WO2008023052A1 - Combinaison d'un fvii et d'un facteur x activable par la thrombine - Google Patents

Combinaison d'un fvii et d'un facteur x activable par la thrombine Download PDF

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Publication number
WO2008023052A1
WO2008023052A1 PCT/EP2007/058797 EP2007058797W WO2008023052A1 WO 2008023052 A1 WO2008023052 A1 WO 2008023052A1 EP 2007058797 W EP2007058797 W EP 2007058797W WO 2008023052 A1 WO2008023052 A1 WO 2008023052A1
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Prior art keywords
factor
thrombin
preparation
factor vii
activable
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PCT/EP2007/058797
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English (en)
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Egon Persson
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Novo Nordisk Health Care Ag
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Publication of WO2008023052A1 publication Critical patent/WO2008023052A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—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
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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)
    • C12N9/6432—Coagulation factor Xa (3.4.21.6)
    • 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)
    • C12N9/647—Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
    • 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)
    • C12Y304/21006—Coagulation factor Xa (3.4.21.6)

Definitions

  • the invention relates to a pharmaceutical composition comprising a preparation of a factor Vl I or factor Vl l-related polypeptide and a preparation of thrombin activable factor X.
  • the invention also relates to a kit-of-parts for treatment of bleeding episodes comprising a preparation of a factor VII or factor Vl l-related polypeptide and a preparation of thrombin activable factor X.
  • the invention also relates to use of a preparation of a factor VII or factor VII- related polypeptide and a preparation of thrombin activable factor X for the preparation of a medicament.
  • the invention relates to methods for treating bleedings, reducing clotting time, enhancing haemostasis, reducing the number of administrations of coagulation factor protein needed to accomplish haemostasis, reducing the amount of administered coagulation factor protein needed to accomplish haemostasis, prolonging clot lysis time, increasing clot strength, and enhancing fibrin clot formation.
  • Blood coagulation factor VII is a plasma coagulation factor.
  • Activated factor VII (FVIIa) initiates the normal haemostatic process by forming a complex with tissue factor (TF), exposed as a result of the injury to the vessel wall, which subsequently activates fac- tors IX and X (FIX and FX) into their activated forms, factors IXa and Xa (FIXa and FXa).
  • Factor Xa converts limited amounts of prothrombin to thrombin on the tissue factor-bearing cell.
  • Thrombin activates platelets and factors V and VIII into factors Va and Villa (FVa and FVIIIa), both cofactors in the further process leading to the full thrombin burst. This process includes generation of factor Xa by thrombin from thrombin-activable factor X and occurs on the surface of activated platelets.
  • Thrombin finally converts fibrinogen to fibrin resulting in formation of a fibrin clot.
  • Factor VII exists in plasma mainly as a single-chain zymogen, which is cleaved by FXa into its two-chain, activated form, FVIIa.
  • Recombinant activated factor Vila (rFVIIa) has been developed as a pro-haemostatic agent.
  • the administration of rFVIIa offers a rapid and highly effective pro-haemostatic response in haemophilic subjects with bleedings who cannot be treated with coagulation factor products due to antibody formation. Also bleeding subjects with factor VII deficiency or subjects having a normal coagulation system but experiencing excessive bleeding can be treated successfully with FVIIa. In these studies, no unfavourable side effects of rFVIIa (in particular the occurrence of thromboembolism) has been encountered.
  • FX is normally the substrate of FIXa or FVIIa.
  • FX is normally the substrate of FIXa or FVIIa.
  • thrombin-activable FX contains an altered activation peptide making it susceptible to activation by thrombin (thrombin-activable FX).
  • thrombin-activable FX thrombin-activable FX
  • WO 02/062376 discloses pharmaceutical compositions comprising factor VII and factor IX and use of the combination to treat bleeding episodes.
  • FVIIa including variants of FVIIa
  • thrombin-activable FX can bypass the need for a functional FX activation complex (FIXa: FVI I Ia) and revert a haemophilic pheno- type.
  • FVIIa directly activates endogenous FX
  • thrombin-activable FX allows for thrombin to activate FX.
  • the combination of FVIIa and thrombin-activable FX exploits two routes of FX activation, and thereby increase the thrombin-generating potential.
  • the combination can be used to treat e.g. massive bleedings in a subject with an otherwise normal haemostatic system, taking advantage of both the great thrombin- generating potential of a FVIIa variant and the ensuing feedback activation of thrombin- activable FX.
