Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-09-20
2002-09-17
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S350000, C530S300000, C435S006120, C435S007100, C536S023100, C514S002600
Reexamination Certificate
active
06451976
ABSTRACT:
This application is a national stage application of PCT/GB 98/00848, filed Mar. 20, 1998, which claims foreign priority to GB 97 05787.1 filed Mar. 20, 1997.
The present invention relates to dendroaspin-based chimeric molecules which have anticoagulant, antiplatelet and other activities. The invention also relates to nucleic acid molecules encoding these chimeric dendroaspin molecules, cloning and expression vectors comprising such nucleic acids and host cells transformed with expression vectors so as to provide recombinant chimeric multifunctional dendroaspin. The invention further relates to pharmaceutical compositions comprising chimeric dendroaspin for use in the prevention or treatment of disease associated with thrombus formation or platelet aggregation. The invention also further relates to the use of a dendroaspin scaffold in the design and generation of chimeric dendroaspin derivatives having inhibitory activity against integrin binding activity of platelets plus some further functionality such as an anticoagulant or antithrombotic action.
The role of blood coagulation is to provide an insoluble fibrin matrix for consolidation and stabilization of a haemostatic plug. Formation of a cross-linked fibrin clot results from a series of biochemical interactions involving a range of plasma proteins.
Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis and peripheral arterial occlusion are caused by either partial or total occlusion of a blood vessel by a blood clot.
The formation of a blood clot within a blood vessel is termed thrombosis and is dependent upon platelet aggregation. In the context of blood vessel injury (such as that which might arise in surgical procedures) the interaction of blood platelets with the endothelial surface of injured blood vessels and with other platelets is a major factor in the course of development of clots or thrombi.
Various agents for preventing formation of blood clots are now available, such as aspirin, dipyridamole and filopidine. These products generally inhibit platelet activation and aggregation, or delay the process of blood coagulation but they have the potential side effect of causing prolonged bleeding. Moreover, the effect of such products can only be reversed by new platelets being formed or provided.
Platelet aggregation is dependent upon the binding of fibrinogen and other serum proteins to the glycoprotein receptor IIb/IIIa complex on the platelet plasma membrane. GP IIb/IIIa is a member of a large family of cell adhesion receptors known as integrins, many of which are known to recognize an Arg-Gly-Asp (RGD) tripeptide recognition sequence.
Heparin and low molecular weight heparins have been used widely 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 haemorrhaging and thrombocytopenia. A more specific and less toxic anticoagulant is therefore required.
Direct thrombin inhibitors are available and examples of these are hirudin, hirugen and hirulog (the latter two being synthetic hirudin derivatives), PPACK (a synthetic tripeptide) and argatroban (an arginine derivative). The actions of these inhibitors are reviewed in Lefkovits J and Topol E J (1994), Circulation 90:1522-1536. Although in theory, the bleeding risk with direct thrombin inhibitors is lower than with other antithrombotics because of their mono-target specificity, absence of direct platelet effects, and short half-life, bleeding still remains as the most concerning adverse effect.
There are a range of other thrombin inhibitors which have been developed (listed in table 1 of Lefkovits J and Topol E J supra) but these have turned out to be just too toxic for clinical use.
Localized narrowing of an artery caused by atherosclerosis is a condition which can usually be remedied surgically by the technique of balloon angioplasty. The procedure is invasive and causes some tissue damage to the arterial wall which can result in thrombus formation. Extracellular proteins such as fibronectin in the arterial wall become exposed to blood in the artery. Platelets bind to the RGD motif of fibronectin via integrin receptors which in turn leads to platelet aggregation and the start of the cascade of clotting reactions. An agent which specifically inhibits platelet aggregation at the sites of damage and which also inhibits clotting at these sites is required. The agent should be non-toxic and free of undesirable side effects such as a risk of generalized bleeding.
