Antithrombosis enzyme from the snake venom of agkistrodon...

Details Antithrombosis enzyme from the snake venom of agkistrodon... Antithrombosis enzyme from the snake venom of agkistrodon...

1998-04-10

2002-12-03

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins from animals other than mammals or birds

Snakes; venom

C530S350000, C435S183000, C435S219000, C424S542000, C424S094100

Reexamination Certificate

active

06489451

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to an antithrombosis enzyme derived from the snake venom of an acutus species.
BACKGROUND OF THE INVENTION
Anti-thrombus drugs extracted from acutus venom have been reported in the literature, e.g., “Preparation and Study of Anti-thrombus Enzymes No. 1, 2, and 3
”, Journal of the Medical Univ. of China
, 1989.18 (special issue); and “Technique for Extracting Definriogenase from the venom of
Agkistrodon acutus
,” CN 92102645.5 (CN 1065680.A). These anti-thrombus drugs are proteinase components extracted from the snake venom. They act like thrombase with hemorrhagic side-effect. In addition, some of these products are not single component proteinase, but a mixture of different components, which limits the pharmaceutical application of these drugs in human.
Other snake venom derived pharmaceutical products include Ancrod, Trigtamin and Integrilin (see Matsuzaki et al.,
Biochem. Biophy. Res. Com
. 220(2):382-387, 1996; Morita et al.,
Natural Toxins II
, pp187-196, Edited by B. R. Singh and A. T. Tu, Plenum Press, New York, 1996; U.S. Pat. Nos. 5,196,403, 5,242,810, 5,453,370, 4,017,012, 5,344,783, 5,686,571, 5,523,292, 5,066,592 and 5,342,830).
SUMMARY OF THE INVENTION
Within the scope of this invention, Applicant has extracted, purified and cloned an antithrombosis enzyme (ATE, also called a fibrinolytic enzyme in the provisional application) from the venom of Southern-Anhui
Agkistrodon acutus
in China. This enzyme degrades both fibrinogen and fibrin, and inhibits platelet aggregation. It is useful for preventing and treating vaso-occulusive and thromboembolic disorders, including, but not limited to, myocardial infarction, restenosis, peripheral anginaphraxis, angiopathic thrombosis, cerebral thrombosis, ischemic cerebral vascular diseases, unstable angina, acute thrombosis, unstable stenocardia and hemiparalysis caused by cerebral thrombosis.
The present invention provides methods and compositions for preventing or treating diseases and processes mediated (caused or aggravated) by undesired and/or uncontrolled thrombosis by administering to a human or animal a composition containing or capable of expressing the antithrombosis enzyme in a dosage sufficient to prevent, reduce, eliminate or inhibit thrombosis. The antithrombosis enzyme may be substantially purified or in a crude extract. The antithrombosis enzyme may be produced from snake venom, chemically synthesized or expressed from a recombinant vector. It may also be combined with a pharmaceutically acceptable excipient or carrier, and optionally sustained-release compounds or compositions, such as biodegradable polymers, to form therapeutic compositions.
The present invention is particularly useful for treating or preventing acute and recurrent cerebral thrombosis, myocardial infarction, restenosis, peripheral anginaphraxis, angiopathic thrombosis, ischemic cerebral vascular thrombosis, unstable angina, unstable stenocardia, and thromboangitis obliterans. Administration of the antithrombosisi enzyme can prevent blood clot formation and reduce, diminish or dissolve blood clot. The antithrombosis enzyme may also be used in combination with other compositions and procedures for the treatment of thrombosis. For example, it may be used in combination with a thrombolytic agent known in the art, which includes, but is not limited to, tissue plasminogen activator purified from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, prourokinase, anisolated streptokinase plasminogen activator complex (ASPAC), animal salivary gland plasminogen activators and known, biologically active derivatives of any of the above. In these combination compositions, the antithrombosis enzyme and other thrombolytic agent work in a complementary fashion to dissolve blood clots, resulting in decreased reperfusion times and increased reocclusion times in patients treated with them. The use of the antithrombosis enzyme in the compositions of this invention advantageously allows the administration of a thrombolytic reagent in dosages previously considered too low to result in thrombolytic effects if given alone. This avoids some of the undesirable side effects associated with the use of thrombolytic S agents, such as bleeding complications. The compositions of this invention may also be used before, concurrent with, or after angioplastic or fibrolytic treatment to prevent or treat restenosis.
Thus, in a first aspect, this invention features an isolated, purified or recombinant antithrombosisi enzyme which has (i) a molecular weight of between about 28 kD and about 32 kD when analyzed by polyacrylamide gel electrophoresis, (ii) an aspartic acid content of between about 2% and about 5%, and (iii) a glutamic acid content of between about 2% and about 5%. This enzyme has the ability to hydrolyze fibrin, dissolve thrombus, inhibit platelet aggregation, and inhibit the formation of thrombus.
In a preferred embodiment, the enzyme has fibrinolytic activity of no less than one fibrinolytic activity unit per mg protein. In another preferred embodiment, the enzyme has fibrinolytic activity of between about one and about three fibrinolytic activity units per mg protein. This enzyme specifically hydrolyzes the A (&agr;) chain of fibrinogen. This enzyme completely or almost completely inhibits human platelet aggregation induced by agonists such as ADP, Epinephrine, Thrombin and collagen. This enzyme has no detectable hydrolysis effect on casein. The enzyme dissolves arterial and venous thrombus in a mammal, prevent thrombosis, reduce blood viscosity, and improve microcirculation. At the same time, this enzyme has minimum effect on the thromosystem, resulting in little possibility of hemorrhage. This enzyme is different from related enzymes from other Acutus species (e.g., IX/X binding proteins, Matsuzaki et al.,
Biochem. Biophy. Res. Com
. 220(2):382-387, 1996; Morita et al.,
Natural Toxins II
, pp187-196, Edited by B. R. Singh and A. T. Tu, Plenum Press, New York, 1996) in that this enzyme has both fibrinolytic activity and antiplatelet aggregation activity, and less hemorrhagic activity.
In other preferred embodiments, this enzyme is purified from Southern-Anhui
Agkistrodon acutus
. The enzyme is a heterodimer of A chain and B chain each with a molecular weight of about 14 KD to about 16 KD. The A chain has at its amino end the following sequence Sequence ID No. 3:
Asp-Cys-Ser-Ser-Asp-Trp-Ser-Ser-Tyr-Glu-Gly-His-Cys-Tyr-Lys-Val-Phe-Lys-Gln-Ser-Lys-Thr-Trp-Thr-Asp-Ala-Glu-Ser-Phe-, and the B chain has at its amino end the following sequence Sequence ID No. 4:
Asp-Cys-Pro-Ser-Glu-Trp-Ser-Ser-Tyr-Glu-Gly-Phe-Cys-Tyr-Lys-Pro-Phe-. Preferrably, the A chain and the B chain are linked by one or more disulfide bond.
In other preferred embodiments, this antithrombosis enzyme contains Ca++ and/or has aspartic acid at its amino terminus.
By “isolated” in reference to a polypeptide is meant a polypeptide isolated from a natural source or synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring amino acid sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is the predominate sequence present (at least 10-20% more than any other sequence) and is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
By “enriched” in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid

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