  • one object of the present invention is to provide compositions, which can effectively be used in the treatment or prophylaxis of bleeding episodes and coagulation disorders.
  • a second object of the present invention is to provide compositions in one dosage form, which can effectively be used in the treatment or prophylaxis of bleeding episodes or as a procoagulant.
  • a further object of the present invention is to provide compositions, methods of treatment or kits exhibiting a synergistic effect.
  • a further object of the present invention is to provide compositions, methods of treatment or kits exhibiting no substantial side effects, such as a high level of systemic activation of the coagulation system.
  • the invention concerns a pharmaceutical composition
  • a pharmaceutical composition comprising a preparation of a factor VII or a factor Vll-related polypeptide, and a preparation of a thrombin- activable factor X.
  • the invention in a second aspect concerns a kit-of-parts containing a treatment for bleeding episodes comprising a) an effective amount of a preparation of a factor VII or factor Vll-related polypeptide and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a preparation of a thrombin-activable factor X and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms.
  • the invention concerns the use of a preparation of a factor VII or factor Vll-related polypeptide in combination with a preparation of a thrombin-activable factor X for the manufacture of a medicament for treating bleeding episodes in a subject.
  • the invention concerns the use of a preparation of a factor VII or factor Vll-related polypeptide in combination with a preparation of a thrombin-activable factor X for the manufacture of a medicament for reducing clotting time
  • the invention concerns the use of a preparation of a factor VII or factor Vll-related polypeptide in combination with a preparation of a thrombin-activable factor X for the manufacture of a medicament for prolonging the clot lysis time.
  • the invention concerns the use of a preparation of a factor VII or factor Vll-related polypeptide in combination with a preparation of a thrombin-activable factor X for the manufacture of a medicament for increasing clot strength.
  • the invention concerns a method for treating bleeding episodes in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amount together are ef- fective to treat bleedings.
  • the invention concerns a method for reducing clotting time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are ef- fective to reduce clotting time.
  • the invention concerns a method to enhance haemostasis in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are ef- fective to enhance haemostasis.
  • the invention concerns method for prolonging the clot lysis time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are ef- fective to prolong the clot lysis time.
  • the invention concerns a method for increasing clot strength in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are ef- fective to increase clot strength.
  • the invention concerns a method for enhancing fibrin clot formation in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to enhance fibrin clot formation.
  • the invention concerns a kit-of-parts containing a treatment for bleeding episodes comprising a) An effective amount of a factor VII or factor Vll-related polypeptide and an effective amount of a thrombin-activable factor X and a pharmaceutically acceptable carrier in a one-unit dosage form; and b) Container means for containing said one-unit dosage form.
  • the factor VII or factor Vll-related polypeptide is a factor Vll-related polypeptide.
  • the factor VII or factor Vll-related polypeptide is a factor VII polypeptide.
  • said factor Vll-related polypeptide is a factor VII amino acid sequence variant.
  • the ratio between the activity of the factor Vll-related polypeptide and the activity of native human factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay" as described in the present description.
  • the factor VII or factor Vll-related polypeptide is a factor VII polypeptide.
  • the factor VII is human factor VII.
  • the factor VII is bovine, porcine, canine, equine, murine or salmon factor VII.
  • the factor VII polypeptide is recombinant factor VII.
  • the factor VII polypeptide is plasma-derived factor VII.
  • the factor VII polypeptide is plasma-derived human factor VII.
  • the factor VII polypeptide is recombinant human factor VII.
  • the factor VII or factor Vll-related polypeptide is in its activated form.
  • the factor VII polypeptide is recombinant human factor Vila.
  • the thrombin-activable factor X polypeptide is recombinant human thrombin-activable factor X.
  • factor VII or factor Vll-related polypeptide and the thrombin- activable factor X or factor-IX related polypeptide are present in a ratio by mass of between about 100:1 and about 1 :100 factor Vll:thrombin-activable factor X.
  • the factor Vll-related polypeptides are amino acid sequence variants having no more than 20 amino acids replaced, deleted or inserted compared to wild- type factor VII (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No.
