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WO1996001312A1 - Derives activateurs du plasminogene non glucosyles et leur utilisation en cas de risque eleve d'hemorragie - Google Patents

Derives activateurs du plasminogene non glucosyles et leur utilisation en cas de risque eleve d'hemorragie Download PDF

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Publication number
WO1996001312A1
WO1996001312A1 PCT/EP1995/002391 EP9502391W WO9601312A1 WO 1996001312 A1 WO1996001312 A1 WO 1996001312A1 EP 9502391 W EP9502391 W EP 9502391W WO 9601312 A1 WO9601312 A1 WO 9601312A1
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WIPO (PCT)
Prior art keywords
plasminogen activator
tissue plasminogen
glycosylated
derivative according
inhibitor
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PCT/EP1995/002391
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German (de)
English (en)
Inventor
Ulrich Kohnert
Anne Stern
Ulrich Martin
Stephan Fischer
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Boehringer Mannheim Gmbh
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Filing date
Publication date
Application filed by Boehringer Mannheim Gmbh filed Critical Boehringer Mannheim Gmbh
Priority to AU28861/95A priority Critical patent/AU2886195A/en
Priority to EP95924298A priority patent/EP0769050A1/fr
Publication of WO1996001312A1 publication Critical patent/WO1996001312A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Non-glycosylated plasminogen activator derivatives and their use when there is an increased risk of bleeding
  • the invention relates to new plasminogen activator derivatives, their production and use of therapeutic agents for the treatment of thromboembolic disorders with an increased risk of bleeding.
  • the invention also relates to processes for the production of these variants and pharmaceutical compositions which contain these variants.
  • Tissue plasminogen activator is a multi-domain serine protease that catalyzes the conversion of plasminogen to plasmin and is used for fibrinolytic therapy.
  • fibrinolysis is regulated in part by the interaction between t-PA and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor from the Se family).
  • PAI-1 plasminogen activator inhibitor 1
  • the binding of PAI-1 to t-PA takes place essentially via amino acids 296-302.
  • a mutation in this region reduces the inhibitory influence of PAI-1 on t-PA (EL Madison et al. (1990) (S).
  • S plasminogen activator inhibitor
  • the mechanism of the interaction between the amino acid region 296-302 of t-PA with PAI-1 has been extensively investigated (cf.
  • tPA variants the use of which results in a reduced bleeding frequency, are described in WO 93/24635 (11) and by BA Keyt et al., Proc. Natl. Acad. Be. USA 91 (1994) 3670-3674 (31). These t-PA variants have an additional glycosylation site at amino acid positions 103-105. In addition, these t-PA variants can have a modification on amino acids 296-302, which increases the fibrin specificity. The object of the present invention was to provide further t-PA derivatives which show reduced bleeding complications when used.
  • the object of the invention is achieved by a non-glycosylated tissue plasminogen activator derivative which contains the essential parts of at least one Kringledomain and a B-chain of t-PA, characterized in that the amino acids 296 - 299 (Lys-His-Arg -Arg (SEQ ID NO: 2)) are each exchanged for the sequence Ala-Ala-Ala-Ala (SEQ ID NO: 3).
  • the derivative according to the invention preferably additionally contains N-terminal at least one amino acid from the sequence Gly Ala Arg Ser Thr Gin Val Ile (amino acids -3 - +5 of the tPA sequence, SEQ ID NO: 4).
  • the derivative according to the invention particularly preferably contains the amino acids +1 to +3 of the tPA sequence (Ser Thr Gin) at the N-terminal.
  • plasminogen activators are far less active in vitro than unmodified derivatives or wild-type plasminogen activators. Surprisingly, however, the use of the plasminogen activator variants according to the invention shows a significantly reduced risk of bleeding and an increased in vivo activity.
  • Unmodified t-PA in its form found in plasma, consists of 527 amino acids and can be split into two chains by plasmin, which are then held together by a disulfide bridge.
  • the A chain also called heavy chain
  • the finger domain (amino acids 1-49) shows certain similarities with the finger structures in fibronectin.
  • the growth factor domain (amino acids 50-86) is to a certain extent homologous to murine and human epidermal growth factors.
  • the two Kringled domains (amino acids 87-175 and 176-262) are largely homologous to the fourth and fifth Kringledomain of plasminogen.
  • the finger and kringle 2 domains of t-PA are particularly involved in fibrin binding and in the stimulation of proteolytic activity by fibrin.
  • the B chain of t-PA (amino acids 276-527) is a serine protease and largely homologous to the B chains of urokinase and plasmin (TJR Harris (1987) (1) and J. Krause (1988) (2) ).
  • the essential parts of the domains are to be understood as the amino acid ranges which are necessary for the biological activity of the plasminogen activator. This is preferably at least 80%, particularly preferably at least 90%, of the domains mentioned.
