Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1998-12-15
2001-04-03
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S216000, C435S325000, C530S350000, C530S381000
Reexamination Certificate
active
06210667
ABSTRACT:
TECHNICAL FIELD
The present invention relates to modifications of streptokinase and other microbial enzymes for use to activate plasminogen in the presence of fibrin, to efficiently dissolve an unwanted blood clot in a subject.
BACKGROUND ART
Clinical studies of acute coronary thrombosis have established that administration of plasminogen (Pg) activators such as recombinant tissue plasminogen activator (t-PA) and streptokinase (SK) saves lives and reduces morbidity. Comparison of the efficacy of SK (administered according to a 30 year old dosing regimen) and t-PA (administered by a variety of different protocols), in the presence and absence of heparin, showed that SK is markedly cheaper and can cause less cerebral bleeding than t-PA, whereas t-PA appears to be slightly better at reducing mortality than SK, at least as it is currently administered (e.g. GUSTO, New Engl. J. Med. 329:1615-1622, 1993).
However, problems with administration of these thrombolytic agents remain. For example, acute myocardial infarction patients receiving early administration of either t-PA or SK failed to show reperfusion within 90 min, and reperfusion was not observed in 45-67% of patients (Karagounis L., Amer. Coll. Cardiol. 19:1-10 (1992), Lincoff A., et al. Am J Cardio; 75(14):871-766 (1995); Simes R., et al. Circulation 91(7):1905-1907 (1995)). Failed reperfusion is associated with an approximately double mortality rate from myocardial infarction compared to successful reperfusion, and significantly increases morbidity in surviving patients. The results of thrombolytic therapy as a treatment for venous thromboembolism are also relatively disappointing. Further, the mortality rate for pulmonary embolism appears not to have changed in ~30 years, and no Pg activator has been shown to change the death rate in these patients (Goldhaber S., Chest 107:45S-51S (1995)). When administered to patients with pulmonary embolism, t-PA restores blood flow to only ~33% of occluded lung segments within 24 hours (Goldhaber S., Lancet 2:886-889 (1986)). Similar results have been described with other agents (UPET, Circulation 47:1-108, 1973). In cases of deep venous thrombosis, about two-thirds of patients treated with SK or t-PA have minimal or no lysis of the clot on repeat venography after 24 hours (Salzman E., et al. Basic Principles and Clinical Practice; 3rd Ed. Uppincott, Philadelphia (1994); Goldhaber S. Am J Med 88:235-240 (1990)), thus neither SK nor t-PA treatments are optimal for thrombotic disease in patients.
One property that diminishes the effectiveness of SK as a therapeutic agent is that the administered SK can complex as an antigen with preexisting anti-SK antibodies that are found generally in the human population as a result of prior streptococcal infection. The presence of anti-SK antibodies reduces the efficacy of SK as a thrombolytic agent by quenching Pg activation and increasing the clearance of the protein (Fletcher, A. J Clin Invest 37:1306-1315 (1959)). Thus, the presence of neutralizing human anti-SK antibodies inhibits the formation of a plasminogen activator complex. Antigenicity of SK can be remediated by identification as and elimination or reduction of epitopes by, e.g., genetic deletion (WO 94/07992) of as many structural features of SK
c
that do not contribute to Pg binding and activation in the presence of fibrin.
Another property of SK that limits its potency and efficacy as a thrombolytic agent is its rapid proteolytic cleavage during Pg activation, which inactivates the SK-plasminogen activator complex (SK-PAC). An alteration by mutation of SK, e.g. one or more point mutations that removes a proteolytic cleavage substrate site (see WO 96/41883, the contents of which are hereby expressly incorporated by reference) can be made to eliminate cleavage and inactivation during Pg activation, and these alterations can be combined also with an alteration that is a deletion of an antigenic site on SK (WO 94/07992) in a region of the protein that is not involved in SK binding to Pg.
Finally, widespread Pg activation by SK throughout the circulation, rather than SK activation limited to association with a clot, squanders the effectiveness of SK administered as a thrombolytic agent A novel modified SK with enhanced activity, particularly with activity limited to activation of Pg in the presence of fibrin, is desirable.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (Cold Spring Harbor Laboratory Press (1989)); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1984));
Bacillus subtilis
and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics (Sonenshein, A et al., American Society for Microbiology, Washington, D.C. (1993)); Genetic Manipulation of Streptomyces: a Laboratory Manual (Hopwood, D. et al., Eds., John Innes Foundation, Norwich, England (1985)); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London (1987)); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds. (1986)).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and material similar or equivalent to those herein can be used in the practice of testing of the present invention, the preferred methods and material are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. In addition, the materials, methods and examples are illustrative only and not intended to be limiting.
SUMMARY OF THE INVENTION
The invention disclosed herein relates to alterations of a bacterial protein, for example, a streptokinase, that render plasminogen activation by the altered protein substantially dependent on the presence of fibrin, for optimization of use as a thrombolytic agent at lower effective dosages than were heretofore possible, which enables more rapid administration with fewer concomitant side effects.
Accordingly, in one embodiment, the invention provides a pharmaceutical composition for dissolving blood clots in a subject, comprising an isolated fibrin-dependent bacterial protease, for example, a modified streptokinase, for example, a modified streptokinase that is substantially pure. A preferred embodiment of the invention is an effective dose of a streptokinase that is substantially pure, and in another embodiment, streptokinase that is substantially pure and in a pharmaceutically acceptable carrier. In another embodiment, the invention provides a pharmaceutical composition wherein the bacterial protease is a modified staphylokinase.
In a preferred embodiment of a modified streptokinase, the streptokinase activation of plasminogen is at least ten-fold greater in the presence of fibrin than in the absence of fibrin, and in another embodiment, the streptokinase activation of plasminogen is at least hundred-fold greater in the presence than in the absence of fibrin. In one embodiment, the modification of the streptokinase is an alteration in the amino terminus selected from the group consisting of mutation, chemical treatment, or enzymatic treatment. The mutation can be selected from the group consisting of deletion, insertion, transposition, and substitution. The enzymatic treatment can be a proteolytic treatment. In a preferred embodiment of a modification of the streptokinase, the mutation is a deletion, for example, a deletion in the amino te
Bromberg & Sunstein LLP
Hutson Richard
Prouty Rebecca E.
The President and Fellows of Harvard College
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