WO1984001786A1 - Derives d'enzymes et leur utilisation dans le traitement de la thrombose - Google Patents

Derives d'enzymes et leur utilisation dans le traitement de la thrombose Download PDF

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Publication number
WO1984001786A1
WO1984001786A1 PCT/GB1983/000273 GB8300273W WO8401786A1 WO 1984001786 A1 WO1984001786 A1 WO 1984001786A1 GB 8300273 W GB8300273 W GB 8300273W WO 8401786 A1 WO8401786 A1 WO 8401786A1
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WO
WIPO (PCT)
Prior art keywords
activator
modified
plasminogen activator
unmodified
human
Prior art date
Application number
PCT/GB1983/000273
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English (en)
Inventor
Jeffery Hugh Robinson
Original Assignee
Beecham Group Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beecham Group Plc filed Critical Beecham Group Plc
Publication of WO1984001786A1 publication Critical patent/WO1984001786A1/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

  • This invention relates to a plasminogen activator, processes for preparing the activator and pharmaceutical compositions containing it.
  • US Patent No. 4 326 033 discloses a method of increasing the biological alf life of the fibrinolytic agent urokinase which consists of chemically treating urokinase to remove or degrade carbohydrate units in the urokinase molecule. The patent states that this treatment results in the retention of 20% of the fibrinolytic activity of the original starting material.
  • Urokinase is one of many glycoproteins, and there is no indication in the above US Patent that the treatment described therein would have any applicability to other glycoproteins.
  • Tissue-type plasminogen activator (hereinafter referred to as t-PA) is, unlike urokinase, similar in its immunolo ical and biological nature to human extrinsic plasminogen activator normally present in human blood. T-PA can be readily isolated from the culture fluid of human melanoma cells, and has been shown to have a potent thrombolytic effect in animals and man.
  • tissue-type plasminogen activator t-PA
  • t-PA tissue-type plasminogen activator
  • ii) Enzymatic treatment of t-PA with a mixture of exo lycosidases, selected for example from (3-galactosidase, ⁇ -mannosidase, -fucosidase, B-N-acetylglucosaminidase or neuraminidase, or one or more endoglycosidases, such as endoglycosidase D (from Diplococcus pneumoniae) or endoglycosidase H (from Streptomyces plicatus).
  • endoglycosidase D from Diplococcus pneumoniae
  • endoglycosidase H from Streptomyces plicatus
  • T-PA can itself be obtained according to the method described in Published European Patent Application No. 0 041 766, which describes the production of a preferred form of t-PA, namely melanoma plasminogen activator, from human melanoma cells.
  • the proportion of retained fibrinolytic activity of the structurally modified t-PA depends to some extent on the method of modification and the efficiency of recovery and purification. We have found that by employing the method of paragraph (i) above, and carefully monitoring the physical conditions of the degradation process, we can obtain 70% or more of retained fibrinolytic activity.
  • modified t-PA of this invention can be formulated into pharmaceutical compositions in accordance with standard procedures.
  • the invention also provides a pharmaceutical composition which comprises structurally modified t-PA as defined herein together with a pharmaceutically acceptable carrier.
  • the composition is preferably adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions of the modified t-PA in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent to keep the modified t-PA in solution and a local anaesthetic such as lignocaine to ease pain at the site of injection.
  • the modified t-PA will be supplied in unit dosage form for example as a dry powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of t-PA in activity units.
  • modified t-PA is to be administered by infusion
  • it will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection'.
  • modified t-PA is to be administered by injection, it is dispensed with an ampoule of sterile water for injection.
  • the injectable or infusable* composition will be made up by mixing the ingredients prior to administration.
  • the quantity of material administered will depend upon the amount of fibrinolysis required and the speed with which it is required, the seriousness of the thromboembolic condition and the position and size of the clot.