Integrins are a family of cell surface receptors that mediate adhesion of cells to each other or to the extracellular matrix (Kieffer N & Philips D R (1990) Annu Rev Cell Biol 6: 329-357; Hynes R O (1992) Cell 69: 11-25; McEver R P (1992) Curr Opin Cell Biol 4: 840-849; Smyth S S et al (1993) Blood 81: 2827-2843; Giancotti F G and Mainiero F (1994) Biochim Biophys Acta 1198: 47-64). They are composed of noncovalently associated &agr; and &bgr; transmembrane subunits. There exist 16 different &agr; and &bgr; different &bgr; subunits that heterodimerize to produce about 20 different kinds of receptors (Clark E A & Brugge J S (1995) Science 268: 233-239). Among the integrins, the platelet membrane integrin &agr;
IIb
&bgr;
3
is one of the best characterized. Upon cell activation, the &agr;
IIb
&bgr;
3
integrin binds several glycoproteins, predominantly through the Arg-Gly-Asp (RGD) tripeptide sequence (Pierschbacher M D and Ruoslahti E (1984) supra; Plow E F et al (1987) Blood 70: 110-115; Pytela R et al (1986) Science 231: 1559-1562) present in fibrinogen (Nachman R L and Nachman L L K (1992) J Clin Invest 69: 263-269), fibronectin (Gardner J M and Hynes R O (1985) Cell 42: 439-448), von Willebrand factor (Ruggeri Z et al (1983) J Clin Invest 72: 1-12), vitronectin (Pytela R M et al (1985) Proc Natl Acad Sci USA 82: 5766-5770), and thrombospondin (Karczewski J et al (1989) J Biol Chem 264: 21322-21326). The nature of the interactions between these glycoprotein ligands and their integrin receptors is known to be complex, and conformational changes occur in both the receptor (Sims P J et al (1991) J Biol Chem 266: 7345-7352) and the ligand (Ugarova T et al (1995) Thromb Haemostasis 74: 253-257).
Recently, many proteins from a variety of snake venoms have been identified as potent inhibitors of platelet aggregation and integrin-dependent cell adhesion. The majority of these proteins which belong to the so-called “disintegrin” family share a high level of sequence homology, are small (4-8 kDa), cysteine-rich and contain the sequence RGD (Gould R J et al (1990) Proc Soc Exp Biol Med 195: 168-171) or KGD (Scarborough R M et al (1991) J Biol Chem 266: 9359-9362). In addition to the disintegrin family, a number of non-disintegrin RGD proteins of similar inhibitory potency, high degree of disulfide bonding, and small size have been isolated from both the venoms of the Elapidae family of snakes (McDowell R S et al (1992) Biochemistry 31: 4766-4772; Williams J A et al (1992) Biochem Soc Trans 21: 73S) and from leech homogenates (Knapp A et al (1992) J Biol Chem 267: 24230-24234). All of these proteins are approximately 1000 times more potent inhibitors of the interactions of glycoprotein ligands with the integrin receptors than simple linear RGD peptides; a feature that is attributed to an optimally favourable conformation of the RGD motif held within the protein scaffold. The NMR structures of several inhibitors including kistrin (Adler M et al (1991) Science 253: 445-448; Adler M and Wagner G (1992) Biochemistry 31: 1031-1039; Adler M et al (1993) Biochemistry 32: 282-289), flavoridin (Senn H and Klaus W (1993) J Mol Biol 234: 907-925), echistatin (Saudek V et al (1991) Biochemistry 30: 7369-7372; Saudek V et al (1991) Eur J Biochem 202: 329-328; Cooke R M et al (1991) Eur J Biochem 202: 323-328; Cooke R M et al (1992) Protein Eng 5: 473-477), albolabrin (Jaseja M et al (1993) Eur J Biochem 218: 853-860), deco
Authi Kalwant S.
Kakkar Vijay V.
Lu Xinjie
Scully Michael F.
Carlson Karen Cochrane
Dorsey & Whitney LLP
Robinson Hope A.
Trigen Limited
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