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Thrombus-Targeted Complexes of Plasminogen Activator and Fibrin Fragments

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Thrombus-Targeted Complexes of Plasminogen Activator and Fibrin Fragments Europa,schesP_ MM M II II II M INI MMMMI Ml J European Patent Office ^ _ _ _ © Publication number: 0 473 689 B1 Office_„. europeen des brevets © EUROPEAN PATENT SPECIFICATION © Date of publication of patent specification: 14.12.94 © Int. CI.5: A61 K 37/54, A61 K 37/547, A61K 37/02, C12N 9/50, © Application number: 90908772.8 C12N 9/70 C12N 9/72 C07K 15/06 ©r\ Daten * of,„■ filing: 21.05.90o, neon © International application number: PCT/US90/02781 © International publication number: WO 90/14102 (29.11.90 90/27) (54) THROMBUS-TARGETED COMPLEXES OF PLASMINOGEN ACTIVATOR AND FIBRIN FRAGMENTS. ® Priority: 24.05.89 US 356978 © Proprietor: Temple University of the Common- wealth System of Higher Education @ Date of publication of application: 11.03.92 Bulletin 92/11 Philadelphia PA 19122 (US) © Publication of the grant of the patent: @ Inventor: BUDZYNSKI, Andrei, Z. 14.12.94 Bulletin 94/50 2050 Wharton Road Glenslde, PA 19038 (US) © Designated Contracting States: Inventor: KNIGHT, Linda, C. AT BE CH DE DK ES FR GB IT LI LU NL SE 158 Oak Creek Road East Windsor, NJ 08520 (US) © References cited: Inventor: HASAN, Ahmed, Ak. US-A- 4 427 646 885 North Easton Road US-A- 4 536 391 Apt. 4A6 US-A- 4 673 573 Glenslde, PA 19038 (US) CIRCULATION, vol. 80, no. 4, suppl. 2, Octo- ber 1989, page II-642; A. HASAN etal.: "Bind- © Representative: W.P. THOMPSON & CO. Ing of one-chain and two-chain t-PA to fibrin Eastcheap House fragments" Central Approach 00 Letchworth, Hertfordshire SG6 3DS (GB) o> TAKEDA ET AL, BIOLOGICAL ABSTRACTS, 00 volume 86, number 7, Issued 01 October CO 1988 00 Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid (Art. 99(1) European patent convention). Rank Xerox (UK) Business Services (3. 10/3.09/3.3.3) EP 0 473 689 B1 BODE ET AL, J. Mol. Cell Cardiol., Issued 1987, vol.19, pages 335-341 BODE ET AL, The Journal of Biological Chemistry, Issued 05 August 1987, volume 262, number 22, see pages 10819-10823 deMUNK ET AL, Biochemistry, Issued 1989, vol.28, pages 7318-7325 NIEUWENHUIZEN ET AL, Biochimica et Blophyslca Acta, Issued 1983, vol. 755, pages 531-533 2 1 EP 0 473 689 B1 2 Description solve thrombi if administered locally, but local ad- ministration requires catheterization, which may of- The invention relates to novel agents for throm- ten preclude administration promptly enough to bolytic therapy, more particularly to hybrids formed provide significant benefit to the patient. Systemic by joining plasminogen activators and fibrin frag- 5 administration of streptokinase in acute myocardial ments. infarction causes an undesirable side effect by A safe, effective method of dissolving vascular degrading fibrinogen and severely depleting plas- thrombi is urgently needed, owing to the life-threat- minogen in the circulation. Mentzer et al., Am. J. ening complications of thromboembolic disease. Cardiol. 57:1220-1226 (1986). The most common of such disorders is the forma- io Another widely used plasminogen activator, tion of thrombi, in a blood vessel or heart cavity urokinase, is available in three forms. So called that remain at the point of formation. Thrombi in "two-chain urokinase" (hereinafter "urokinase") can heart vessels, for example, can restrict blood flow, be extracted from human urine or prepared from resulting in myocardial infarction. Coronary ar- kidney cell cultures. Another form, "single-chain teriographic studies indicate that 87% of transmural 75 urokinase" or "pro-urokinase" (hereinafter "scu- myocardial infarctions are caused by occlusive PA"), a precursor of urokinase, has been isolated coronary artery thrombosis. DeWood et al., N. Eng. from human fluids and also obtained by recom- J. Med. 303:397-902 (1980). binant DNA technology. A low molecular weight In addition, parts of a thrombus may dislodge form of urokinase is obtained from long plasmin from its point of attachment and move through the 20 digestion of two-chain urokinase. blood vessels until it reaches a point where pas- Although urokinase is not antigenic, its mecha- sage is restricted. The resulting blockage is known nism of plasminogen activation parallels that of as a thromboembolism. streptokinase, resulting in a significant Plasminogen activators function mainly by con- fibrinogenolytic response in the circulating blood. verting the zymogen plasminogen into plasmin, 25 Single-chain urokinase has promising characteris- which is regarded as the end stage non-specific tics. However, it has not yet been adequately test- proteolytic enzyme in fibrinolysis. Plasmin lyses ed in man. In a small study of normal human the fibrin matrix and acts on other components of subjects, scu-PA displayed fibrinolytic