Inhibitors of thrombin

Details Inhibitors of thrombin Inhibitors of thrombin

1992-02-10

1995-07-18

Warden, Jill

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

In an organic compound

436804, 514 13, 514 12, 514 14, 530324, 530326, A61K 3800, C07K 700

Patent

active

054339405

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD OF INVENTION

This invention relates to novel biologically active molecules which bind to and inhibit thrombin. Specifically, these molecules are characterized by a thrombin anion-binding exosite associating moiety (ABEAM); a linker portion of at least 18.ANG. in length; and a thrombin catalytic site-directed moiety (CSDM). This invention also relates to compositions, combinations and methods which employ these molecules for therapeutic, prophylactic and diagnostic purposes.


BACKGROUND ART

Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood system thromboses constitute major health risks. Such diseases are caused by either partial or total occlusion of a blood vessel by a blood clot, which contains fibrin and platelets.
Current methods for the treatment and prophylaxis of thrombotic diseases involve therapeutics which act in one of two different ways. The first type of therapeutic inhibits thrombin activity or thrombin formation, thus preventing clot formation. These drugs also inhibit platelet activation and aggregation. The second category of therapeutic accelerates thrombolysis and dissolves the blood clot, thereby removing it from the blood vessel and unblocking the flow of blood [J. P. Cazenave et al., Agents Action, 15, Suppl., pp. 24-49 (1984)].
Heparin, a compound of the former class, has been widely used to treat conditions, such as venous thromboembolism, in which thrombin activity is responsible for the development or expansion of a thrombus. Although effective, heparin produces many undesirable side effects, including hemorrhaging and thrombocytopenia. This has led to a search for a more specific and less toxic anticoagulant.
Hirudin is a naturally occurring polypeptide which is produced by the blood sucking leech Hirudo medicinalis. This compound, which is synthesized in the salivary gland of the leech, is the most potent natural inhibitor of coagulation known. Hirudin prevents blood from coagulating by binding tightly to thrombin (K.sub.d =2.sup.-11 M) in a 1:1 stoichiometric complex [S. R. Stone and J. Hofsteenge, "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28 (1986)]. This, in turn, inhibits thrombin from catalyzing the conversion of fibrinogen to fibrin (clot), as well as inhibiting all other thrombin-mediated processes [J. W. Fenton, II, "Regulation of Thrombin Generation and Functions", Semin. Thromb, Hemost., 14, pp. 234-40 (1988)].
The actual binding between hirudin and thrombin is a two-step process. Initially, hirudin binds to a "low" affinity site on the thrombin molecule (K.sub.d =1.times.10.sup.-8 M) which is separate from the catalytic site. This binding involves interaction of structure from the C-terminus of hirudin with an "anion-binding exosite" (ABE) in thrombin [J. W. Fenton, II et al., "Thrombin Anion Binding Exosite Interactions with Heparin and Various Polyanions", Ann New York Acad. Sci., 556, pp. 158-65 (1989)]. Following the low affinity binding, the hirudinthrombin complex undergoes a conformational change and hirudin then binds to the "high" affinity site on thrombin IS. Kono et al., "Analysis of Secondary Structure of Hirudin and the Conformational Change Upon Interaction with Thrombin" Arch Biochem Biophys., 267, pp. 158-66 (1988)]. This latter site corresponds to the active site of thrombin.
The isolation, purification and chemical composition of hirudin are known in the art [P. Walsmann and F. Markwardt, "Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie, 36, pp. 653-60 (1981)]. More recently, the complete amino acid sequence of the polypeptide has been elucidated [J. Dodt et al., "The Complete Covalent Structure of Hirudin: Localization of the Disulfide Bonds" Biol. Chem. Hoppe-Seyler, 366, pp. 379-85 (1985); S. J. T. Mao et al., "Rapid Purification and Revised Amino Terminal Sequence of Hirudin: A Specific Thrombin Inhibitor of the Blood-Sucking Leech" Anal Biochem, 161,

REFERENCES:
S. Bajusz et al., "Thrombin Inhibition by Hirudin Fragments: Possible Mechanism of Hirudin-Thrombin Interaction" In Peptides 1984, Ragnarsson (ed.), Almqvist and Wiksell International, pp. 473-476 (1984).
J.-Y. Chang, "The Functional Domain of Hirudin, a Thrombin--Specific Inhibitor", FEBS Lett., 164, pp. 307-313 (1983).
J. DiMaio et al., "Bifunctional Thrombin Inhibitors Based on the Sequence of Hirudin.sup.45-65 ", J. Biol. Chem., 265, pp. 21698-21703 (1990).
C. Kettner et al., "D-Phe-Pro-ArgCH.sub.2 Cl--A Selective Affinity Label for Thrombin", Thromb. Res., 14, pp. 969-973 (1979).
J. L. Krstenansky et al., "Antithrombin Properties of C-Terminus of Hirudin Using Synthetic Unsulfated N.sup..alpha. -acetyl-hirudin.sub.45-65 ", FEBS Lett., 211, pp. 10-16 (1987).
J. M. Maraganore et al., "Anticoagulant Activity of Synthetic Hirudin Peptides", J. Biol. Chem., 264 pp. 8692-8698 (1989).
F. Markwardt. "Pharmacology of Selective Thrombin Inhibitors", Nouv. Rev. Fr. Hematol., 30, pp. 161-165 (1988).
Rauber, P., Wikstrom, P., and Shaw, E. (1988) Analytical Biochem. 168,259-264.
Dodt, J., Muller, H.-P., Seemuller, U., and Chang, J.-Y. (1984) FEBS Lett. 165, 180-184.

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