  • the precise dose to be employed and mode of administration may be decided according to the circumstances by the physician supervising treatment.
  • a patient being treated for a medium size thrombus will generally receive a daily dose of from 0.10 to 1.0 mg kg" 1 of body weight either by injection in up to eight doses or by infusion.
  • the invention provides a therapeutic or prophylactic method of treating thrombosis in human or non-human animals, which comprises treating an animal with an effective, non-toxic amount of the structurally modified plasminogen activator of the invention.
  • Melanoma plasminogen activator was purified to 70-90% purity from cultured Bowes melanoma cells by chromatography using zinc-chelate agarose, and lysine - Sepharose.
  • the purified MPA was then subjected to carbohydrate degradation, based on published procedures (Biochem. Biophys. Res. Commun. , j57_, 55, 1974) which were modified in order to obtain a high degree of recovery of- enzyme activity.
  • Purified MPA was dissolved in a buffer containing 0.1 M sodium phosphate, 0.15M sodium chloride, 0.01% Tween 80, pH 6.0 at a concentration of about 0.5-1.0 mg/mi. Any contaminating ions present in the MPA preparation can be removed by prior gel filtration or dialysis. To this solutiorr ⁇ was added a solution of 0.1 M sodium periodate in the above buffer to a final concentration of 10 mM. The solution was kept at 4 ⁇ C in the dark for one hour. During this period the enzymic activity, as measured by chromogenic substrate assay, ' dropped to 70% of the original.
  • the modified activator was found to be equally active as the native activator.
  • Chromogenic substrate assay - MPA was assayed by spectrophotometric measurement of the rate of release of p-nitroaniline from the substrate S-2288 (Kabi Diagnostica) when incubated with the enzyme in 0.1 M triethanolamine buffer, pH 8.0 at 25 ⁇ C.
  • the substrate concentration was 1 mM.
  • mice Male Sprague-Dawley rats (300-400 g) were anaesthetized with pentobarbitone sodium (60 mg/kg i.p.). One carotid artery was cann ⁇ lated for collection of blood samples. One femoral vein was cannulated for injection of heparin (50 U/kg) and compound under test. Approximately 5 min after heparinization, a pre-dose blood sample (0.8 ml) was taken and mixed with 0.1 volumes 129 mM trisodium citrate. The compound under test was then injected (1 ml/kg) over 10s. Further blood samples were taken exactly 1,2,4,8,16,30 and 60 min later.
  • Heparin treatment 50 U/kg was repeated after the 30 min sample to maintain cannula patency. All citrated blood samples were kept on ice until the end of each experiment, then centrifuged at 1700 g for 15 min at 4 ⁇ to obtain plasma. The euglobulin fraction was precipitated by adding 0.1 ml each plasma to 1.82 ml ice-cold 0.011% (v/v) acetic acid in water. After 30 min standing in ice, all tubes were centrifuged at 1700 g for 15 min at 4°.
  • Fibrin plates were prepared from 0.4% (w/v) human fibrinogen (Kabi, Grade L, Flow Laboratories, Scotland) dissolved in 0.029 M barbitone in 125 mM NaCl, pH 7.4, pipetted (9 ml) into 10 x 10 cm square plastic dishes (Sterilin) and clotted by rapid mixing with 0.3 ml bovine thrombin (50 NIH units/ml, Parke-Davis, UK). Plates were incubated at 370 for 18-24h usually, but longer if required, and stained with aqueous bro ophenol blue. For each lysis zone, two diameters perpendicular to each other were measured using Vernier calipers.
  • Fig. 1 shows the clearance of native and modified MPA from the bloodstream of the rat.
  • the modified MPA is cleared less rapidly than the native material.
  • Thrombolysis in vivo is generally considered to be a prolonged event which requires significant concentrations of activator to be present over a long period; the initial rapid clearance phase (which is essentially identical for both modified and native MPA) is of less importance than the second phase.
  • Fig. 1 shows that after the first ca. 10 minutes, the plasma, concentration of modified MPA is on average six times that of native MPA and that the rate of clearance is significantly slower for the modified form.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

On modifie structuralement un activant plasminogène de type tissulaire de façon à enlever ou à dégrader au moins une partie de la portion hydrate de carbone de la molécule activante. L'activant modifié possède une activité fibrinolytique conservée au moins à 50 % en comparaison avec l'activant non modifié, couplée avec une demi vie biologique accrue.
PCT/GB1983/000273 1982-10-28 1983-10-25 Derives d'enzymes et leur utilisation dans le traitement de la thrombose WO1984001786A1 (fr)

