Pentapeptide With Morphine- Like Activity

Enkephalin, a natural opiate receptor agonist in brain, has been identified [see Hughes et al., Nature, 256, 577(1975)] as a mixture of two pentapeptides: H-Tyr-Gly-Gly-Phe-Met-OH (methionine-enkephalin) and H-Tyr-Gly-Gly-Phe-Leu-OH (leucine-enkephalin). Both peptides mimic the ability of morphine to block electrically evoked contractions of mouse vas deferens and guinea pig ileum, and both inhibit the stereospecific receptor binding of the opiate antagonist 3H-naloxone in brain homogenates.

It has been proposed that enkephalin receptors may be sites at which morphine-like drugs exert their analgesic activities, and that enkaphalin may be the modulator or transmittor in brain systems for pain suppression or analgesia. It has been reported that methionine-enkephalin and leucine-enkephalin, when administered by injection in the brain ventricle in rats, induce a profound analgesia that is fully reversible by naloxone. [See Beluzzi et al., Nature, 260, 625(1976)]. The enkephalins are inactive peripherally, however, and it is believed that the enkephalins are rapidly destroyed by blood enzymes and/or are poorly transported across the blood-brain barrier.

The amino acid sequence of methionine-enkephalin is identical to that of the N-terminal portion of the C-fragment (.beta.-endorphin or .beta.-LPH[61-91]) of the peptide .beta.-lipotropin, which is found in large concentrations in the pituitary and in much lower concentrations in the brain. Other naturally-occuring fragments of .beta.-lipotropin are known, for example: .alpha.-endorphin (.beta.-LPH[61-76]) and .gamma.-endorphin (.beta.-LPH[61-77]). Both .beta.-lipotropin and the endorphins show morphine-like properties in various test systems, and it has been suggested that methionine-enkephalin is a breakdown product of the large opiate-like peptides. Enkephalin, its relationship to .beta.-lipotropin and the endorphins, and the pharmacological properties thereof, are reviewed in an article by Iversen et al., Nature, 262, 738(1976). Recent developments are also described in detail in the "Proceedings of the International Narcotics Research Club Meeting, Abderdeen, U.K., July 19-22, 1976," published in OPIATES AND ENDOGENOUS OPIOID PEPTIDES, North Holland Publishing Company, Amsterdam, 1976.

Various structural variations of methionine-enkephalin and leucine-enkephalin are described in the literature. For example, the pentapeptide H-Tyr-Gly-Gly-Phe-Thr-OH, wherein the fifth amino acid residue (methionine or leucine) is replaced by threonine, is described by Chang et al., Life Sciences, 18, 1473 (1976).

The present invention relates to a structural modification of methionine-enkephalin and of leucine-enkephalin wherein the fifth amino acid (methionine or leucine, respectively) is replaced by serine. In accordance with the invention there is provided a pentapeptide of the Formula:

or a non-toxic salt thereof. All chiral amino acid residues identified in Formula I, and in the other formulae depicted herein, are in the natural or L-configuration.

The pentapeptide of Formula I, or a non-toxic salt thereof, exerts an analgesic effect as demonstrated in rats using the rat-tail flick method of D'Amour and Smith [D'Amour and Smith, J. Pharmacol. Exp. Ther., 72, 74 (1941)] when the pentapeptide is injected directly into the lateral brain ventricle. In this test method, which is a standard test for analgesia, a light beam is focused on the tip of the rat's tail, and the pain threshold of the animal is measured by the latency of the rat to remove his tail from the noxious heat source. When tested by the rat-tail flick method by intracerebroventricular injection, according to the procedure of Belluzzi et al., Nature, 260, 625 (1976), the pentapeptide H-Tyr-Gly-Gly-Phe-Ser-OH produced the effects set forth in Table I.

The results in Table I show that injection of the pentapeptide H--Tyr--Gly--Gly--Phe--Ser--OH into the lateral brain ventricle produced an increase in pain threshold as indicated by the significant difference between the mean % change in latency for the drug (drug minus baseline) and the mean % change in latency for the vehicle (Ringer's solution minus baseline). Since it has been found that the pentapeptide exhibits weak binding to opiate receptors in vitro and blocks the electrically-induced contraction of guinea pig ileum (which effects are blocked by naloxone), it is suggested that the pentapeptide exerts an opiate-like analgesic effect.