  • the finger and / or the growth factor domain are preferably deleted in the plasminogen activator derivatives according to the invention.
  • the Kringled domains are either both preserved, only one domain is obtained (preferably the K2 domain ne) or one of the domains (preferably Kj or K2) is present multiple times (preferably doubled).
  • the t-PA variants according to the invention can be produced by the methods familiar to the person skilled in the art.
  • the compounds according to the invention are preferably produced by genetic engineering. Such a method is described, for example, in WO 90/09437 (25), EP-A 0 297 066 (26), EP-A 0 302 456 (27), EP-A 0 245 100 (28) and EP-A 0 400 545 (29), which are the subject of the disclosure for such manufacturing processes.
  • the mutations at position 296-299 are then introduced into the cDNA of t-PA or a derivative thereof by "oligonucleotide-directed site-specific mutagenesis".
  • the "site-specific mutagenesis” is, for example, by Zoller and Smith (1984) (12), modified from T.A. Kunkel (1985) (13)) and Morinaga et al. (1984) (19).
  • the method of PCR mutagenesis which is described, for example, in Ausubel et al. (1991) (30).
  • nucleic acid sequence of the protein according to the invention can additionally be modified. Such modifications are, for example:
  • the nucleic acid obtained in this way is used to express the t-PA derivative according to the invention if it is present on an expression vector suitable for the host cell used.
  • non-glycosylated t-PA derivatives according to the invention are produced either in eukaryotic host cells, the glycosylated product initially obtained being obtained by Methods familiar to the person skilled in the art must be deglycosylated, or preferably by expression in non-glycosylating host cells, particularly preferably in prokaryonic host cells.
  • E. coli, Streptomyces spec. are prokaryotic host organisms. or Bacillus subtilis.
  • the prokaryotic cells are fermented in a customary manner and, after the bacteria have been digested, the protein is isolated in a customary manner. If the protein is obtained in inactive form (inclusion bodies), it is solubilized and naturalized according to the methods familiar to the person skilled in the art. It is likewise possible, according to the methods familiar to the person skilled in the art, to secrete the protein as active protein from the microorganisms.
  • An expression vector which is suitable for this purpose preferably contains a signal sequence which is suitable for the secretion of proteins in the host cells used, and the Nucleic acid sequence which codes for the protein.
  • the protein expressed with this vector is secreted either into the medium (for gram-positive bacteria) or into the periplasmic space (for gram-negative bacteria).
  • a sequence coding for a cleavage site which allows the protein to be split off either during processing or by treatment with a protease.
  • the selection of the base vector into which the nucleic acid (preferably DNA) coding for the t-PA derivative according to the invention is introduced depends on the host cells used later for expression. Suitable plasmids and the minimum requirements placed on such a plasmid (e.g. origin of replication, restriction sites) are known to the person skilled in the art. In the context of the invention, a cosmid, the replicative double-stranded form of phage ( ⁇ , Ml 3) or other vectors known to the person skilled in the art can also be used instead of a plasmid.
  • the t-PA derivatives according to the invention are preferably purified in the presence of L-arginine, in particular at an arginine concentration of 10-1000 mmol 1.
  • Foreign proteins are preferably separated off by affinity chromatography and particularly preferably by means of an adsorber column on which ETI (Erythrina Trypsin Inhibitor) is immobilized.
  • Sepharose® for example, is used as the carrier material.
  • Cleaning via an ETI adsorber column has the advantage that the ETI adsorber column material can be loaded directly from the concentrated renaturation batch even in the presence of arginine concentrations as high as 0.8 mol / 1 arginine.
  • the plasminogen activators according to the invention are preferably purified via an ETI adsorber column in the presence of 0.6-0.8 mol / 1 arginine.
  • the solution used here preferably has a pH of over 7, particularly preferably between 7.5 and 8.6.
  • the elution of the t-PA derivatives according to the invention from the ETI column is carried out by lowering the pH both in the presence and in the absence of arginine under conditions in which the tPA derivatives according to the invention are readily soluble.
  • the pH is preferably in the acidic range, particularly preferably between pH 4.0 and 5.5.
  • the t-PA variants according to the invention can be formulated for the production of therapeutic agents in a manner familiar to those skilled in the art, the compounds according to the invention usually being combined with a pharmaceutically acceptable carrier.
  • Such compositions typically contain an effective amount of 0.1-7 mg / kg, preferably 0.3-7 mg / kg, particularly preferably 0.7-5 mg / kg body weight as a dose. A dose of 1-3 mg / kg proved to be particularly suitable.
