Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2005-06-07
2005-06-07
Pak, Michael (Department: 1646)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C424S243100, C435S220000, C514S002600, C530S350000
Reexamination Certificate
active
06902733
ABSTRACT:
Methods for the identification, production and use of staphylokinase derivatives characterized by a reduced immunogenicity after administration in patients. The derivatives of the invention are 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; culturing the host cell under conditions suitable for expressing the DNA fragment; and purifying the expressed staphylokinase derivative to homogeneity. Preferably the DNA fragment is a 453 bp EcoRI-HindIII fragment of the plasmid pMEX602sakB, (pMEX.SakSTAR), the in vitro site-directed mutagenesis is performed by spliced overlap extension polymerase chain reaction and the mutated DNA fragment is expressed inE. colistrain 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.
REFERENCES:
Collen et al.,Circulation, vol. 94, pp. 197-206, 1996.
Collen et al.,Fibrinolysis, vol. 6, pp. 226-231, 1992.
Collen et al., “Comparative Thrombolytic and Immunogenic Properties of Staphylokinase and Streptokinase”,Fibrinolysis, vol. 6, pp. 232-242, 1992.
Collen et al.,Circulation, vol. 87, pp. 996-1006, 1993.
Desire′ Jose′ Collen
Leuven Research & Development VZW
Pak Michael
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
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