  • the factor Vila variants have no more than 15 amino acids replaced, deleted or inserted; in a further embodiment, the factor VII variants have no more than 10 amino acids replaced, deleted or inserted; in one embodiment, the factor VII variants have no more than 8 amino acids replaced, deleted or inserted; in one embodiment, the factor VII variants have no more than 6 amino acids replaced, deleted or inserted; in a further embodiment, the factor VII variants have no more than 5 amino acids replaced, deleted or inserted; in a further embodiment, the factor VII variants have no more than 3 amino acids replaced, deleted or inserted compared to wild-type factor VII.
  • the factor VII variants are selected from the list of L305V-FVIIa, L305V/M306D/D309S-FVIIa, L305I- FVIIa, L305T-FVIIa, F374P-FVIIa, V158T/M298Q-FVIIa, V158D/E296V/M298Q-FVIIa, K337A-FVIIa, M298Q-FVIIa, V158D/M298Q-FVIIa, L305V/K337A-FVIIa, V158D/E296V/M298Q/L305V-FVIIa, V158D/E296V/M298Q/K337A-FVIIa, V158D/E296V/M298Q/L305V/K337A-FVIIa, V158D/E296V/M298Q/L305V/K337A-FVIIa, K157A
  • the factor VII or factor Vll-related polypeptides have increased tissue factor-independent activity compared to native human coagulation factor Vila.
  • the increased activity is not accompanied by changes in the substrate specificity.
  • the binding of the factor VII or factor Vll- related polypeptides to tissue factor should not be impaired and the factor VII or factor Vll- related polypeptides should have at least the activity of wild-type factor Vila when bound to tissue factor.
  • the factor VII or factor Vll-related polypeptide and the thrombin- activable factor X are recombinant human factor Vila and recombinant human thrombin- activable factor X.
  • the clotting time is reduced in mammalian blood.
  • the haemostasis is enhanced in mammalian blood.
  • the clot lysis time is prolonged in mammalian blood.
  • the clot strength is increased in mammalian blood.
  • the fibrin clot formation is enhanced in mammalian blood.
  • the mammalian blood is human blood.
  • the mammalian blood is normal blood; in a further embodiment, the mammalian blood is blood having a normal level of coagulation factor proteins; in a further embodiment, the mammalian blood is blood having a normal level of thrombin-activable factor X; in a further embodiment, the blood is normal human blood; in one embodiment, the blood is blood from a subject having an impaired thrombin generation. In one embodiment, the blood is blood from a subject having a deficiency of one or more coagulation factors; in a further embodiment, the blood is blood from a subject having inhibitors against one or more coagulation factors. In one embodiment, the blood is from a subject having a lowered concentration of fibrinogen. In one embodiment, the blood is thrombin-activable factor X- deficient human blood.
  • the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are the sole haemostatic agents employed. In one embodiment, the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are the sole active haemostatic agents employed. In a further embodiment, the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are the sole coagulation factors employed. In one embodiment of the invention, the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are the sole active agents employed.
  • sole agents or factors as used herein refers to situations in which the factor VII or factor VII- related polypeptide and the thrombin-activable factor X, taken together, are the only haemostatic agents, active haemostatic agents, or coagulation factors, as applicable, contained in the pharmaceutical composition or kit, or are the only haemostatic agents, active haemo- static agents, or coagulation factors, as applicable, administered to the patient in the course of a particular treatment, such as, e.g., in the course of a particular bleeding episode. It will be understood that these situations encompass those in which other haemostatic agents or coagulation factors, as applicable, are not present in either sufficient quantity or activity so as to significantly influence one or more coagulation parameters.
  • the pharmaceutical composition is formulated for intravenous administration.
  • the composition further contains a pharmaceutical acceptable excipient.
  • the composition is in single-dosage form wherein the single-dosage form contains both coagulation factors.
  • the composition is in the form of a kit-of-parts comprising a preparation of a factor VII or factor Vl l-related polypeptide as a first unit dosage form and a preparation of a thrombin-activable factor X as a second unit dosage form, and comprising container means for containing said first and second dosage forms.
  • the composition or kit as applicable, further contains directions for the administration of the composition or separate components, respectively.
  • the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are administered in single-dosage form. In one embodiment of the invention, the factor VII or factor Vll-related polypeptide and the thrombin- activable factor X are administered in the form of a first unit dosage form comprising a preparation of a factor VII or factor Vll-related polypeptide and a second unit dosage form comprising a preparation of a thrombin-activable factor X.
