Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Patent
1985-01-03
1986-01-14
Schain, Howard E.
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
435188, 435215, 424 94, C12N 972, C12N 1102, A61K 3748
Patent
active
045645965
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to bio-organic chemistry and, more particularly, to novel urokinase derivatives possessing affinity for thrombus material and displaying a thrombolytic activity.
BACKGROUND OF THE INVENTION
Known in the art are various urokinase derivatives, for example, water-soluble urokinase derivatives suitable for therapeutic purposes comprising complexes of the enzyme of urokinase with heparin or dextran sulphate (cf. U.S. Pat. No. 4,106,992; 1978; Japanese Pat. No. 159387, 1977).
These urokinase derivatives are, however, unstable.
When penetrating into blood circulation, they cause destruction of electrostatic interactions with the formation of substantially a mixture of urokinase with the vehicle, which is accompanied by a rapid inactivation of the enzyme and discontinuation of the thrombolytic effect.
Also known in the art are urokinase derivatives comprising urokinase covalently bonded with dextran (cf. Japanese Pat. No. 54-113488, 1979).
These derivatives are water-soluble and feature an enhanced stability. However, such modification of the enzyme does not ensure its affinity for the thrombus material, wherefore an enhanced fibrinolytic activity of blood with the use of such derivatives still does not guarantee an effective process of thrombolysis.
DISCLOSURE OF THE INVENTION
The present invention is directed to novel urokinase derivatives featuring an increased affinity for fibrin and possessing a prolonged fibrinolytic effect owing to a higher stability thereof.
The urokinase derivatives according to the present invention are novel and hitherto unknown in the literature.
The derivatives of urokinase according to the present invention comprise urokinase covalently bonded with fibrinogen, or urokinase covalently bonded, through an aliphatic diamine, with fibrinogen and corresponding to the general formula: ##STR3## wherein P is fibrinogen, E is urokinase, R is either absent or stands for ##STR4## where n is 1 to 12; with a molecular mass of 360,000-440,000D, protein content of 10-30% by mass, and an esterase catalytic activity of 30 to 60%.
The compounds according to the present invention comprise a product of bonding of urokinase to fibrinogen which is employed as a vehicle for immobilization of urokinase.
Owing to proper selection of a matrix of a high-molecular vehicle (fibrinogen) the compounds according to the present invention have an increased tropism to the thrombus material. This ensures an effective progress of the thrombolytic process. At the same time, a preliminary modification of fibrinogen with aliphatic diamines hinders destruction of the protein matrix in the course of fibrinolysis. The addition of urokinase of fibronogen imparts an increased stability to the compounds which can also ensure prolongation of the fibrinolytic activity of such derivatives. As regards their kinetic parameters, the compounds according to the present invention are close to the native enzyme and in their fibrinolytic effect they are nearly two times more effective as compared to the starting urokinase.
DETAILED DESCRIPTION OF THE INVENTION
Properties of the novel compounds according to the present invention have been studied in in vitro experiments.
The experiment was carried out in parallel with the use of native urokinase and the following urokinase derivatives according to the present invention: compound I (urokinase attached to fibrinogen by means of 1,12-dodecamethylenediamine), compound 2 (urokinase added to fibrinogen by means of 1.10-decamethylenediamine), compound 3 (urokinase added to fibrinogen by means of 1,7-heptamethylenediamine), compound 4 (urokinase added to fibrinogen by means of 1,4-tetramethylene diamine), compound 5 (urokinase added to fibrinogen).
The study of kinetic parameters of an enzymatic hydrolysis of methyl ether of acetylglycyl lysine (AGLMe) by the compounds according to the present invention has shown that these compounds are very close to the native enzyme (urokinase). The data of the experiment are shown in Table 1 hereinbe
REFERENCES:
patent: 3544427 (1970-12-01), Sloane
patent: 4011142 (1977-03-01), Jacobi
patent: 4029767 (1977-06-01), Vairel et al.
patent: 4244943 (1981-01-01), Yamahira et al.
patent: 4286063 (1981-08-01), Suyama
patent: 4349630 (1982-09-01), Maximenko et al.
patent: 4381346 (1983-04-01), Huasin et al.
Chazov Evgeny I.
Maximenko Alexandr V.
Smirnov Vladimir N.
Tischenko Elena G.
Torchilin Vladimir P.
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