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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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • 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)
  • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • 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)
  • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• European Patent No 0009879 discloses derivatives of in vivo fibrinolytic enzymes which are useful therapeutic agents for treating venous thrombosis.
• the derivatives are characterised by the active catalytic site on the enzymes being blocked by a group which is removable by hydrolysis such that the pseudo first order rate constant for hydrolysis is in the range 10 — Pi to 10 — J - ⁇ sec—1.
• EP-A-0297882 discloses a hybrid plasminogen activator which comprises the five kringle domains of plasminogen linked to the B-chain of t-PA or u-PA via an amino acid sequence comprising, respectively, the t-PA cleavage site between residues 275 and 276 and the cysteine residue 264 of t-PA or the u-PA cleavage site between residues 158 and 159 and the cysteine residue 148 of u-PA, the catalytic site of the t-PA or u-PA B-chain being optionally blocked by a removable blocking group.
• 4-methoxybenzoyl plasminogen 1-5 1/t-PA 262-527 including one and two chain variants, lys ⁇ g and glu- ⁇ variants, and mixtures thereof.
• the invention provides glu- ⁇ , single chain plasminogen 1-541/t-PA 262-527.
• This variant is preferably present at a level of at least 60%, more preferably 80% relative to the other chain variants.
• the unacylated hybrid PA is first prepared by expressing DNA encoding the hybrid PA in host cell and recovering the hybrid PA product.
• the DNA is prepared conventionally by the condensation of appropriate mono-, di- or oligomeric nucleotide units as described in EP-A-0297882.
• the host cell is prepared conventionally by transformation with a replicable expression vector capable, in the host cell, of expressing the coding DNA.
• CHO cells were trypsinised and plated out at 5 x 10 * ⁇ per
• the mixture was buffer-exchanged into 0.02M Tris/0.2M NaCl/0.2M L-arginine/0.01% Tween 80 pH 7.4 using a Sephadex G25 PD10 column.
• the buffer-exchanged product was stored at -40°C.
• the deacylation half-life of the product in human, plasma at 37°C was 34 min.

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• Health & Medical Sciences (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Diabetes (AREA)
• Animal Behavior & Ethology (AREA)
• Hematology (AREA)
• Veterinary Medicine (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Enzymes And Modification Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (2)

Family

ID=10677652

Family Applications (1)

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Cited By (1)

Citations (3)

Family Cites Families (1)

• 1990
  • 1990-06-14 GB GB909013345A patent/GB9013345D0/en active Pending
• 1991
  • 1991-06-12 ZA ZA914520A patent/ZA914520B/xx unknown
  • 1991-06-12 WO PCT/GB1991/000945 patent/WO1991019793A2/en not_active Application Discontinuation
  • 1991-06-12 CA CA002085224A patent/CA2085224A1/en not_active Abandoned
  • 1991-06-12 JP JP91510746A patent/JPH05506363A/ja active Pending
  • 1991-06-12 NZ NZ238506A patent/NZ238506A/xx unknown
  • 1991-06-12 IE IE199991A patent/IE911999A1/en unknown
  • 1991-06-12 AU AU80681/91A patent/AU648567B2/en not_active Ceased
  • 1991-06-12 EP EP91910869A patent/EP0535037A1/en not_active Withdrawn
  • 1991-06-12 KR KR1019920703217A patent/KR930701607A/ko not_active Application Discontinuation
  • 1991-06-13 IL IL98478A patent/IL98478A0/xx unknown
  • 1991-06-14 PT PT97983A patent/PT97983A/pt not_active Application Discontinuation

Patent Citations (3)

Non-Patent Citations (2)

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