgesic, antidiuretic, or antidiarrheal activity. These findings suggest that [Meten- jections were made into a lateral tail vein. Rats were prepared kephalin-Arg6-Phe7 may be at least as important as the en- for the i.v. infusion of drug by cannulation of the right external kephalins in the postulated enkephalin system mediating pain jugular vein. and analgesia. Analgesic (Antinociceptive) Assay. Analgesic activity after i.c.v. administration of opioid was measured by the radiant-heat The pharmacologic characterization of the opiate-like penta- tail-flick procedure of D'Amour and Smith (11) as modified by peptides [Met]enkephalin and [Leu]enkephalin revealed that Dewey et al. (12). A control latency of 2-4 sec and a cutoff time intracerebralventricular (1, 2) or spinal subarachnoid (3) ad- of 10 sec were used. The response was calculated as the per- ministration can produce a behaviorally defined analgesia in centage of maximal response by the following formula (13): response to nociceptive stimuli. The opiate actions of these [(test - control)/(10 - control)] X 100 = % maximal possible peptides suggest that they may be endogenous ligands for the effect. Dose-response curves were constructed by plotting the opiate receptor. The distribution of enkephalin-containing percent maximal possible effect against the logarithm of the neurons, the transient analgesic action of [Metlenkephalin and dose on probit paper. The median effective dose (ED5o), slope, [Leu]enkephalin in laboratory animals, and reports of en- ratios with their 95% confidence limits kephalin-like material in human cerebrospinal fluid during and relative potency stimulation produced analgesia have led some workers to pro- were calculated by the method of Litchfield and Wilcoxon (14). pose that the enkephalins are involved in brain systems mod- At least eight mice were tested at each dose with three or four ulating the perception of pain (4, 5). Although the enkephalins dose levels used to determine each ED5o. were first isolated from mammalian brain (6), they are now Antidiarrheal Assay. Antidiarrheal activity after i.v. ad- known to be present in other tissues, including intestine and ministration of opioid was assessed by the suppression of pros- adrenal medulla (7). Recent studies of the biosynthetic pathway taglandin E2-induced diarrhea, by the method of Sanner of the enkephalins by Stern et al. (7) led to the isolation from (15). adrenal chromaffin granules of several novel enkephalin-con- Antidiuretic Assay. Antidiuretic activity after i.v. admin- taining peptides and proteins. One of these, the heptapeptide istration of opioid was assessed by the prolongation of the ex- [Met]enkephalin-Arg6-Phe7, which is also present in beef stri- cretion of intraperitoneally administered water, by the method atum, human putamen, and human globus pallidus (8), has been of Inturrisi and Fujimoto (16). chemically synthesized in amounts sufficient for pharmaco- logical characterization. RESULTS AND DISCUSSION MATERIALS AND METHODS The analgesic dose-response curves after i.c.v. administration of the compounds of interest are given in Fig. 1. Each produced Male Swiss Webster mice (20-30 g) and male Sprague-Dawley a dose-related inhibition of the tail-flick response of mice. The rats (200-250 g), purchased from Taconic Farms (Germantown, effects of [Met]enkephalin-Arg6-Phe7 and [Metlenkephalin NY), were used in these experiments. 10 The anal- [Met]Enkephalin was purchased from Boehringer peaked at 2-4 min and were dissipated by min. Mannheim, camel f3-endorphin (i3-endorphin) from Peninsula gesic effect of morphine and (3c-endorphin reached a peak later Laboratories (San Carlos, CA), and morphine sulfate from (10-15 min) and lasted substantially longer (60 min). Analysis Mallinckrodt. Naloxone-HCl was a gift from Endo Laboratories of the data revealed no significant deviation from parallelism; the ED50 and relative potency estimates are presented in Table was The publication costs of this article were defrayed in part by page 1. On a molar basis, [Met]enkephalin-Arg6-Phe7 approxi- charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: ic.v., intracerebralventricular; i.v., intravenous; this fact. 3C-endorphin, camel 3-endorphin; EDso, median effective dose. 5512 Downloaded by guest on September 26, 2021 Neurobiology: Inturrisi et al. Proc. Natl. Acad. Sca. USA 77 (1980) 5513 100

