Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1993-04-09
1996-12-24
Sayala, Chhaya D.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
514 12, 530350, 530416, 530858, A61K 3858
Patent
active
055873600
DESCRIPTION:
BRIEF SUMMARY
The present invention is concerned with leech-derived proteins or polypeptides capable of binding to biological substrates such as collagen, as well as other substrates (typically strongly charged substrates, such as ionically negatively charged substrates) and inhibiting the adhesion of platelets.
Therapeutic intervention at the level of platelet adhesion/aggregation is useful for the prevention and treatment of most thrombotic diseases. There are many drugs with inhibitory effects on platelet function, such as aspirin, which interferes with the arachidonic pathway by inhibition of the enzyme cyclooxygenase. In the latter pathway, the arachidonic acid (produced by the collagen induced stimulation of phospholipase A.sub.2 within the platelets) is converted through the action of cyclooxygenase to thromboxane A.sub.2.
Leech saliva contains several known agents capable of inhibiting platelet aggregation, notable amongst these being: Hirudo medicinalis. Hirudin prevents thrombin-induced platelet aggregation by binding tightly to thrombin in a 1:1 stoichiometric complex. This in turn inhibits thrombin-catalysed conversion of fibrinogen to fibrin. antagonist derived from saliva of Hirudinidae having inhibitory activity against platelet aggregation induced by aggregating agents such as PAF-acether is disclosed in EP-A-0348208A. hydrolyric scission of peptide bonds in helical regions of the collagen molecule is disclosed in WO87/00860. et al
in Comp. Blochem. Physiol. 87B, pp.567-574, 1987.
However, a substance which can bind to the surface of a biological substrate such as collagen and which has a non-endopeptidase interaction with the biological substrate, such that collagen induced platelet aggregation is inhibited, has not previously been disclosed.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a typical example of a high performance cation exchange chromatography, using a NaCl gradient.
FIG. 2 shows a SDS-PAGE analysis of the product of Type I collagen treated with the protein.
FIG. 3 shows results from assays for activity (by incubation with chromogenic substrate) of crude leech saliva fractions collected from a gel filtration column.
FIG. 4 shows the time dependent flocculation of Type I collagen particles caused by the protein.
FIG. 5A shows the normal pattern of aggregation in human plasma by collagen.
FIG. 5B shows that the doses of the protein (1000-5000 units) have an effect on collagen within 2 mins. such that it is consequently unable to induce platelet aggregation in human plasma.
The present invention is concerned with a protein, derived from leech tissue or leech secretions, having a non-endopeptidase binding mechanism with collagen.
According to the present invention, therefore, there is provided a protein or polypeptide derived from leech tissue or leech secretions, which is capable of binding directly or indirecfiy to collagen, the nature of the protein-collagen binding interaction being such that substantial cleavage of the pepfide bonds present in the collagen molecules does not occur on SDS-PAGE. The binding may be direct to collagen or to an intermediary such as a ligand or Von Willebrand's factor, which intermediary is itself bound to a collagen.
When referring to the protein being "derived" from leech tissue or leech secretions, we intend to encompass not only the isolation of the protein from leech tissue or leech secretions, but also the production of synthetic equivalents by techniques such as recombinant DNA synthesis which involve isolation of the protein and then using the isolated material as the starting material for quantity production of the equivalent protein (and also shortened polypeptide analogues with comparable binding properties).
The present invention therefore further comprises a recombinant or protein engineered equivalent to the leech derived protein according to the invention.
The protein according to the invention has a molecular weight of approximately 65 kilodaltons in reduced form (that is about 60-70 kilodaltons) and is derived from the saliva or tissue of blood
REFERENCES:
patent: 4390630 (1983-06-01), Sawyer et al.
patent: 4588587 (1986-05-01), Gasic
patent: 4832849 (1989-05-01), Cardin
patent: 5114922 (1992-05-01), Maschler et al.
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patent: 5182113 (1993-01-01), Rigbi et al.
patent: 5246715 (1993-09-01), Orevi et al.
Munro et al., Blood Coagulation and Fibrinolysis, vol. 2, pp. 179-184, 1991.
Munro et al., Thrombosis & Haemostasis, vol. 3, pp. 366-369, 1989.
Bagdy et al., "Methods in Enzymology", vol. 45, pp. 669-678, 1976.
Biological Abstracts, vol. 79, No. 11, 1985 (Philadelphia, PA, US) I. P. Baskova et al.: "Secretion of the salivary glands of Hirudo medicinalis inhibits the ADP-induced aggregation of human platelets and platelet adhesion to a collagen-coated surface", abstract 92228, & Byull. Eksp. Biol. Med. 97 (6): 696-699, 1984.
Chemical Abstracts, vol. 107, No. 23, 7 Dec. 1987 (Columbus, Ohio, US) I. P. Baskova et al.: "Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis", abstract 211673n, & Biokhimiya (Moscow) 1987, 52(9), 1461-1468.
Merck Patent GmbH
Sayala Chhaya D.
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