  • the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are administered simultaneously. In a further embodiment, the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are administered sequentially. In one embodiment, the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are administered with a time separation of no more than 15 minutes, 10, 5, or 2 minutes. In one embodiment, the factor VII or factor Vll- related polypeptide and the thrombin-activable factor X are administered with a time separation of up to 2 hours, fromi to 2 hours, up to1 hour, from 30 minutes to1 hour, up to 30 minutes, or from15 to 30 minutes.
  • the effective amount of the factor VII or factor Vll-related polypeptide is an amount from about 0.05 mg/day to about 500 mg/day (70-kg subject). In one embodiment, the effective amount of a preparation of a thrombin-activable factor X is from about 0.01 mg/day to about 500 mg/day (70-kg subject). In one embodiment the factor VII or factor Vll-related polypeptide and thrombin- activable factor X are present in a ratio by mass of between about 100:1 and about 1 :100 factor Vll:thrombin-activable factor X.
  • the pharmaceutical composition is in single-dosage form and consists essentially of a preparation of a factor VII or factor VII- related polypeptide and a preparation of a thrombin-activable factor X, and one or more of the components selected from the list of pharmaceutical acceptable excipients or carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and protease inhibitors.
  • the subject is a human; in one embodiment, the subject has an impaired thrombin generation; in one embodiment, the subject has a lowered plasma concentration of fibrinogen (e.g., a multi-transfused subject); in one embodiment, the subject has a lowered plasma concentration of thrombin-activable factor X.
  • fibrinogen e.g., a multi-transfused subject
  • the subject has a lowered plasma concentration of thrombin-activable factor X.
  • the pharmaceutical composition is for home treatment
  • the invention concerns the use of a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin-activable factor X for the preparation of a medicament for the treatment of bleedings in a subject suffering from a thrombin- activable factor X-responsive syndrome.
  • the invention concerns the use of a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin-activable factor X for the preparation of a medicament for the treatment of bleedings in a subject having a reduced level of throm- bin-activable factor X.
  • the invention concerns a method to enhance haemostasis in a subject suffering from a thrombin-activable factor X responsive syndrome compared to when the subject is treated with thrombin-activable factor X as the only coagulation protein, the method comprising administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective to enhance haemostasis.
  • the invention concerns a method to enhance haemostasis in a subject having a reduced level of thrombin-activable factor X compared to when the subject is treated with thrombin-activable factor X as the only coagulation protein, the method compris- ing administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin- activable factor X, wherein the first and second amounts together are effective to enhance haemostasis.
  • the invention concerns a method to enhance formation of thrombin in a subject, the method comprising administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective to enhance formation of thrombin.
  • the invention concerns a method to enhance formation of thrombin in a subject suffering from a thrombin-activable factor X responsive syndrome compared to when the subject is treated with thrombin-activable factor X as the only coagulation protein, the method comprising administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective to enhance formation of thrombin.
  • the invention concerns a method to enhance formation of thrombin in a subject having a reduced level of thrombin-activable factor X compared to when the subject is treated with thrombin-activable factor X as the only coagulation protein, the method com- prising administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin- activable factor X, wherein the first and second amounts together are effective to enhance formation of thrombin.
  • the invention concerns a method for reducing the number of admini- strations of coagulation factor protein needed to accomplish haemostasis in a subject suffering from a thrombin-activable factor X responsive syndrome compared to the number of administrations needed when thrombin-activable factor X is administered to the subject as the only coagulation factor protein, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective to reduce the number of administrations of coagulation factor protein.
  • the invention concerns a method for reducing the number of administrations of coagulation factor protein needed to accomplish haemostasis in a subject having a reduced level of thrombin-activable factor X compared to the number of administrations needed when thrombin-activable factor X is administered to the subject as the only coagulation factor protein, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts to- gether are effective to reduce the number of administrations of coagulation factor protein.
  • the invention concerns a method for reducing the amount of administered coagulation factor protein needed to accomplish haemostasis in a subject suffering from a thrombin-activable factor X responsive syndrome compared to the amount of administered coagulation factor protein needed when thrombin-activable factor X is adminis- tered to the subject as the only coagulation factor protein, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll- related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective to reduce the amount of administered coagulation factor protein.
