Leuven Research & Development Vzw

Other

Kabivitrum Ab

Leuven Research & Development Vcw

Desire J Collen

Henri R Lijnen

Dingeman C Rijken

Jean-Marie Stassen

Osamu Matsuo

Methods for the identification, production and use of derivatives of the invention obtained by preparing a DNA fragment comprising at least the part of the coding sequence of staphylokinase that provides for its biological activity; performing in vitro site-directed mutagenesis on the DNA fragment to replace one or more codons for wild-type amino acids by a codon for another amino acid; cloning the mutated DNA fragment in a suitable vector; transforming or transfecting a suitable host cell with the vector; and culturing the host cell under conditions suitable for expressing the DNA fragment. Preferably the DNA fragment is a 453 bp EcoRI-HindIII fragment of the plasmid pMEX602sakB, the in vitro site-directed mutagenesis is performed by spliced overlap extension polymerase chain reaction and the mutated DNA fragment is expressed in E. coli strain TG1 or WK6. The invention also relates to pharmaceutical compositions comprising at least one of the staphylokinase derivatives according to the invention together with a suitable excipient, for treatment of arterial thrombosis.

Identification, production and use of new staphylokinase derivatives

A method for producing the derivatives of the invention by preparing a DNA fragment comprising at least the part of the coding sequence of staphylokinase that provides for its biological activity; performing in vitro site-directed mutagenesis on the DNA fragment to replace one or more codons for wild-type amino acids by a codon for another amino acid; cloning the mutated DNA fragment in a suitable vector; transforming or transfecting a suitable host cell with the vector; and culturing the host cell under conditions suitable for expressing the DNA fragment. Preferably the DNA fragment is a 466 bp EcoRI-HINDIII fragment of the plasmid pMEX602SAK, the in vitro site-directed mutagenesis is performed by an oligonucleotide-directed mutagenesis system using the plasmid pMa/c and the repair deficient E. coli strain WK6MutS, and the mutated DNA fragment is cloned in E. coli strain-WK6. The invention also relates to pharmaceutical compositions comprising at least one of the staphylokinase derivatives according to the invention together with a suitable excipient, for treatment of arterial thrombosis.

Staphylokinase derivatives

The present invention relates to the use of staphylokinase for the preparation of a pharmaceutical composition for treating arterial thrombosis, such as e.g. myocardial infarction. It is demonstrated that staphylokinase can be particularly useful as a thrombolytic agent for the lysis of platelet-rich clots. Furthermore it is demonstrated that staphylokinase, unlike streptokinase, does not induce antibody induction upon repeated administration.

Use of staphylokinase for the preparation of a pharmaceutical

Plasminogen activators of the tissue-type and plasminogen activators of the urokinase-type appear to have a synergistic action in vivo when administered together as thrombolytic agents. Use in pharmaceutical compositions and in methods for the preparation and use thereof are set forth.

Pharmaceutical composition having thrombolytic activity

A new plasminogen activator which is very similar to the plasminogen activator from blood can be isolated in good amounts from the culture fluid of human melanoma cells. This new plasminogen activator has a strong thrombolytic effect and pharmaceutical compositions thereof may be used in the therapeutic treatment of thrombosis disorders.

Plasminogen activator and pharmaceutical composition having thrombolytic

Novel chemical compounds which are heparin fragments with a molecular weight of from 2,000 to 5,500 covalently bound to antithrombin III, and their use in medicine.

Novel composition of matter of antithrombin III bound to a heparin

A newly isolated constituent of human blood plasma, called antiplasmin, is disclosed together with its isolation method. Further, the invention relates to antiplasmin, a newly isolated constituent of the human blood plasma and to its method of isolation. Further, an antiplasmin-antiserum together with its preparation and utilization, and a method of determining antiplasmin in blood samples are disclosed.

Antiplasmin and antiserum

A thrombosis test is based on the presence of specific, newly-found, enzyme-inhibitor complexes (such as plasmin-antiplasmin, plasmin-.alpha..sub.2 -macroglobulin, thrombin-antithrombin-III) in blood samples having an activated blood coagulation and/or fibrinolytic system. The test is immunochemical and uses a purified antiserum which has been generated against the enzyme-inhibitor complex. The antiserum is purified by incubating the antiserum with plasminogen and fresh blood plasma and isolating a gamma globulin fraction containing antibodies specific to the enzyme-inhibitor complex. A reagent for the test is prepared by contacting a blood cell suspension or a latex of particles of synthetic resin with the purified antiserum to obtain a suspension or latex in which antibodies from the antiserum are present on the surface of the cells or particles.

Thrombosis-test

A test for determining the anti-coagulation level in blood samples is based on ascertaining the presence of heparin-antithrombin-III complex therein. This may be done with the aid of antibodies against thrombin-antithrombin-III complex, such antibodies being used e.g. in the form of a latex reagent or a bloodcell reagent.

Determination of heparin in blood plasma

The invention relates to antiplasmin, a newly isolated constituent of the human blood plasma and to its method of isolation. Further, an antiplasmin-antiserum together with its preparation and utilization, and a method of determining antiplasmin in blood samples are disclosed.

Antiserum which has been generated against an enzyme-inhibitor-complex is purified by immunoprecipitating a freshly generated antiserum with fresh human bloodplasma to form a precipitate and a fluid, and subjecting the fluid to chromatographic separation over an insolubilized enzyme-inhibitor complex against which the antiserum has been generated. A reagent for immunochemical assay is prepared by contacting the purified antiserum with red bloodcells or synthetic resin particles to obtain on the surface of the red bloodcells or synthetic resin particles antibodies from the antiserum. In a diagnosis test, blood plasma is contacted with the purified antiserum and with red bloodcells or synthetic resin particles which bear the enzyme-inhibitor complex used to prepare the antiserum, and whether or not agglutination occurs is ascertained. In an alternative diagnosis test, blood plasma is contacted with red bloodcells or synthetic resin particles having on their surface antibodies from the antiserum, and whether or not agglutination occurs is ascertained.

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Winksele, Belgium

Desire J Collen