  • the therapeutic compositions are usually in the form of sterile, aqueous solutions or sterile, soluble dry formulations such as lyophilisates.
  • the compositions usually contain a suitable amount of a pharmaceutically acceptable salt used to prepare an isotonic solution.
  • Buffers such as arginine buffers and phosphate buffers can also be used to stabilize a suitable pH (preferably 5.5-8.0, particularly preferably 5.5-7.5).
  • the amount of the dosage of the compounds according to the invention can be readily determined by any person skilled in the art. It depends, for example, on the type of application (infusion or bolus) and the duration of the therapy. Because of their longer half-life, the compounds according to the invention are particularly suitable for a bolus application (single bolus, multiple bolus).
  • a suitable form for a bolus application is, for example, an ampoule which contains 5-1000 mg, preferably 25-1000 mg, of the compound according to the invention, arginine and buffer.
  • the compounds according to the invention are preferably used as a multiple bolus. Suitable time intervals are between 20 and 180 minutes, an interval between 30 and 90 minutes is particularly preferred and an interval between 30 and 60 minutes is particularly preferred.
  • the compounds according to the invention are particularly suitable for the treatment of all thromboembolic diseases, such as e.g. acute heart attack, cerebral infarction, pulmonary embolism, deep leg vein thrombosis, acute arterial occlusion, etc.
  • the compounds according to the invention are used with particular preference for the treatment of subchronic thromboembolic diseases in which prolonged thrombolysis has to be carried out.
  • the compounds of the invention in combination with an anticoagulant such as. B. heparin or hirudin and / or an inhibitor of platelet aggregation, whereby the vascular opening effect is increased with minor side effects.
  • an anticoagulant such as. B. heparin or hirudin and / or an inhibitor of platelet aggregation, whereby the vascular opening effect is increased with minor side effects.
  • Figure 3 shows the inhibition of CHO-tPA (curve I), r-PA (curve II) and r-PA
  • the starting plasmid pA27fd contains the following components: tac promoter, lac operator Region with an ATG start codon, the coding region for the t-PA derivative rPA, consisting of the N-terminal amino acids +1 to +3 of tPA, the Kringle2 domain (K2) and the protease domain (P), and the fd transcription terminator; the starting vector is the plasmid pKK223-3, which is described in EP-A 0 382 174 (17).
  • Fragment B Plasmid pA27fd is linearized with the restriction enzyme Pvul and also obtained preparatively after gel electrophoresis. The following oligonucleotide is synthesized for mutagenesis:
  • fragment A, fragment B (450 fmol each) and the oligonucleotide (75 pmol) are mixed and first in the presence of 50 mmol of 1 NaCl, 10 mmol / 1 Tris-HCl pH 7.5 and 10 mmol / l MgSÜ4 incubated for three minutes at 100 ° C and immediately transferred to ice. The DNA is renatured for 30 minutes at 60 ° C. The following is added to the heteroduplex for repair synthesis:
  • E. coli (1989) (20) and EP-A 0 373 365 (22) (these publications being the subject of the disclosure) transformed into E. coli (for example C600 + , see EP-A 0 373 365 (22)).
  • the transformants are selected by adding ampicillin and kanamycin (50 ⁇ g / ml each) to the nutrient medium.
  • the plasmid obtained is designated pA27Ala. It differs from the starting plasmid in that the codons for the amino acids KHRR are replaced by AAAA and by an additional PvuII site at the mutagenesis site.
  • the E. coli strain transformed with the plasmids pA27Ala and pUBS520 is incubated in LB medium (Sambrook et al., 1989, Molecular Cloning, Cold Spring Harbor) (14) in the presence of ampicillin and kanamycin (50 ⁇ g each / ml) up to an OD at 550 nm of 0.4.
  • Expression is initiated by adding 5 mmol of 1 IPTG (isopropyl- ⁇ -D-H-thiogalactoside). The culture is incubated for an additional 4 hours.
  • IPTG isopropyl- ⁇ -D-H-thiogalactoside
  • coli cells are then collected by centrifugation and resuspended in buffer (50 mmol / 1 Tris-HCl pH 8, 50 mmol / 1 EDTA); the cells are lysed by sonication.
  • the insoluble protein fractions are collected by renewed centrifugation and resuspended in the above-mentioned buffer by sonication.
  • 1/4 suspension of application buffer 250 mmol / l Tris-HCl pH 6.8, 10 mmol / l EDTA, 5% SDS, 5% mercaptoethanol, 50% glycerol and 0.005% bromophenol blue
  • a 12.5 % SDS polyacrylamide gels analyzed is added to the suspension and a 12.5 % SDS polyacrylamide gels analyzed.
  • the same preparation is carried out with a culture of E. coli with the two plasmids pA27Ala and pUBS520, which has not been treated with IPTG, and applied in the polyacrylamide gel.
  • IPTG-induced culture after staining the gel with Coomassie Blue R250 (dissolved in 30% methanol and 10% acetic acid), a clear band with a molecular weight of about 40 kD can be seen. This band is not present in the control preparation.
  • IB's 100 g IB's (wet weight) are dissolved in 450 ml 0.1 mol / 1 Tris-HCl / 6 mol / 1 guanidine HCl / 0.2 mol / 1 DTE (1,4-dithioerythritol) / 1 mmol / 1 EDTA pH 8, 6 suspended and stirred at 25 ° C for 2.5 h.