  • the invention concerns a method for reducing the amount of administered coagulation factor protein needed to accomplish haemostasis in a subject having a reduced level of thrombin-activable factor X compared to the amount of administered coagulation factor protein needed when thrombin-activable factor X is administered to the subject as the only coagulation factor protein, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective to reduce the amount of administered coagulation factor protein.
  • the invention concerns a method of treating bleedings in a subject suf- fering from a thrombin-activable factor X responsive syndrome, the method comprising administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective in treating bleedings.
  • the invention concerns a method of treating bleedings in a subject hav- ing a reduced level of thrombin-activable factor X, the method comprising administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective in treating bleedings.
  • the subject has a reduced level of thrombin-activable factor X. In one embodiment the subject suffers from a thrombin-activable factor X-responsive syn- drome. In one embodiment the thrombin-activable factor X responsive syndrome is haemophilia B.
  • the reduced thrombin-activable factor X level is 90% of normal level or below, in a further embodiment the thrombin-activable factor X level is 80% or below, in one embodiment 50% or below, in a further embodiment 40% or below, in a further embodiment 30% or below, in a further embodiment 20% or below, in a further embodiment 10% or below, in a further embodiment 5% or below, in a further embodiment 2% or below.
  • the terms may, where appropriate, be used interchangeably.
  • the thrombin-activable factor X level is below 30 % of normal level.
  • the invention concerns a method of treating bleedings in a subject suffering from a thrombin-activable factor X responsive syndrome, the method comprising administering to the subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amounts together are effective in treating bleedings.
  • the factor VII is human recombinant factor Vila (rFVIIa).
  • the rFVIIa is NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark).
  • the thrombin-activable factor X is human recombinant thrombin- activable factor X.
  • the pharmaceutical composition is formulated for intravenous administration.
  • the composition further comprises an inhibitor of the fibrinolytic system, including, without limitation, aprotinin, ⁇ -aminocaproic acid or tranexamic acid.
  • moderate bleedings may lead to complications if they require the administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation defects, etc.) because this is associated with the risk of transferring human viruses (e.g., hepatitis, HIV, parvovirus, or other, by now unknown viruses) as well as non-viral pathogens.
  • Extensive bleedings requiring massive blood transfusions may lead to the development of multiple organ failure including impaired lung and kidney function.
  • Subjects with thrombocytopenia also have an impaired thrombin generation as well as a defective stabilization of the fibrin plugs resulting in haemostatic plugs prone to premature dissolution.
  • subjects subjected to major trauma or organ damage and who, as a consequence, have obtained frequent blood transfusions often have lowered platelet counts as well as lowered levels of fibrinogen, factor VIII, and other coagulation proteins.
  • These subjects experience an impaired (or lowered) thrombin generation.
  • These subjects therefore, have a defective, or less efficient, haemostasis leading to the formation of fibrin plugs that are easily and prematurely dissolved by proteolytic enzymes, such enzymes in addition being extensively released in situations characterized by extensive trauma and organ damage.
  • a patient experiencing a major loss of blood becomes clinically unstable. Such patient are in risk of experiencing auricular fibrillation, which may lead to a fatal stop of cardiac activity; impaired renal function; or fluid extravasations in lungs (so-called "wet lungs” or ARDS).
  • haematomas Bleedings in tissues may also lead to the formation of haematomas.
  • the sizes of (in particular intercranial and spinal) haematomas are closely correlated to the extent of loss of neurological function, rehabilitation difficulties, and/or the severity and degree of permanent impairments of neurological function following rehabilitation.
  • the most severe consequences of haematomas are seen when they are located in the brain where they may even lead to the death of the patient.
  • compartment syndrome is a clinical condition caused by heavy bleeding internally into an extremity. In arms and legs the muscles and bones are externally confined by an almost inelastic collagen sheet called the fascia.
  • necrotic tissue will to a large extent, during the event of healing, be transformed into connective tissue, which is contracted compared to the original muscle tissue.
  • Such contractures make the subject liable to experience impaired motility of affected joints which again leads to the need of corrective surgery.
  • Severe haematomas may furthermore lead to formation of pseudo cysts which may be likened to benign tumours in that such cysts, like tumours, erode the affected muscle or bone tissues. Again, surgery is needed to remove such pseudo cysts.
  • Formation of haematomas furthermore increases the frequency of infections in a subject. So does infusion of blood products such as, e.g., red blood cells. Infusions of red blood cells lead to a risk of formation of antibodies in the subject. When antibodies to blood type antigens have been formed transfusion of the subject are difficult as it will be increasingly difficult to find suitable types of blood.
