Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-09-17
2004-08-10
Wax, Robert A. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000, C424S094640
Reexamination Certificate
active
06774107
ABSTRACT:
SUMMARY OF THE INVENTION
A naturally occuring protein isolated from the saliva of the medicinal leech
Hirudo medicinalis
is described which strongly binds to collagen thus acting as an inhibitor of natural platelet adhesion to collagen. The protein has a molecular weight of about 12 000, an acidic isoelectric point and contains six cysteins. The protein was sequenced and the gene was cloned from a
H. medicinalis
cDNA-library. Procedures for producing such polypeptide by recombinant techniques are disclosed. The recombinant and the natural occuring proteins are potent inhibitors of collagen-dependent platelet adhesion and therefore useful for the therapeutic treatment of various conditions related to hard disease and diseases of the circulation system. Furthermore the protein is useful for coating natural or artificial collagen surfaces in order to render them nonadhesive for cells and prevent the activation of cells.
FIELD OF INVENTION
In haemostasis or thrombosis platelets adhere to the cell-extracellular matrix of an injured vessel and cover the surface of the damaged area. Preventing this important initial step in the pathogenesis of thrombosis and arterial occlusion should be of therapeutic benefit in the effort of to prevent thrombotic diseases. Collagen is considered to be the most thrombogenic surface component and has been shown to be a strong stimulant for platelet adhesion, aggregation and the release of their granules leading to the recruitment of (Ruggeri, Z. M. et al.; Seminars in Hematology, 1994, 31, 229-39) additional platelets to this area to form aggregates or a thrombus. The initial contact of the platelets to the vessel surface is mediated by collagen bound van Willebrand Factor (vWF) and a specific vWF receptor on platelets, the glycoprotein Ib-V-IX complex. In addition ADP, epinephrine and circulating clotting factors drive the further activation process of platelets while simultaneously an increase in thrombin activity contributes to the formation of the cross-linked fibrin clot. Platelet-platelet aggregation supports this process and is mainly driven by fibrinogen as a mediator that bridges cells through the glycoprotein IIb/IIIa receptor.
This normal physiological response is quite critical in the course of the pathological process where platelets adhere to collagenes exposend in sclerotic lesions (Van der Rest M. et al.; FASEB Journal, 1991, 5, 2814-23) and start to bild-up occlusions. Depending on the location and extent of the occlution severe complications such as myocardial infarction, stroke, inflammation or pulmonary embolism may be the severe outcome of this process.
As a direct acting antithrombotic agent heparin which blocks the thrombin activity, thus preventing the formation of fibrin rich thrombi, is the currently most well known drug used in anti-thrombotic interventions. Heparin is widely used in indications such as: unstable angina and acute myocardial infarction. However despite the wide use several severe short comings such as intravenous application, requirement for anti-thrombin-III as a cofactor, reduced affinity for clot-bound thrombin, it's inactivation by several plasma proteins, the occasional induction of thrombocytopenia and it's biological heterogeneity remain unresolved. As a consequence the results of using heparin in the clinical setting have not been overvelming sofar.
Recent development of low molecular weight heparin has contributed a version for subcutaneous application, however the therapeutic benefit over the standard heparin has been modest. Unfortunately the same applies to the other directly acting antithrombins such as Hirudin, Hirulog and Warfarin. It turned out, that one of the major problems seems to be related to the increased production of thrombin under antithrombotic treatment (Rao, A. K et al., Circulation, 1996, 94, 389-2395).
Other recent strategies have therefore been focussed to the process of prothrombin activation which is driven by Factor Xa. The major challenge is the design of appropriate inhibitors directed to this factor. In summary on would therefore argue that the full therapeutic potential of this type of intervention has not yet been realized.
Another pannel of therapeutics is represented by the thrombolytic regimens and has been focussed on the development of staphylokinase, streptokinase, urokinase type Plasminogen Activator, tissue type Plasminogen Activator and anisoylated-plasminogen-streptokinase activator complex. The differences in time necessary to inducing reperfusion is remarkable different for each of these thrombolytic agents, however the contribution in terms of reducing the overall mortality is equal for all the products. In addition reocclusion or prolonged bleeding are frequent complications. This might be due to relatively low specificity for fibrin and the short plasma half-life of these compounds. Currently various application regimens and combinations of different fibrinolytic principles are tested in order to overcome some of the current short comings in thrombolytic therapie. The improvement which are expected are however rather small.
Recently a new group of patients occured with probelms such as acute thrombotic occlusion and late restenosis due to procedures such as angioplasty, atherectomy, arterial grafting or vessel wall stenting. The possible therapeutic interventions comprise anti-platelet, antithrombotic and thrombolytic strategies. Various other agents such as ticlopdine acting as ADP antagonists or Calcium ionophore A-23187 and especially Asprin have a direct influence on the platelet function and have been suggested or used to prevent or minimize platelet aggregation. The new anti-platelet adhesion substance according to this invention could as well help to overcome these clinical complications when applied during surgery.
Another complication related to this topic arises if artificial surfaces come in contact with blood, then their is increased tendency to induce thrombotic events by activation of platelets and/or induction of coagulation. These effects may cause failure of vascular grafts, cardiac valves, stents, catheters or any other blood contacting device or material. The ability of the protein disclosed here to create non-thrombogenic surfaces may therefore be further exploited by immobilization of this protein to the materials and devices described above Such a treatment should render such materials or devices biocompatible and thromboresistant.
Due to the limitations associated with the available antithrombotic agents there is an actual need for new alternative strategies and therapeutics.
BACKGROUND OF THE INVENTION
A potential for future improvements in the treatement of caridvacular disorders may be contributed by approaches as disclosed in this invention which directely interfere with the collagen and/or vWF factor induced platelet adhesion.
Several novel inhibitors which prevent platelet adhesion are monoclonal to antibodies directed to vWF. It has as well been suggested that glycoprotein IIb/IIIa inhibitors may be beneficial in inhibiting platelet adhesion.
Some of these inhibitors like the monoclonal Ab c7E3 have already been tested clinically while others like the KGD- and RGDF-inhibitors are still under study. However, the specificity of most of these new inhibitors is not very well studied, thus the spectrum of side effects that will be induced by using this inhibitors is still open and deserves carefull examination.
A rich source for the screening of new compounds that interfere with collagen induced platelet adhesion is given in nature through blood-sucking animals. Several inhibitors have been isolated from nature as described in the literature: A 65 kD protein called Calin isolated from
Hirudo medicinalis
(U.S. Pat. No. 5,587,360 WO 92/07005) (Munro, R. et al., Blood Coagulation and Fibrinolysis, 1991, 2, 179-184) and a 16 kD (LAPP) protein isolated from the salivary glands of the leech
Haementera officinalis
(U.S. Pat. No. 5,324,715). Both proteins have been described as aggregation inhibitors as tested in static
Fotev Zisi
Güssow Detlef
Hemberger Jürgen
Hofmann Uwe
Scheuble Bernhard
Merck Patent GmbH
Wax Robert A.
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