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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0178105A2 (fr) * 1984-10-01 1986-04-16 Genzyme Corporation Techniques d'ADN recombinant et produits ainsi obtenus
EP0225286A2 (fr) * 1985-11-18 1987-06-10 Ciba-Geigy Ag Agent fibrinolytique
EP0238304A2 (fr) * 1986-03-18 1987-09-23 Genentech, Inc. Activateur de plasminogène tissulaire humain modifié et sa préparation
WO1988000615A1 (fr) * 1986-07-16 1988-01-28 Celltech Limited Procede de purification d'un activateur de plasminogene
US4753879A (en) * 1984-08-27 1988-06-28 Biogen N.V. Modified tissue plasminogen activators
EP0292009A1 (fr) * 1987-05-22 1988-11-23 Zymogenetics, Inc. Protéines fibrinolytiques
EP0293936A2 (fr) * 1987-06-04 1988-12-07 Zymogenetics, Inc. Analogues d'un activateur tissulaire du plasminogène ayant des domaines modifiés d'un facteur de croissance
EP0293934A1 (fr) * 1987-06-04 1988-12-07 Zymogenetics, Inc. t-PA mutant avec remplacement de kringle
WO1989004368A1 (fr) * 1987-11-06 1989-05-18 Genentech, Inc. Nouvelle variante d'activateurs de plasminogene de tissus humains
WO1989007641A1 (fr) * 1988-02-10 1989-08-24 Genzyme Corporation Exaltation des proprietes therapeutiques d'une glycoproteine
WO1989008702A1 (fr) * 1988-03-11 1989-09-21 Codon Procedes d'obtention d'activateurs plasminogenes tissulaires a l'aide d'agents chaotropiques
US4935237A (en) * 1988-03-21 1990-06-19 Genentech, Inc. Processes for the preparation of t-PA mutants
US5002887A (en) * 1986-01-31 1991-03-26 Genetics Institute, Inc. Truncated thrombolytic proteins
US5037646A (en) * 1988-04-29 1991-08-06 Genentech, Inc. Processes for the treatment of vascular disease
US5071972A (en) * 1986-01-31 1991-12-10 Genetics Institute, Inc. DNA sequences encoding novel thrombolytic proteins
US5242688A (en) * 1990-12-24 1993-09-07 Eli Lilly And Company Method of treating thromboembolic disorders by administration of diglycosylated t-pa variants
US5244806A (en) * 1985-08-26 1993-09-14 Eli Lilly And Company DNA encoding novel tissue plasminogen activator derivatives having kringles 1 and 2 deleted, vectors and host cells
JPH05268959A (ja) * 1986-01-31 1993-10-19 Genetics Inst Inc 新しい血栓溶解タンパク質
US5258298A (en) * 1986-01-31 1993-11-02 Genetics Institute, Inc. Deletion and glycosylation mutant of human tissue plasminogen activator
US5425943A (en) * 1987-08-10 1995-06-20 Eisai Co., Ltd. Modified tPA-containing injection composition having increased solubility
US5439679A (en) * 1985-12-23 1995-08-08 Chiron Corporation Peptide plasminogen activators
US5589361A (en) * 1986-03-18 1996-12-31 Genentech, Inc. Human tissue-type plasminogen activator variant
WO1997020042A1 (fr) * 1995-11-30 1997-06-05 Eisai Co., Ltd. Derives d'activateur tissulaire du plasminogene presentant une deficience au niveau de la chaine de sucre
US5648254A (en) * 1988-01-15 1997-07-15 Zymogenetics, Inc. Co-expression in eukaryotic cells
US5648250A (en) * 1987-08-03 1997-07-15 Fujisawa Pharmaceutical Co., Ltd. Tissue plasminogen activator
US5837518A (en) * 1986-01-31 1998-11-17 Genetics Institute, Inc. Thrombolytic proteins
US6994988B1 (en) 1987-02-12 2006-02-07 Genetech, Inc. Methods and deoxyribonucleic acid for the preparation of tissue factor protein
US7084251B1 (en) 1987-02-12 2006-08-01 Genentech, Inc. Methods and Deoxyribonucleic acid for the preparation of tissue factor protein