  • Guanidine hydrochloride (solid) is introduced so that after final dilution of the above dialysate with 10 mmol / 1 HCl the concentration of guanidine HCl is 6 mol / 1.
  • the mixture is preincubated at 25 ° C. for 1.5 h, then oxidized glutathione (GSSG) is added to a final concentration of 0.1 mol / 1 and Tris-HCl to a final concentration of 0.05 mol / 1 and the pH value is 5 mol / 1 NaOH titrated to pH 9.3.
  • the mixture is stirred at 25 ° C for 3.5 h.
  • dialysis against 10 mmol / 1 HCl (3 x 100 1, 48 h, 4 ° C.) is carried out. After dialysis, centrifugation is carried out and the clear supernatant is processed further.
  • a 10 1 reaction vessel is filled with 0.1 mol / 1 Tris-HCl, 0.8 mol 1 L-arginine, 2 mmol / l GSH (glutathione, reduced form), 1 mmol / 1 EDTA pH 8.5.
  • the naturation is carried out at 20 ° C by adding 3 times each 100 ml of derivative (mixed disulfide see above) at intervals of 24 h.
  • the renaturation preparation can be concentrated using a hemodialyzer.
  • r-PA r-PA
  • r-PA r-PA (KHRR296-299AAAA)
  • CHO-t-PA Actilyse®, recombinant glycosylated t-PA with the complete sequence according to Pennica et al. (1983) (18), produced from CHO cell lines) diluted with 0.01 M Tris HCl pH 7.5, 0.015% Tween 80® in such a way that a comparable increase in extinction was achieved after 2 h in the plasminogenetic assay.
  • the plasminogenolytic activity was determined by the method described in H. Lill (1987) (23), the content of this publication being the subject of the disclosure.
  • r-PA, r-PA (KHRR296 - 299 AAAA) and CHO-t-PA were adjusted with buffer to the concentrations given in the table and in the figures and their activity was determined in the clot lysis assay. Carrying out the clot-lysis assay
  • the sample is adjusted to the protein concentration required in each case by adding buffer (0.06 M Na2HP04, pH 7.4, 5 mg / ml BSA (bovine serum albumin), 0.01% Tween®).
  • buffer 0.06 M Na2HP04, pH 7.4, 5 mg / ml BSA (bovine serum albumin), 0.01% Tween®.
  • 0.1 ml of the sample is mixed with 1 ml of human fibrinogen solution (IMCO) (2 mg / ml 0.006 M Na2HP ⁇ 4, pH 7.4, 0.5 mg / ml BSA, 0.01% Tween 80®) and 5 incubated min at 37 ° C.
  • IMCO human fibrinogen solution
  • CHO-t-PA, r-PA and r-PA (KHRR296 - 299 AAAA) were dialyzed against 0.5 M arginine H 3 PO 4, pH 7.2 and adjusted to a protein concentration of 0.15 mg / ml. All samples were adjusted to a protein concentration of 1.5 ⁇ g ml with 0.05 M Tris HCl, pH 7.5, 0.15 M NaCl, 0.01% Tween 80®.
  • each of the samples were mixed with 770 ⁇ l test buffer, 100 ⁇ l fibrinogen (final concentration as shown in the figure), 10 ⁇ l BSA (100 mg / ml H2O), 10 ⁇ l thrombin (100 units / ml), 10 ⁇ l aprotinin (3, 75 mg / ml H2O) mixed and incubated for 1 h at 37 ° C.
  • the clot formed was separated by centrifugation (13,000 rpm, Sigma centrifuge) and the amount of enzyme present in the supernatant was determined by ELISA. A separate calibration curve was created for each protein.
  • r-PA and r-PA have identical fibrin binding, which differs significantly from the fibrin binding of CHO-t-PA (Fig. 2).
  • r-PA, Actilyse® and r-PA were diluted with 0.01 M Tris / HCl, pH 7.5, 0.015% Tween 80® so that they had an absorbance of 0.7 in the plasminogenolytic assay - 0.9 (wavelength 405 nm) reached after 2 h.
  • 40 ⁇ l of the diluted sample were mixed with 40 ⁇ l PAI-1 and incubated at 25 ° C. for 15 min. 25 ⁇ l of the sample were used in the plasminogenolytic test. The determination was made according to Lill et al. (1987) (23).
  • the rabbit model of neck vein thrombolysis established by D. Collen (1983) (21) was used to test the thrombolytic potency and efficiency.
  • Alteplase recombinant glycosylated tissue plasminogen activator from CHO cells, commercially available as Actilyse® from Thomae, Biberach, Germany
  • r-PA KHRR296 - 299AAAA
  • solvents 0.2 M arginine phosphate buffer
  • r-PA KHRR296 - 299AAAA
  • the solvent was applied in a volume dose of 26.7 ml. Additional experiments were carried out with iv bolus injection of r-PA (KHRR296 - 299AAAA) (1 mg / kg; after 2 h measurement of thrombolysis). Plasma samples were obtained repeatedly before, during and after the infusion or bolus injection. The plasminoge- nolytic activity was measured with a spectrophotometric test according to Lill (1987) (23). The half-life was taken from the semi-logarithmic plasma concentration time curve using graphical methods. Fibrinogen was determined according to Clauss
  • r-PA (KHRR296 - 299AAAA) had a dominant half-life of 15 min after iv bolus injection of 1 mg / kg in rabbits. This half-life is five times longer than the half-life of Alteplase (3 min) after iv bolus injection of 1 mg / kg (Martin et al., Thromb Res 1991; 62: 137-146) (24).
  • r-PA (KHRR296 - 299AAAA) is a thrombolytically active protein that has a 5-times longer half-life compared to Alteplase.
  • r-PA KHRR296 - 299AAAA
  • r-PA KHRR296 - 299AAAA
  • therapy with r-PA did not show any other usual bleeding from the cut wounds on the neck, although the fibrin specificity of r-PA (KHRR296 - 299AAAA) did not differ from that of Alteplase.
  • This property is of great importance for improving the safety profile of new thrombolytically active substances, since it reduces or even eliminates the side effects of bleeding that are otherwise common with thrombolytics, such as, for example, Alteplase.

