Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1991-02-12
1994-05-31
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435 696, 435 697, 43525231, 4353201, 536 232, 536 234, 935 10, 935 14, 935 29, 935 47, 935 73, C12N 972, C12N 1558, C12N 1562, C12N 1570
Patent
active
053169344
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to methods for the preparation of human prourokinase-like polypeptides i.e., substantially pure human prourokinase mutein, with binding ability through covalent bonds with blood clots, by means of gene engineering. Prourokinase mutein functions as a thrombolytic agent.
DESCRIPTION OF RELATED ART
Human urokinase is an enzyme present in human urine in a trace amount, and can convert the inactive plasminogen to the active plasmin. The formed plasmin can lyse blood clots. Owing to this fact, human urokinase is clinically used widely as a thrombolytic agent.
However, human urokinase has low affinity with blood clot and is of the active form. Therefore it activates not only plasminogen at or in the vicinity of the blood clot but also plasminogen in the circulation, resulting in formation of a large amount of plasmin in the circulation. The extensive plasmin formation in the circulation modifies platlet function (Blood: Vol. 68, p 275, 1986) and degrades circulating fibrinogen and clotting factors V and VIII (Blood: Vol. 68, p 1280, 1986) the extensive plasmin formation also carried the risk of grave side effect of systemic hemorrhage, which often results in death (Journal of American Colleague Cardiology: Vol. 10, p 970, 1987).
The ideal thrombolytic agent should be capable of attacking the components of a thrombus while sparing the circulating clotting proteins and platlets and have an appropriate half life in vivo. In recent years, human prourokinase and tissue plasminogen activator (hereinafter referred to as tPA) are under development as the second generation thrombolytic agents. These agents activate the plasminogen praticularlly in close proximity to a thrombus. Therefore, excessive administration of these thrombolytic agents carries less risk of grave side effect of systemic hemorrhage. However, human prourokinase has better affinity with thrombus than human urokinase, but has not yet been shown to be of practical interest. The tPA has such defects as a large amount of inhibitor (plasminogen activator inhibitor I) present in the blood, a short half life so that therapeutic effects are not realized unless a large amount is administered, and an occurrence of reocclusion after artery recanalization by thrombolysis in many cases (Circulation: Vol. 73, p 347, 1986; New England Journal of Medicine: Vol. 317, p 581, 1987). Substances which are not inactivated irreversibly by plasminogen activator inhibitor I and which have better affinity with blood clot than human prourokinase are believed to be ideal thrombolytic agents. Attempts have been made to produce such substances.
A first of such substances is a chimeric substance in which the NH.sub.2 -terminal region of tPA is fused with the COOH-terminal region of human prourokinase having an enzymatic activity to activate the plasminogen, and produced by recombinant DNA technology, with recognition that the NH.sub.2 -terminal region of tPA has high affimity with the blood clot (The Journal of Biological Chemistry, Vol. 268, p 10855, 1987; Thrombosis and Haemostasis, Vol. 54, p 893, 1985). However, contrary to the expectation, this substance has less affinity to blood clot as that of tPA.
A second of the substances is a chimeric substance in which about 560 amino acids at the NH.sub.2 -terminal side which has affinity to the blood clot is combined chemically with the COOH-terminal region of human prourokinase, same as that described above (Biochemistry, Vol. 25, p 3603, 1986). A third is a chimeric substance made by chemically combining an antibody against fibrin which is a major component of blood clot, with the COOH-terminal region of human prourokinase, same as that described above (Clinical Research Abstract, Vol. 36, p 265A, 1989). The second and third substances have improved affinity to blood clot; therefore, there substances would be expected to improve the thrombolytic effect. Economical manufacturing of these second and third substances is however difficult.
Further, urokinase derivatives are reported (PCT/
REFERENCES:
patent: 4892826 (1990-01-01), Homandberg et al.
Mosher, D. F., et al., 1980, The Journal of Biological Chemistry, 255(3): 1181-1188.
Ichinose A. et al., 1983, FEBS Letters 153(2): 369-371.
Hirosawa, S., et al, 1988, Proceedings of the National Academy of Sciences, USA, 85(18): 6836-40 and 86(5): 1612-1613.
Holmes, W. E., et al., 1987, Journal of Biological Chemistry, 262(4): 1659-1654.
Kornblihtt, A. R., et al., 1985, The EMBO Journal, 4(7): 1755-1759.
Oswald, R. E., et al., 1989, Nature 337:579-582.
Bogusky, M. J., et al., 1989, Biochemistry, 28:6728-6735.
Ichinose A., et al., 1990, The Journal of Biological Chemistry, 265(23): 13411-13414.
Kobayashi Yoh-ichi
Mukohara Yukuo
Nakamura Hiroaki
Satoh Masayuki
Watabe Ken
Mason, Jr. Joseph C.
Moore William W.
Nippon Soda Co. Ltd.
Smith Ronald E.
Wax Robert A.
LandOfFree
Effective thrombosis mediated by human prourokinase-like polypep does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Effective thrombosis mediated by human prourokinase-like polypep, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Effective thrombosis mediated by human prourokinase-like polypep will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1627561