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2454809A1 (fr) * 1979-04-26 1980-11-21 Asahi Chemical Ind Procede de production d'un activateur de plasminogene
EP0037687A2 (fr) * 1980-04-03 1981-10-14 Abbott Laboratories Acide désoxyribonucléique recombinant codant pour activateur de plasminogène et méthode pour la production de protéine étant un activateur de plasminogène à partir de celui-ci
EP0041766A2 (fr) * 1980-06-11 1981-12-16 Leuven Research & Development V.Z.W. Nouveau activateur de plasminogène et composition pharmaceutique à activité thrombolytique
US4326033A (en) * 1980-05-05 1982-04-20 Abbott Laboratories Modified urokinase having extended activity and method of making

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2454809A1 (fr) * 1979-04-26 1980-11-21 Asahi Chemical Ind Procede de production d'un activateur de plasminogene
EP0037687A2 (fr) * 1980-04-03 1981-10-14 Abbott Laboratories Acide désoxyribonucléique recombinant codant pour activateur de plasminogène et méthode pour la production de protéine étant un activateur de plasminogène à partir de celui-ci
US4326033A (en) * 1980-05-05 1982-04-20 Abbott Laboratories Modified urokinase having extended activity and method of making
EP0041766A2 (fr) * 1980-06-11 1981-12-16 Leuven Research & Development V.Z.W. Nouveau activateur de plasminogène et composition pharmaceutique à activité thrombolytique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Nature, Vol. 301, January 1983, D. PENNICA et al.: "Cloning and Expression of Human Tissue-Type Plasminogen Activator cDNA in E. Coli" pages 214-221, see the entire document *
Proc. Natl. Acad. Sci. USA, Vol. 80, January 1983, T. EDLUND et al.: "Isolation of cDNA Sequences Coding for a part of Human Tissue Plasminogen Activator", pages 349-352, see the entire document *