activity com- the thrombus as well. The efficiency of the lytic parable to urokinase, and a lower systemic action activity of exogenous plasminogen activator de- 30 than urokinase. Trubestein et al., Hemostasis pends significantly on the amount of plasmin it 17:238-244 (1987). generates from endogenous plasminogen, which is Non-urokinase plasminogen activators, which present in free-form in circulating blood and bound include tissue plasminogen activator ("t-PA"), are form on the fibrin surfaces of thrombi and hemo- physiologic activators of plasminogen which may static plugs. In any thrombolytic therapy, it is desir- 35 be manufactured by cell culture and genetic en- able to generate the maximum amount of plasmin gineering. t-PA is capable of binding fibrin, and is on the fibrin surface i.e., plasmin immobilized on believed to convert plasminogen to plasmin at the the thrombus, with a minimum quantity of plasmin fibrin surface. Thus, it works more efficiently than (if possible, none at all) in the circulating blood. streptokinase or urokinase when administered sys- While immobilized plasmin efficiently carries out 40 temically. However, initial clinical trials indicate that the lysis of the thrombus and helps establish reper- lysis of fibrinogen is still significant during admin- fusion, circulating plasmin exhibits fibrinogenolytic istration of t-PA. Sherry, N. Engl. J. Med. activity, degrades platelet receptors, digests throm- 313:1014-1017 (1985). bospondin, and consequently decreases the plate- Although it has high specificity for fibrin, t-PA let aggregation capacity of the patient. The result is 45 has certain limitations. It has a very short biological the so called "lytic state", which is accompanied half-life time in the circulation of humans, on the by various undesired complications. order of five minutes. Cellular clearance of t-PA by A variety of plasminogen activators have been the liver is believed responsible for this limited utilized to promote dissolution of thrombi and res- biological half-life time. Rapid decrease of t-PA in toration of blood circulation to blocked vessels. 50 the blood after intravenous administration consti- However, the available plasminogen activators are tutes a major difficulty in drug therapy with respect far from ideal. Streptokinase, which is of bacterial to maintaining an adequate concentration of the origin, is antigenic. A loading dose is needed to activator. neutralize preexisting anti-streptokinase antibodies. Plasma contains plasminogen activator inhibi- Streptokinase treatment must be limited in duration 55 tors which can bind and inactivate plasminogen because of an increase in such antibody levels. activators. Continuous infusion of large doses of t- Conard et al., Semin. Throm. Haemostas. 13:212- PA are therefore necessary to overcome the effect 222 (1987). Streptokinase works efficiently to dis- of these inhibitors. At physiologic levels, free t-PA 3 3 EP 0 473 689 B1 4 is bound by the plasma inhibitor, PAI-1. Wun et al., Fibrinogen must be treated with thrombin, in Blood 69:1354-1362 (1987). The latter has been order to maximize targeting to thrombi. Thus, the shown to be present in elevated levels in the plas- fibrinogen-urokinase hybrid of Patent 4,564,596 re- ma of patients with myocardial infarction, Hamsten quires the presence of a thrombotic state for op- et al., N. Engl. J. Med. 313:1557-1563 (1985); 5 timal performance. Moreover, fibrinogen is an ad- recurrent deep venous thrombosis, deJong et al., hesive protein, since it contains the amino acid Thromb. Haemostas. 57:140-143 (1987); and sequence Arg-Gly-Asp. Fibrinogen binds to a vari- coronary artery disease, Paramo et al., Br. J. Med. ety of cells, providing a bridge between cells. Con- 291:573-574 (1985). When purified t-PA is added to sequently, much of the injected fibrinogen- plasma to create higher-than physiologic levels to io urokinase hybrid may bind to cells in the circulat- overcome the inhibitory effect of PAI-1, it com- ing blood, making less hybrid available to bind to plexes with other inhibitors present in plasma, such thrombi. as C1 -inhibitor, alpha2-antiplasmin and macro- Human fibrin fragment Ei binds specifically to globulin. Thorsen et al., Blochem. Blophys. Acta polymers of fibrin, Olexa et al., Proc. Natl. Acad. 802:111-118 (1984); Rijken et al., J. Lab. Clin. is Scl. USA 77:1374-1378 (1980), and has the ability Med. 101:285-294 (1983); Kruithoff et al., Blood to bind to aged as well as fresh thrombi. Knight et 64:907-913 (1984). al., J. Clin. Invest. 72:2007-2013 (1983). ^-label- Administration of urokinase, streptokinase and ed fragment Ei has been used to detect venous t-PA results in an increased tendency for bleeding, thrombi in patients. Knight et al., Radiology limiting the usefulness of these agents, particularly 20 156:509-514 (1985).
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