Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors
• • 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)
  • C12N9/6464—Protein C (3.4.21.69)
• • 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

• Fig.2 illustrates impact of human protein S on the effect of APC (wt and.mutant) in an APTT assay.
• the following APC variants were examined: wt APC (•) and APC mutant QGNSEDY (ALL) (D).
• such variants encompass only a few modified amino acid residues, and possibly only one amino acid residue, in order to preserve substantial homology with respect to the wild-type substance. This is of particular importance in connection with use of the present variants for treatment in vivo to avoid, or at least reduce, a possible immune response to the variant used for treatment.
• the present variants are substantially homologous to the corresponding wild-type substance and contain only point mutations, e. g. one or a few single amino acid residue substitutions, deletions and/or insertions.
• the variants contain more than one amino acid residue modification and could contain as many as up to 10 amino acid residue modifications for use in vivo.
• recombinant DNA technique is used to prepare the present DNA segments comprising a modified structural gene.
• a DNA segment of the present invention comprising a structural gene encoding a modified PC/APC can be obtained by modification of the said recombinant DNA molecule to introduce desired amino acid residue changes, such as substitutions (replacements), deletions and/or insertions (additions), after expression of said modified recombinant DNA molecule.
• desired amino acid residue changes such as substitutions (replacements), deletions and/or insertions (additions
• One convenient method for achieving these changes is by site-directed mutagenesis, e.g. performed with PCR-technology. PCR is an abbreviation for Polymerase Chain Reaction, and was first reported by Mullis and Faloona (1987).
• Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPVl/pML2d (International Biotechnologies, Inc.), pXTl available from Stratagene (La Jolla, California), pJ5E ⁇ available from The American Type Culture Collection (ATCC; Rockwille, MD) as accession number ATCC 37722, pTDTl (ATCC 31255) and the like eukaryotic expression vectors.
• pRc/CMV available from Invitrogen, California, U.S.A. has been used to obtain expression plasmids for use in adenovirus-transfected human kidney cells.
• the present invention is also concerned with methods for inhibiting coagulation in an individual, e. g. a human, said method comprising administering to said individual a composition comprising a therapeutically effective amount of a variant PC/APC of the present invention. Conditions that could be treated are disclosed elsewhere in this specification.
• the transfected cells from (b) were incubated for 16 hours, whereafter the medium was replaced with complete medium containing 10% calf serum and the cells were incubated for additional 48-72 hrs.
• the cells were then trypsinized and seeded into 10-cm dishes contaning selection medium (DMEM comprising 10% serum, 400 ⁇ g/ml G418, 2 mM L- glutamine, 100 U/ml penicillin, 100 U/ml streptomycin and 10 ⁇ g/ml vitamin Ki) (Grinnell, et al. 1990).
• G418-resistant colonies were obtained after 3-5 weeks selection. From each DNA transfection procedure, 24 colonies were selected and grown until confluence.
• Example 2(b)(i) Experiments with protein S deficient plasma like those described in Example 2(b)(i), were also performed, the thromboplastin system of Example 2(c)(i) being used. The results thereby obtained were similar to those described for the APTT system in Example 2(b)(ii).In brief, it was found that the QGNSEDY variant is active in the absence of protein S, but yet, its activity is potentiated by protein S.

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• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 2002
  • 2002-03-01 EP EP02701843A patent/EP1370649A1/en not_active Withdrawn
  • 2002-03-01 JP JP2002570708A patent/JP2004527239A/ja active Pending
  • 2002-03-01 WO PCT/SE2002/000363 patent/WO2002070681A1/en not_active Ceased
  • 2002-03-01 AU AU2002235073A patent/AU2002235073B2/en not_active Ceased
  • 2002-03-01 CA CA002440092A patent/CA2440092A1/en not_active Abandoned
• 2003
  • 2003-09-02 US US10/652,947 patent/US20050059132A1/en not_active Abandoned

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