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4753879A (en) * 1984-08-27 1988-06-28 Biogen N.V. Modified tissue plasminogen activators
US5344773A (en) * 1984-10-01 1994-09-06 Genzyme Corporation Human uterine tissue plasminogen activator produced by recombinant DNA
EP0178105A3 (fr) * 1984-10-01 1988-11-17 Genzyme Corporation Techniques d'ADN recombinant et produits ainsi obtenus
EP0178105A2 (fr) * 1984-10-01 1986-04-16 Genzyme Corporation Techniques d'ADN recombinant et produits ainsi obtenus
US5244806A (en) * 1985-08-26 1993-09-14 Eli Lilly And Company DNA encoding novel tissue plasminogen activator derivatives having kringles 1 and 2 deleted, vectors and host cells
US4960702A (en) * 1985-09-06 1990-10-02 Codon Methods for recovery of tissue plasminogen activator
EP0225286A2 (fr) * 1985-11-18 1987-06-10 Ciba-Geigy Ag Agent fibrinolytique
EP0225286B1 (fr) * 1985-11-18 1992-07-22 Ciba-Geigy Ag Agent fibrinolytique
US5976530A (en) * 1985-12-23 1999-11-02 Chiron Corporation Peptide plasminogen activators
US5656269A (en) * 1985-12-23 1997-08-12 Chiron Corp. Peptide plasminogen activators
US5501853A (en) * 1985-12-23 1996-03-26 Chiron Corporation Peptide plasminogen activators
US5439679A (en) * 1985-12-23 1995-08-08 Chiron Corporation Peptide plasminogen activators
US5071972A (en) * 1986-01-31 1991-12-10 Genetics Institute, Inc. DNA sequences encoding novel thrombolytic proteins
US5002887A (en) * 1986-01-31 1991-03-26 Genetics Institute, Inc. Truncated thrombolytic proteins
US5258298A (en) * 1986-01-31 1993-11-02 Genetics Institute, Inc. Deletion and glycosylation mutant of human tissue plasminogen activator
JPH05268959A (ja) * 1986-01-31 1993-10-19 Genetics Inst Inc 新しい血栓溶解タンパク質
US5837518A (en) * 1986-01-31 1998-11-17 Genetics Institute, Inc. Thrombolytic proteins
US5589361A (en) * 1986-03-18 1996-12-31 Genentech, Inc. Human tissue-type plasminogen activator variant
EP0238304A2 (fr) * 1986-03-18 1987-09-23 Genentech, Inc. Activateur de plasminogène tissulaire humain modifié et sa préparation
EP0238304A3 (en) * 1986-03-18 1988-07-06 Genentech, Inc. Modified human tissue-type plasminogen activator and its preparation
FR2597883A1 (fr) * 1986-03-18 1987-10-30 Genentech Inc Nouveaux polypeptides activateurs du plasminogene de type tissulaire humain
US5714144A (en) * 1986-03-18 1998-02-03 Genentech, Inc. Human tissue-type plasminogen activator variant
DE3708681A1 (de) * 1986-03-18 1987-10-01 Genentech Inc Human-gewebeplasminogen-aktivator, verfahren zu seiner herstellung und seine verwendung als arzneistoff
WO1988000615A1 (fr) * 1986-07-16 1988-01-28 Celltech Limited Procede de purification d'un activateur de plasminogene
US6994988B1 (en) 1987-02-12 2006-02-07 Genetech, Inc. Methods and deoxyribonucleic acid for the preparation of tissue factor protein
US7084251B1 (en) 1987-02-12 2006-08-01 Genentech, Inc. Methods and Deoxyribonucleic acid for the preparation of tissue factor protein
EP0292009A1 (fr) * 1987-05-22 1988-11-23 Zymogenetics, Inc. Protéines fibrinolytiques
EP0293936A2 (fr) * 1987-06-04 1988-12-07 Zymogenetics, Inc. Analogues d'un activateur tissulaire du plasminogène ayant des domaines modifiés d'un facteur de croissance
EP0293934A1 (fr) * 1987-06-04 1988-12-07 Zymogenetics, Inc. t-PA mutant avec remplacement de kringle
EP0293936A3 (en) * 1987-06-04 1989-09-20 Zymogenetics Inc. Tissue plasminogen activator analogs having modified growth factor domains
US5648250A (en) * 1987-08-03 1997-07-15 Fujisawa Pharmaceutical Co., Ltd. Tissue plasminogen activator
US5840533A (en) * 1987-08-03 1998-11-24 Fujisawa Pharmaceutical Co., Ltd. Tissue plasminogen activator
US5425943A (en) * 1987-08-10 1995-06-20 Eisai Co., Ltd. Modified tPA-containing injection composition having increased solubility
AU658909B2 (en) * 1987-11-06 1995-05-04 Genentech Inc. Novel human tissue-type plasminogen activator variant
EP0754753A1 (fr) * 1987-11-06 1997-01-22 Genentech, Inc. Nouvelle variante d'activateurs de plasminogène de tisses humains
WO1989004368A1 (fr) * 1987-11-06 1989-05-18 Genentech, Inc. Nouvelle variante d'activateurs de plasminogene de tissus humains
US5648254A (en) * 1988-01-15 1997-07-15 Zymogenetics, Inc. Co-expression in eukaryotic cells
WO1989007641A1 (fr) * 1988-02-10 1989-08-24 Genzyme Corporation Exaltation des proprietes therapeutiques d'une glycoproteine
WO1989008702A1 (fr) * 1988-03-11 1989-09-21 Codon Procedes d'obtention d'activateurs plasminogenes tissulaires a l'aide d'agents chaotropiques
US4935237A (en) * 1988-03-21 1990-06-19 Genentech, Inc. Processes for the preparation of t-PA mutants
US5037646A (en) * 1988-04-29 1991-08-06 Genentech, Inc. Processes for the treatment of vascular disease
US5242688A (en) * 1990-12-24 1993-09-07 Eli Lilly And Company Method of treating thromboembolic disorders by administration of diglycosylated t-pa variants
WO1997020042A1 (fr) * 1995-11-30 1997-06-05 Eisai Co., Ltd. Derives d'activateur tissulaire du plasminogene presentant une deficience au niveau de la chaine de sucre

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