00 0~

40-

0 0.01 0.05 0.1 0.5 1 5 10 50 100 500 1000 Dose, nmol/mouse

FIG. 1. Dose-response curves after i.c.v. administration to mice of3,B-endorphin (0), morphine (3), [Met]enkephalin-Arg6-Phe7 (a), and [Met]enkephalin (X).

mately 8 times more active than [Metlenkephalin but 1/63rd in mice [Met]enkephalin has little or no systemic antidiarrheal as potent as morphine and 1/1156th as potent as flc-endorphin. activity whereas j3-endorphin is active. In rats the i.v. infusion In vivo estimates of relative potency are the result of receptor of 0.10 mg of [Met]enkephalin-Arg6-Phe7 per min for 30 min interactions and pharmacokinetic factors. Thus, it is not sur- did not produce analgesia or gross behavioral changes. Un- prising that the more labile pentapeptides and presumably doubtedly, the absence of effects is due predominantly to rapid more labile [Met]enkephalin-Arg6-Phe7 have a shorter duration degradation of [Met]enkephalin-Arg6-Phe7 by blood and tissue of action and are less potent when compared to morphine or as is the case with the enkephalins. f3-endorphin (17). [Met]Enkephalin-Arg-Phe7 is present in human putamen Pretreatment with naloxone, an opiate antagonist, diminished and beef striatum in amounts comparable to that of [Leu]- or prevented the response to approximately equianalgesic doses enkephalin and about one-fourth that of [Metlenkephalin (8). of [Met]enkephalin-Arg6-Phe7 and morphine (Table 2). Pre- Although [Met]enkephalin-Arg6-Phe7 can be converted in vitro treatment with naloxone or saline followed by i.c.v. adminis- to [Metlenkephalin by digestion with trypsin and carboxy- tration of saline was without effect (not shown in Table 2). A peptidase B, the greater analgesic potency of the heptapeptide larger dose of naloxone was required to block the analgesic in vivo argues against its action being merely that of a metabolic response to [Metjenkephalin-Arg6-Phe7 compared to morphine. intermediate. Thus, in the radioreceptor binding assay using This suggests that different receptors or binding affinities may neuroblastoma-glioma hybrid cells with [3H]tyrosine-labeled be involved. [Leu]enkephalin as the competing ligand, the concentration Because [Metjenkephalin-Arg6-Phe7 is present in the chro- of [Metlenkephalin and [Leujenkephalin necessary for 50% maffin granules of the adrenal medulla and stimulation of the inhibition is 3.5 nM and that of [Met]enkephalin-Arg6-Phe7 is adrenal causes release of enkephalins (8) and their precursors, 6.8 nM (8). In addition to [Met]enkephalin-Arg6-Phe7, several a consideration of the systemic action seemed appropriate. We opioid hexapeptides and another heptapeptide have been iso- found that neither [Met]enkephalin-Arg6-Phe7 nor [Met]en- lated from the adrenal medulla and demonstrated in brain as kephalin produced characteristic opiate effects after systemic well (8). administration. Doses of the heptapeptide up to 11.4 Mmol/kg Whatever their physiologic roles, [Met]enkephalin-Arge-Phe7 (10 mg/kg) did not produce analgesic, antidiuretic, or antidi- and the other opioid heptapeptides and hexapeptides contribute arrheal effects in mice whereas equimolar doses of morphine to the mapping of the enkephalin pathway in the central ner- produced a significant effect in each of these opiate tests. These results are consistent with the report of Dairman et al. (18) that Table 2. Naloxone blockade of analgesia