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Abstract

L'invention concerne un dérivé activateur tissulaire non glucosylé du plasminogène, renfermant les parties essentielles d'au moins un domaine de Kringle et une chaîne B à activité sérine-protéase de l'activateur tissulaire du plasminogène, caractérisé en ce que les amino-acides 296-299 (Lys-His-Arg-Arg) sont remplacés chacun par une alanine. En raison du faible risque d'hémorragie associé à son emploi, ce dérivé est particulièrement approprié pour le traitement d'affections thrombo-emboliques subchroniques.
PCT/EP1995/002391 1994-07-05 1995-06-20 Derives activateurs du plasminogene non glucosyles et leur utilisation en cas de risque eleve d'hemorragie WO1996001312A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU28861/95A AU2886195A (en) 1994-07-05 1995-06-20 Non-glycosylated plasminogen activator derivatives and their use in conditions involving a high risk of bleeding
EP95924298A EP0769050A1 (fr) 1994-07-05 1995-06-20 Derives activateurs du plasminogene non glucosyles et leur utilisation en cas de risque eleve d'hemorragie

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DE19944423574 DE4423574A1 (de) 1994-07-05 1994-07-05 Nicht-glykosylierte Plasminogenaktivator-Derivate und ihre Verwendung bei erhöhtem Blutungsrisiko
DEP4423574.7 1994-07-05

