Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Patent
1990-09-28
1993-06-29
Draper, Garnette D.
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
43525233, 4353201, 435226, 435 696, 536 232, 424 9464, 935 14, C12N 1558, C12N 964, A61K 37547, A61K 3754
Patent
active
052232565
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a new thrombolytically active protein, a DNA sequence which codes for the new thrombolytically active protein, expression plasmids which contain a DNA sequence which codes for the thrombolytically active protein as well as a process for the preparation of such plasmids, a process for the production of the thrombolytically active protein and an agent for dissolving blood clots which contains the thrombolytically active protein.
Coagulated blood contains polymeric fibrin which is the main component of the protein matrix. Fibrin is dissolved under physiological conditions by a fibrinolytic system in a reaction cascade which is similar to that of blood coagulation. The central reaction in this is the activation of plasminogen to plasmin which is for example mediated by the tissue-type plasminogen activator t-PA. Plasmin, in turn, dissolves fibrin which is the main component of the protein matrix of coagulated blood. The enzymatic activity of natural t-PA or t-PA obtained from eukaryotes by genetic engineering, i.e. the catalytic activation of plasminogen to plasmin, is very low in the absence of fibrin or fibrinogen cleavage products, but it can be substantially increased in the presence of these proteins, namely by more than ten-fold.
T-PA is cleaved by proteases present in the blood into an A-chain and a B-chain. Both parts of the chain remain bound via a cysteine-bridge. The ability to stimulate the activity of t-PA is a significant advantage in comparison with other known plasminogen activators such as, for example urokinase or streptokinase (cf. for example M. Hoylaerts et al., J. Biol. Chem. 257 (1982), 2912-2919; W. Nieuwenhuizen et al., Biochem. Biophys. Acta, 755 (1983), 531-533).
The mechanism of action of t-PA in vivo is described for example in Korniger and Collen, Thromb. Hamostasis 46 (1981), 561-565. The focus of enzymatic activity on the fibrin surface would seem to make it a suitable agent for the treatment of pathological vascular occlusions (for example myocardial infarction) which has been confirmed to a large extent by clinical trials (Collen et al., Circulation 70 (1984), 1012; Circulation 73 (1986), 511).
A disadvantage of t-PA is however the rapid decrease in its plasma concentration (clearance). As a result, a relatively large amount of t-PA is necessary to achieve an effective lysis of thrombi. On the other hand, high therapeutic doses result in side effects such as for example bleeding.
A natural degradation product of t-PA is described in EP 0 196 920 which only contains the kringle II and protease domains, and whose N-terminus begins with alanine 160 (enumeration system according to the amino acid sequence cited by Pennica et al. in Nature 301 (1983), 214-221).
The clearance rate of this product of t-PA degradation does not, however, differ significantly from that of the natural t-PA. Only a chemical modification of the catalytic domain via attachment of a blocking group can result in an improvement.
It is therefore the object of the present invention to modify t-PA such that the resultant derivative has a much reduced clearance rate and thus a longer half-life in blood plasma. In this process the ability to lyse thrombi as well as the ability to be stimulated by fibrin should be preserved.
The embodiment of the present invention is therefore a tissue-type plasminogen activator (t-PA derivative) which is characterized in that it is not glycosylated and consists of the following amino acid sequence: ##STR2## which can be extended by M at the amino end i.e. at the amino acid No. 1=S.
It was established that, surprisingly, deletion of the other domains which are present in native t-PA had no effect on the thrombolytic efficacy of the protein and that the fibrin-dependent stimulatability of the mutein was comparable to that of native t-PA. Although it was determined that the thrombolytically active protein according to the present invention lacked the property to bind to fibrin it however, surprisingly, exhibited a thrombolytic efficacy in vivo which was
REFERENCES:
patent: 4511502 (1985-04-01), Builder et al.
patent: 4620948 (1986-11-01), Builder et al.
patent: 4753879 (1988-06-01), Rosa et al.
patent: 4766075 (1988-08-01), Goeddel et al.
patent: 4777043 (1988-10-01), Bennett et al.
patent: 4898825 (1990-02-01), Morii et al.
patent: 4908205 (1990-03-01), Bennett et al.
patent: 4933434 (1990-06-01), Rudolph et al.
patent: 4935237 (1990-06-01), Higgins et al.
patent: 4963357 (1990-10-01), Bell et al.
patent: 4978620 (1990-12-01), Morii et al.
patent: 5034225 (1991-07-01), Bennett et al.
patent: 5037752 (1991-08-01), Bell et al.
patent: 5073494 (1991-12-01), Heyneker et al.
Saito, Y. et al., Annals New York Academy of Sciences, vol. 613:452-454, 1990.
World Patent Index Abstract, Accession No. C89-017728 for Niwa et al. patent family including EPO 302456 and DK 8804319.
Heussen, C. et al., J. Biol. Chem., 259 (19):11635-11638, 1984.
Scopes, R. K. Protein Purification, Springer-Verlag, New York, 1987, pp. 15-20 and 133-144.
Cleary, S. et al., Biochemistry, 25:1884-91, 1989.
Fischer Stephan
Kohnert Ulrich
Martin Ulrich
Rudolph Rainer
Stern Anne
Allen Marianne Porta
Boehringer Mannheim GmbH
Draper Garnette D.
LandOfFree
Thrombolytically active non-glycosylated protein does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Thrombolytically active non-glycosylated protein, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Thrombolytically active non-glycosylated protein will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1753676