Table 1. Analgesic activity after i.c.v. administration Pretreatment* Analgesic testedt MPEt Time of Saline Morphine, 0.87 nmol 61 peak Naloxone, 2 mg/kg Morphine, 0.87 nmol 0 effect, ED5o,* Relative Saline [Met]Enkephalin-Arg6-Phe7, 72 Drug min nmol/mouse potency 57.4 nmol Naloxone, 2 mg/kg [Met]Enkephalin-Arg6-Phe7, 27 Morphine 20 0.63 (0.29-0.87) 1 57.4 nmol [Met]Enkephalin-Arg6- Naloxone, 4 mg/kg [Met]Enkephalin-Arg6-Phe7, 5 Phe7 2-4 38.5 (23.7-62.3) 0.016 57.4 nmol [Met]Enkephalin 4 330.0 (248-439) 0.0019 * 15 0.034 (0.028-0.046) 18.5 Naloxone was administered subcutaneously. #,-Endorphin t Doses are expressed as nmol/mouse i.c.v. * 95% confidence limits are given in parentheses. MPE, maximal possible effect. Downloaded by guest on September 26, 2021 5514 Neurobiology: Inturrisi et al. Proc. Natt. Acad. Sci. USA 77 (1980) vous system which is carried out by immunohistochemical 8. Stern, A. S., Lewis, R. V., Kimura, S., Rossier, J., Stein, procedures directed toward the pentapeptide sequence. S. & Udenfriend, S. (1980) Arch. Biochem. Biophys., in press. This work was supported in part by National Institute of Drug Abuse 9. Pedigo, N. W., Dewey, W. L. & Harris, L. S. (1975) J. Pharmacol. Grants DA-01457 and GM-26145. J.G.U. is the recipient of an Insur- ance Medical Scientist Scholarship Fund award from the Prudential Exp. Ther. 193,845-852. Insurance Company. 10. Haley, T. J. & McCormick, W. G. (1957) Br. J. Pharmacol. 12, 12-15. 1. Belluzzi, J. D., Grant, N., Garsky, V., Sarantakis, D., Wise, C. D. 11. D'Amour, F. E. & Smith, D. L. (1941) J. Pharmacol. Exp. Ther. & Stein, L. (1976) Nature (London) 260,625-626. 72,72-79. 2. Roemer, D., Buescher, H. H., Hill, R. C., Pless, J., Bauer, W., 12. Dewey, W. L., Harris, L. S., Howes, J. F. & Nuite, J. A. (1970) Cardinaux, F., Closse, A., Hauser, D. & Huguenin, R. (1977) J. Pharmacol. Exp. Ther. 175,435-442. Nature (London) 268,547-549. 13. Harris, L. S. & Pierson, A. K. (1964) J. Pharmacol. Exp. Ther. 143, 3. Yaksh, T. L., Huang, S. P., Rudy, T. A. & Frederickson, R.C.A. (1977) Neuroscience 2,593-596. 141-148. 4. Hokfelt, T., Ljungdahl, A., Terenius, L., Elde, R. & Nilsson, G. 14. Litchfield, J. T., Jr. & Wilcoxon, F. (1949) J. Pharmacol. Exp. (1977) Proc. Natl. Acad. Sci. USA 74,3081-3085. Ther. 96,99-113. 5. Akil, H., Richardson, D. E., Hughes, J. & Barchas, J. D. (1978) 15. Sanner, J. H. (1972) Intra-Sci. Chem. Rep. 6, 1-9. Science 201, 463-465. 16. Inturrisi, C. E. & Fujimoto, J. M. (1968) Toxicol. Appl. Phar- 6. Hughes, J., Smith, L. W., Kosterlitz, H. W., Fothergill, L. A., macol. 13,251-257. Morgan, B. A. & Morris, H. R. (1975) Nature (London) 258, 17. Doneen, B. A., Chung, D., Yamashiro, D., Law, P. Y., Loh, H. 577-579. H. & Li, C. H. (1977) Bwochem. Blophys. Res. Commun. 74, 7. Stern, A. S., Lewis, R. V., Kimura, S., Rossier, J., Gerber, L. D., 656-662. Brink, L., Stein, S. & Udenfriend, S. (1979) Proc. Nati. Acad. Sci. 18. Dairman, W., Kappell, B. & Smolen, M. (1979) Pharmacologist USA 76,6680-6683. 21, 198. Downloaded by guest on September 26, 2021