  • compositions, uses and methods of treatment for treatment of bleeding episodes in subjects in need of such treatment.
  • the compositions, uses and methods may be associated with beneficial effects such as less blood loss before haemostasis is obtained, less blood needed during surgery, blood pressure kept at an acceptable level until haemostasis is obtained, faster stabilisation of blood pressure, shorter recovery time for the treated patient, shorter rehabilitation time for the treated patient, diminished formation of haematomas or formation of smaller haematomas, including haematomas in the brain, less formation of pseudo cysts, less formation of muscle contractures, faster arrest of bleedings, reduction in the number of injections needed to stop bleeding and maintain haemostasis, reduction in the amount of coagulation protein usage for arresting bleeding and maintaining haemostasis.
  • a preparation of a factor VII or factor Vll-related polypeptide, e.g., factor Vila, in combination with a preparation of a thrombin-activable factor X provides a shortened clotting time compared to the clotting time when either factor Vila or thrombin- activable factor X is administered alone.
  • a preparation of a factor VII or factor Vll-related polypeptide, e.g., factor Vila, in combination with a preparation of a thrombin-activable factor X also provides for a reduced total amount of coagulation factor usage to arrest bleeding and maintain haemostasis in a subject in need of such treatment compared to the protein usage when ei- ther factor Vila or thrombin-activable factor X is administered alone.
  • a preparation of a factor VII or factor Vll-related polypeptide, e.g., factor Vila, in combination with a preparation of a thrombin-activable factor X also provides for a reduced time to obtain bleeding arrest and a reduced number of injections to maintain haemostasis compared to the situation when either factor Vila or thrombin-activable factor X is administered alone.
  • the administration of a preparation of a factor VII or factor Vll-related polypeptide, e.g., factor Vila, in combination with a preparation of a thrombin- activable factor X will also provide for a reduced number of injections to ensure full haemostasis compared to the situation when either thrombin-activable factor X or factor Vila is administered alone.
  • the present invention provides a pharmaceutical composition comprising a combination of a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin-activable factor X.
  • the composition may be in the form of a single composition or it may be in the form of a multi-component kit (kit-of-parts).
  • composition according to the present invention is useful as a therapeutic and prophylactic procoagulant in mammals, including primates such as humans.
  • the present invention further provides a method for treating (including prophylactically treating or preventing) bleeding episodes in a subject, including a human being.
  • a combination of a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin-activable factor X is an advantageous product ensuring short clotting times and rapid formation of haemostatic plugs.
  • the present inventors will show that a combination of factor Vila and thrombin-activable factor X can shorten the clotting time of normal human plasma more effectively than factor Vila or thrombin-activable factor X alone. Thus, by shortening the clotting time a more effective treatment of bleedings in subjects can be obtained. Moreover, patients may be treated with lower total amounts of factor VII and thrombin-activable factor X.
  • Factor VII Polypeptides In practicing the present invention, any factor VII polypeptide may be used that is effective in preventing or treating bleeding. This includes factor VII polypeptides derived from blood or plasma, or produced by recombinant means.
  • the present invention encompasses factor VII polypeptides, such as, e.g., those having the amino acid sequence disclosed in U.S. Patent No. 4,784,950 (wild-type human factor VII).
  • the factor VII polypeptide is human factor Vila, as disclosed, e.g., in U.S. Patent No. 4,784,950 (wild-type factor VII).
  • factor VII polypeptides include polypeptides that exhibit at least about 10%, at least about 30%, at least about 50%, or at least about 70%, of the specific biological activity of human factor Vila.
  • factor VII polypeptides include polypeptides that exhibit at least about 90%, at least about 100%, at least about 120%, at least about 140%, or at least about 160%, of the specific biological activity of human factor Vila. In one series of embodiments, factor VII polypeptides include polypeptides that exhibit at least about 70 %, at least about 80 %, at least about 90 %, or at least about 95 %, of identity with the sequence of wild-type factor VII as disclosed in U.S. Patent No. 4,784,950. As used herein, "factor VII polypeptide” encompasses, without limitation, factor VII, as well as factor Vll-related polypeptides.
