Peptides with anticoagulant activity

Details Peptides with anticoagulant activity Peptides with anticoagulant activity

1993-07-29

1995-02-28

Hill, Jr., Robert J.

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

8 to 10 amino acid residues in defined sequence

A61K 3702, C07K 500, C07K 700, C07K 1500

Patent

active

053938739

DESCRIPTION:

BRIEF SUMMARY
Hirudin is a natural substance which has anticoagulant properties and is produced in the salivary glands of the leech Hirudo medicinalis. Hirudin is a mixture of polypeptides with 64-66 amino acids. The main component of this mixture is a polypeptide which has 65 amino acids and which has O-sulfate on tyrosine 63: SEQ. NO. 1 -Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gl y-Cys-Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-G lu-Glu-Ile-Pro-Glu-Glu-Tyr(SO.sub.3 H)-Leu-Gln
Hirudin binds very strongly and with high specificity to thrombin, which is the key enzyme in the coagulation cascade. The thrombin-hirudin complex is no longer able to cleave fibrinogen, the natural substrate for thrombin.
The anticoagulant action suggests that hirudin is a potential drug. However, natural (O-sulfate) hirudin can be isolated from the leeches only in very small yields and with great effort. Recombinant hirudin contains no sulfated tyrosine and has a distinctly less satisfactory binding constant for thrombin.
Hirudin intended for medical uses and isolated from cellular material must be purified with particular care. Residual DNA, foreign proteins, toxins or viral contaminations must be eliminated in all circumstances. In addition, reproducible oral or transdermal administration of a polypeptide of this size is scarcely conceivable, even using pharmaceutical aids.
For the stated reasons, early attempts were made to derive from the hirudin sequence oligopeptides which likewise have anticoagulant properties but can be prepared by complete synthesis and thus at reasonable cost and in high purity.
Many of these peptides disclosed in the literature in fact have thrombin-inhibiting properties (J. Med. Chem. 31 (1988) 1009; EP 291981). However, the potency of these compounds decreases greatly as the chain length is reduced. The EC.sub.100 of the C-terminal decapeptide hirudin.sub.56-65 is about 2000 times that of hirudin.
We have found that with suitable substitution it is possible for the potency of the thrombin-inhibiting action of short peptides derived from the C terminus of hirudin to reach the same order of magnitude as that of hirudin.
The present invention relates to peptides of the formula I ##STR2## where X is H, acetyl, succinyl, maleoyl, phthaloyl or fumaroyl, Glu-NH.sub.2, D-Glu-OH or D-Glu-NH.sub.2 and
The conventional three-letter code is used for abbreviating the amino acids; Cha is cyclohexylalanine and Hyp is trans-4-hydroxyproline. Unless otherwise specified, the amino acids have the L configuration. The unnatural amino acids which are derived from phenylalanine and contain W can have either the L or D configuration.
In formula I, X is preferably succinyl, A.sup.1 is preferably Tyr, A.sup.2 is preferably Glu, A.sup.3 is preferably Pro or Hyp, A.sup.4 is preferably Ile, Val or Cha, A.sup.5 is preferably Hyp, A.sup.6 is preferably Glu, A.sup.7 is preferably Leu, Ile or Cha and Y is preferably OH, Gln-OH, D-Gin-OH, Glu-OH or D-Glu-OH.
Physiologically tolerated acids which may be particularly mentioned are: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonicacid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine.
Suitable physiologically tolerated bases are hydroxides and bicarbonates of the following substances: aluminum, calcium, potassium, lithium, magnesium, sodium, diethanolamine, ethylenediamine and meglumine.
The novel compounds can be prepared by methods known in peptide chemistry.
Thus, the peptides can be assembled sequentially from amino acids or by linking suitable peptide fragments. In the case of sequential assemblage, the peptide chain is extended stepwise by one amino acid each time, starting at the C terminus. In the case of fragment coupling it is possible to link together fragments of different lengths, it being possi

REFERENCES:
Marseigne et al., J. Org. Chem., vol. 53, p. 23621 (1988).
Journal of Medicinal Chemistry, vol. 31, 1988, Jan.-Jun., pp. 1009-1011.

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