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WO1996001312A1 true WO1996001312A1 (fr) 1996-01-18

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714145A (en) * 1988-09-02 1998-02-03 Genentech, Inc. Tissue plasminogen activator having zymogenic or fibrin specific properties
US5728567A (en) * 1988-09-02 1998-03-17 Genentech, Inc. Tissue plasminogen activator having zymogenic or fibrin specific properties
WO2005026341A3 (fr) * 2003-09-12 2005-09-01 Paion Deutschland Gmbh Activateurs plasminogenes a capacite de liaison lysine reduite
US8071091B2 (en) 2001-11-02 2011-12-06 Paion Deutschland Gmbh Non-neurotoxic plasminogen activating factors for treating stroke

Families Citing this family (3)

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US6346510B1 (en) 1995-10-23 2002-02-12 The Children's Medical Center Corporation Therapeutic antiangiogenic endostatin compositions
DE60034434T2 (de) * 1999-02-10 2008-01-10 The Children's Medical Center Corp., Boston Deglykosilierte fragmente der kringle 1-5 region von plasminogen und verwendungsverfahren
US7157556B1 (en) 1999-02-10 2007-01-02 The Children's Medical Center Corporation Deglycosylated kringle 1-3 region fragments of plasminogen and methods of use

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EP0382174A1 (fr) * 1989-02-07 1990-08-16 Roche Diagnostics GmbH Dérivé de l'activateur tissulaire de plasminogène
WO1991013149A1 (fr) * 1990-03-01 1991-09-05 Genentech, Inc. Activateur tissulaire du plasminogene presentant des proprietes specifiques de fibrine
WO1993024635A1 (fr) * 1992-06-03 1993-12-09 Genentech, Inc. Variantes d'activateur de plasminogene tissulaire obtenues par glycosylation, presentant de meilleures proprietes therapeutiques

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EP0382174A1 (fr) * 1989-02-07 1990-08-16 Roche Diagnostics GmbH Dérivé de l'activateur tissulaire de plasminogène
WO1991013149A1 (fr) * 1990-03-01 1991-09-05 Genentech, Inc. Activateur tissulaire du plasminogene presentant des proprietes specifiques de fibrine
WO1993024635A1 (fr) * 1992-06-03 1993-12-09 Genentech, Inc. Variantes d'activateur de plasminogene tissulaire obtenues par glycosylation, presentant de meilleures proprietes therapeutiques

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Title
CHEMICAL ABSTRACTS, vol. 119, no. 21, 22 November 1993, Columbus, Ohio, US; abstract no. 217011, PAONI, NICHOLAS F. ET AL: "Making tissue-type plasminogen activator more fibrin specific" *
EASTMAN, DONNA ET AL: "A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators", BIOCHEMISTRY (1992), 31(2), 419-22 CODEN: BICHAW;ISSN: 0006-2960 *
PROTEIN ENG. (1993), 6(5), 529-34 CODEN: PRENE9;ISSN: 0269-2139 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714145A (en) * 1988-09-02 1998-02-03 Genentech, Inc. Tissue plasminogen activator having zymogenic or fibrin specific properties
US5728567A (en) * 1988-09-02 1998-03-17 Genentech, Inc. Tissue plasminogen activator having zymogenic or fibrin specific properties
US5770426A (en) * 1988-09-02 1998-06-23 Genentech, Inc. Tissue plasminogen activator having zymogenic or fibrin specific properties
US8071091B2 (en) 2001-11-02 2011-12-06 Paion Deutschland Gmbh Non-neurotoxic plasminogen activating factors for treating stroke
US8119597B2 (en) 2001-11-02 2012-02-21 Paion Gmbh Non-neurotoxic plasminogen activating factors for treating of stroke
WO2005026341A3 (fr) * 2003-09-12 2005-09-01 Paion Deutschland Gmbh Activateurs plasminogenes a capacite de liaison lysine reduite
EA011156B1 (ru) * 2003-09-12 2009-02-27 Пайон Дойчланд Гмбх Активаторы плазминогена, имеющие сниженную способность связывать лизин
EP2368984A1 (fr) * 2003-09-12 2011-09-28 Paion Deutschland GmbH Activateurs plasminogenes a capacite de liaison lysine reduite

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EP0769050A1 (fr) 1997-04-23
AU2886195A (en) 1996-01-25

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