  • factor VII is intended to encompass, without limitation, polypeptides having the amino acid sequence 1-406 of wild-type human factor VII (as disclosed in U.S. Patent No. 4,784,950), as well as wild-type factor VII derived from other species, such as, e.g., bovine, porcine, canine, murine, and salmon factor VII, said factor VII derived from blood or plasma, or produced by recombinant means. It further encompasses natural allelic variations of factor VII that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment.
  • Factor VII is also intended to encompass Factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor Vila. Typically, Factor VII is cleaved between residues 152 and 153 to yield Factor Vila.
  • Factor Vll-related polypeptides include, without limitation, factor VII polypeptides that have either been chemically modified relative to human factor VII and/or contain one or more amino acid sequence alterations relative to human factor VII (i.e., factor VII variants), and/or contain truncated amino acid sequences relative to human factor VII (i.e., factor VII fragments). Such factor Vll-related polypeptides may exhibit different properties relative to human factor VII, including stability, phospholipid binding, altered specific activity, and the like.
  • factor Vll-related polypeptides are intended to encompass such polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated "factor Vila-related polypeptides" or "activated factor Vll-related polypeptides"
  • factor Vll-related polypeptides encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild- type human factor VII. These polypeptides include, without limitation, factor VII or factor Vila that has been chemically modified and factor VII variants into which specific amino acid sequence alterations have been introduced that modify the bioactivity of the polypeptide.
  • polypeptides with a slightly modified amino acid sequence for instance, polypeptides having a modified N-terminal end including N-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified relative to human factor Vila.
  • Factor Vll-related polypeptides including variants of factor VII, whether exhibiting substantially the same or better bioactivity than wild-type factor VII, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type factor VII, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wild-type factor VII by insertion, deletion, or substitution of one or more amino acids.
  • Factor Vll-related polypeptides encompass those that exhibit at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, or at least about 130%, of the specific activity of wild-type factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.
  • Factor Vll-related polypeptides including variants, having substantially the same or improved biological activity relative to wild-type factor Vila encompass those that exhibit at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 110%, at least about 120%, or at least about 130% of the specific activity of wild-type factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.
  • Factor VII variants having a substantially modified biological activity relative to wild- type factor VII include, without limitation, factor VII variants that exhibit TF-independent Factor X proteolytic activity and those that bind TF but do not cleave Factor X.
  • the factor VII polypeptides are factor Vll-related polypeptides, in particular variants, wherein the ratio between the activity of said factor VII polypeptide and the activity of native human factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay” (see “Assays", below); in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0.
  • the factor VII polypeptides are factor Vll-related polypeptides, in particular variants, wherein the ratio between the activity of said factor VII polypeptide and the activity of native human factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Proteolysis Assay” (see “Assays", below); in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0; in further embodiments, the ratio is at least about 8.0.
  • the factor VII polypeptide is human factor VII, as disclosed, e.g., in U.S. Patent No. 4,784,950 (wild-type factor VII).
  • the factor VII polypeptide is human factor Vila.
  • the factor VII polypeptides are factor Vll-related polypeptides that exhibit at least about 10%, at least about 30%, at least about 50%, or at least about 70%, of the specific biological activity of human factor Vila.
  • the factor VII polypeptides have an amino acid sequence that differs from the sequence of wild-type factor VII by insertion, deletion, or substitution of one or more amino acids.
  • improved biological activity refers to FVII polypeptides with i) substantially the same or increased proteolytic activity compared to recombinant wild type human Factor Vila or ii) to FVII polypeptides with substantially the same or increased TF binding activity compared to recombinant wild type human Factor Vila or iii) to FVII polypeptides with substantially the same or increased half life in blood plasma compared to recombinant wild type human Factor Vila.
  • PEGylated human Factor Vila means human Factor Vila, having a PEG molecule conjugated to a human Factor Vila polypeptide.
  • the PEG molecule may be attached to any part of the Factor Vila polypeptide including any amino acid residue or carbohydrate moiety of the Factor Vila polypeptide.
  • the term "cysteine-PEGylated human Factor Vila” means Factor Vila having a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced in human Factor Vila.
  • Non-limiting examples of Factor VII variants having substantially the same or in- creased proteolytic activity compared to recombinant wild type human Factor Vila include S52A-FVIIa, S60A-FVIIa ( Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); FVIIa variants exhibiting increased proteolytic stability as disclosed in U.S. Patent No. 5,580,560; Factor Vila that has been proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng.
