EP0125254A1 - 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

Info

Publication number
EP0125254A1
EP0125254A1 EP19830903395 EP83903395A EP0125254A1 EP 0125254 A1 EP0125254 A1 EP 0125254A1 EP 19830903395 EP19830903395 EP 19830903395 EP 83903395 A EP83903395 A EP 83903395A EP 0125254 A1 EP0125254 A1 EP 0125254A1
Authority
EP
European Patent Office
Prior art keywords
activator
modified
plasminogen activator
unmodified
human
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP19830903395
Other languages
German (de)
English (en)
Inventor
Jeffery Hugh Robinson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beecham Group PLC
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 EP0125254A1 publication Critical patent/EP0125254A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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.

Landscapes

  • 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.
EP19830903395 1982-10-28 1983-10-25 Derives d'enzymes et leur utilisation dans le traitement de la thrombose Withdrawn EP0125254A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8230801 1982-10-28
GB8230801 1982-10-28

Publications (1)

Publication Number Publication Date
EP0125254A1 true EP0125254A1 (fr) 1984-11-21

Family

ID=10533890

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19830903395 Withdrawn EP0125254A1 (fr) 1982-10-28 1983-10-25 Derives d'enzymes et leur utilisation dans le traitement de la thrombose

Country Status (2)

Country Link
EP (1) EP0125254A1 (fr)
WO (1) WO1984001786A1 (fr)

Families Citing this family (28)

* 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
EP0178105B1 (fr) * 1984-10-01 1997-04-02 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
GB8528321D0 (en) * 1985-11-18 1985-12-24 Ciba Geigy Ag Modified fibrinolytic agents
ES2031826T3 (es) * 1985-12-23 1993-01-01 Chiron Corporation Procedimiento para producir nuevos peptidos activadores del plasminogeno.
US5258298A (en) * 1986-01-31 1993-11-02 Genetics Institute, Inc. Deletion and glycosylation mutant of human tissue plasminogen activator
WO1987004722A1 (fr) * 1986-01-31 1987-08-13 Genetics Institute, Inc. Nouvelles proteines thrombolytiques
US5837518A (en) * 1986-01-31 1998-11-17 Genetics Institute, Inc. Thrombolytic proteins
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
US5589361A (en) * 1986-03-18 1996-12-31 Genentech, Inc. Human tissue-type plasminogen activator variant
IE81073B1 (en) * 1986-03-18 2000-01-12 Genentech Inc Modified human tissue-type plasminogen activator and its preparation
WO1988000615A1 (fr) * 1986-07-16 1988-01-28 Celltech Limited Procede de purification d'un activateur de plasminogene
US7084251B1 (en) 1987-02-12 2006-08-01 Genentech, Inc. Methods and Deoxyribonucleic acid for the preparation of tissue factor protein
US6994988B1 (en) 1987-02-12 2006-02-07 Genetech, Inc. Methods and deoxyribonucleic acid for the preparation of tissue factor protein
NO882226L (no) * 1987-05-22 1988-11-23 Zymogenetics Inc Fibrinolytiske proteiner.
EP0293934B1 (fr) * 1987-06-04 1994-08-31 Zymogenetics, Inc. t-PA mutant avec remplacement de kringle
NO179874C (no) * 1987-06-04 1997-01-02 Zymogenetics Inc Fremgangsmåte for fremstilling av en t-PA-analog, DNA-sekvens som koder for t-PA-analogen, samt ekspresjonsvektor omfattende DNA-sekvensen og vertcelle omfattende denne
US4935237A (en) * 1988-03-21 1990-06-19 Genentech, Inc. Processes for the preparation of t-PA mutants
IL87276A (en) * 1987-08-03 1995-07-31 Fujisawa Pharmaceutical Co Analog of tissue plasminogen activator comprising only the kringle 2 and protease domain dna encoding the same processes for the preparation thereof and pharmaceutical compositions containing the same
JP2708749B2 (ja) * 1987-08-10 1998-02-04 エーザイ株式会社 修飾型ｔＰＡ含有注射用組成物
IL88247A0 (en) * 1987-11-06 1989-06-30 Genentech Inc Novel human tissue-type plasminogen activator variant
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
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

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3015699C2 (de) * 1979-04-26 1982-07-15 Asahi Kasei Kogyo K.K., Osaka Herstellung eines Plasminogen-Aktivators
US4370417A (en) * 1980-04-03 1983-01-25 Abbott Laboratories Recombinant deoxyribonucleic acid which codes for plasminogen activator
US4326033A (en) * 1980-05-05 1982-04-20 Abbott Laboratories Modified urokinase having extended activity and method of making
NL8003402A (nl) * 1980-06-11 1982-01-04 Leuven Res & Dev Vzw Nieuwe plasminogeen-activator en farmaceutisch preparaat met trombolytische werking.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8401786A1 *

Also Published As

Publication number Publication date
WO1984001786A1 (fr) 1984-05-10

Similar Documents

Publication Publication Date Title
EP0125254A1 (fr) Derives d'enzymes et leur utilisation dans le traitement de la thrombose
EP0155387B1 (fr) Protéine hybride à activité fibrinolytique, son procédé de préparation et composition pharmaceutique
Castellino et al. [29] Human plasminogen
Jesty et al. [11] The activation of bovine coagulation factor X
JP3459416B2 (ja) 修飾されたファクター▲ｖｉｉ▼
EP0199574B1 (fr) Mutants d'activateur de plasminogène tissulaire humain, méthodes et intermédiaires pour ceux-ci et compositions utilisant ces mutants
EP0196920B1 (fr) Espèce dégradée d'activateur de plasminogène tissulaire, composition pharmaceutique et sa méthode de préparation
EP0233013A2 (fr) Enzyme modifié
EP0125269A1 (fr) Composes pharmaceutiquement actifs
Markland Jr [19] Crotalase
Husain Single-chain urokinase-type plasminogen activator does not possess measurable intrinsic amidolytic or plasminogen activator activities
US6706512B2 (en) Antithrombotic thrombin variants
EP0151593A1 (fr) Derives enzymatiques.
US5811279A (en) Method for activating prothrombin with polyethylene glycol
CA1308377C (fr) Complexe forme de polyethyleneglycol et d'activateur du plasminogene tissulaire
US4999194A (en) Two-chain urokinase plasminogen activators for treatment of thrombotic disease
EP0275606A1 (fr) Activateurs hybrides du plasminogène avec des propriétés thrombolytiques et médicaments contenant ces activateurs du plasminogène
EP0247674B1 (fr) Activateur du plasminogène et composition thrombolytique
EP0336508A1 (fr) Mutant recombinant d'une urokinase du type activateur du plasminogène constitué d'une seule chaîne et produite par une mutagénèse remplaçant la lysine 158 par de l'histidine
Li et al. Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator
Lijnen et al. Structural and functional characterization of mutants of recombinant single‐chain urokinase‐type plasminogen activator obtained by site‐specific mutagenesis of Lys158, Ile159 and Ile160
EP0250071A1 (fr) Enzymes fibrinolytiques, leurs procédés de préparation et leur utilisation
Cederholm‐Williams Molecular biology of plasminogen activators and recombinant DNA progress
US4880776A (en) Plasmin A-chain urokinase B-chain hybrid protein
Collen et al. K1K2Pu, a recombinant t-PA/u-PA Chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Designated state(s): CH DE FR GB LI NL

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19841002

RIN1 Information on inventor provided before grant (corrected)

Inventor name: ROBINSON, JEFFERY, HUGH