When administered peripherally, the pentapeptide shows either weak or no analgesia as determined in the rat-tail flick test. By subcutaneous (s.c.) injection (10 mg/kg) in saline, the pentapeptide of Formula I produced a weak analgesic effect, as set forth in Table II:

By intravenous (i.v.) injection (5 mg/kg.), the pentapeptide showed no analgesic activity.

Also contemplated by this invention are the salts of the pentapeptide of Formula I with non-toxic, pharmaceutically acceptable acids. Suitable acids, both organic and inorganic, will be readily apparent to one skilled in the art, for example: hydrochloric, hydrobromic, sulfonic, phosphoric, maleic, acetic, citric, benzoic, succinic, malic, ascorbic, and the like. The salts are prepared and isolated by conventional methods.

The symbols used for representing the amino acid residues in Formula I and in the other formulae employed herein are defined according to the IUPAC-IUB Commission on Biochemical Nomenclature Recommendations (1971), Archives of Biochemistry and Biophysics, 150, 1 - 8 (1972).

The pentapeptide of Formula I is prepared by solid-phase techniques well-known in the art of peptide chemistry. The method of synthesis is illustrated in the following examples.

Chloromethylated polystyrene resin (Lab Systems, Inc.) was esterified with Boc-Ser(Bzl)-OH according to Gisin, Helv. Chim. Acta., 56, 1976 (1973). The polystyrene ester (20 g.) was treated according to schedule A for the incorporation of Boc-Phe-OH, Boc--Gly--OH, and Boc-Tyr(Cl.sub.2 Bzl)Gly-OH to give the titled resin.

1. wash with CH.sub.2 Cl.sub.2 .times. 3;

2. Treat with TFA--CH.sub.2 Cl.sub.2 --DTE (1:2:0.5%) for 5min,;

3. Treat with TFA--CH.sub.2 Cl.sub.2 --DTE (1:2:0.5% for 25 min.;

4. Wash with CH.sub.2 Cl.sub.2 .times. 3;

5. Wash with DMF;

6. treat with 12% TEA in DMF twice for 3 min.;

7. Wash with DMF;

8. wash with CH.sub.2 Cl.sub.2 .times. 3;

9. Treat with 4 equivalents of the corresponding amino acid derivative in CH.sub.2 Cl.sub.2 -DMF and 4 equivalents of N-hydroxybenzotriazole and stir for 5 min.;

10. Add in two portions 5 equivalents of DIC dissolved in CH.sub.2 Cl and over a period of 30 minutes. Reaction time 12-18 hours;

11. Wash with DMF .times. 3;

12. wash with CH.sub.2 Cl.sub.2 .times. 3; and

13. Test ninhydrin reaction according to Kaiser et al.,

Annal. Biochem., 34, 595 (1970). In case of incomplete reaction repeat lines 9 to 13 as above.

The abbreviations for solvents or reagents employed in Schedule A are as follows:

Tea: triethylamine

Dic: diisopropyl-carbodiimide

Tfa: trifluoroacetic acid

Dte: dithioerythritol

Dmf: dimethylformamide

The peptidoresin of the previous example (30 g.) was mixed with 30 ml. anisole and then treated with liquid hydrogen fluoride with exclusion of air for 45 minues at room temperature. The excess hydrogen fluoride was removed in vacuo as fast as possible and the residue was extracted with dilute aq. acetic acid then filtered. The filtrate was extracted with ethyl ether to remove some anisole and the aqueous layer was lyophylized to give a white solid. This solid was chromatographed through a column (2.5 .+-. 60 cm.) of Sephadex G-10 and eluted with 15% aq. acetic acid. The fractions contained in tubes 41 to 60 were pooled and lyophylized to yield 400 mg. of the title compound.

Tlc (n-butanol-water-gl. acetic acid, 4:5:1) 0.60

(n-butanol-gl. acetic acid-water-pyridine, 30:60:24:20) 0.65

Amino acid analysis: Ser (1) 1.18, Gly (2) 1.9, Tyr (1) 1, Phe (1) 1.