  • Non-limiting examples of FVII variants having increased biological activity compared to wild-type FVIIa include FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, WO 03/027147, WO 04/029090, WO 05/075635, and European patent application with application number 05108713.8 (Novo Nordisk A/S), WO 02/38162 (Scripps Re- search Institute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).
  • variants of factor VII include, without limitation, P1 OQ-FVII, K32E-FVII, P10Q/K32E-FVII, L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P- FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,
  • substitution variants in a factor VII polypeptide include, without limitation substitutions in positions P10, K32, L305, M306, D309, L305, L305, F374, V158, M298, V158, E296, K337, M298, M298, S336, S314, K316, K316, F374, S52, S60, R152, S344, T106, K143, N145, V253, R290, A292, G291 , R315, V317, and substitutions, additions or deletions in the amino acid sequence from T233 to N240 or from R304 to C329; or from 1153 to R223, or combinations thereof, in particular variants such as P10Q, K32E, L305V, M306D, D309S, L305I, L305T, F374P, V158T, M298Q, V158D, E296V, K337A, M298Q, M298K, S336G, S314E, K316H, K
  • factor VII biological activity biological activity of factor VII polypeptides
  • factor VII biological activity may be quantified by measuring the ability of a preparation to promote blood clotting using factor Vl l-deficient plasma and thromboplastin, as described, e.g., in U.S. Patent No. 5,997,864.
  • biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "factor VII units” by comparison with a pooled human serum standard containing 1 unit/ml factor VII activity.
  • factor Vila biological activity may be quantified by
  • factor VII biological activity or "factor VII activity” is intended to include the ability to generate thrombin; the term also includes the ability to generate thrombin on the surface of activated platelets in the absence of tissue factor.
  • a factor Vila preparation that may be used according to the invention is, without limitation, NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark).
  • Thrombin-activable factor X polypeptides Thrombin-activable factor X polypeptides:
  • the present invention encompasses thrombin-activable factor X polypeptides, such as, e.g., those disclosed in, J.Biol Chem 280 No50 p41352 (2005) and in WO 03/035861
  • any thrombin-activable factor X polypeptide may be used that is effective in preventing or treating bleeding.
  • Such thrombin-activable factor X polypeptides are derived by recombinant means as described in J.Biol Chem 280 No50 p41352 (2005) and in WO 03/035861.
  • amino acids mentioned herein are L-amino acids. It is to be understood, that the first letter in, for example, K337 represent the amino acid naturally present at the indicated position wild-type factor VII, and that, for example, K337A-FVIIa designates the FVII-variant wherein the amino acid represented by the one-letter code K naturally present in the indicated position is replaced by the amino acid represented by the one-letter code A.
  • factor VII Factor VII
  • Fact Vila Factor Vila
  • FVIIa Factor VII
  • TAFX thrombin- activable factor X
  • subjects with an impaired thrombin generation means subjects who cannot generate a full thrombin burst on the activated platelet surface and includes subjects having a generation of thrombin less that the thrombin-generation in subjects having a fully functioning, normal haemostatic system, including a normal amount and function of coagulation factors, platelets and fibrinogen, and includes, without limitations, subjects lacking thrombin-activable factor X; subjects with a lowered number of platelets or platelets with a defective function (e.g., thrombocytopenia or thrombasthenia Glanzmann or subjects with excessive bleeds); subjects having lowered levels of prothrombin, FX, FVIII, FIX or FVII; sub- jects having a lowered level of several coagulation factors (e.g., due to excessive bleeding as a consequence of trauma or extensive surgery); and subjects with lowered plasma concentrations of fibrinogen (e.g., multitransfused subjects).
  • the term "enhancement of the haemostatic system” means an enhancement of the ability to generate thrombin.
  • the term “enhancing haemostasis” is intended to encompass the situations when the measured thrombin generation for a test sample containing a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin- activable factor X is prolonged relative to the individual thrombin generation of a control sample containing only the factor VII or factor Vll-related polypeptide or the thrombin- activable factor X, respectively, when tested in the same thrombin generation assay.
  • the thrombin generation may be assayed as described in the thrombin generation assay of the present description (see “assay part").
  • Clot lysis time, clot strength, fibrin clot formation, and clotting time are clinical parameters used for assaying the status of patient's haemostatic system. Blood samples are drawn from the patient at suitable intervals and one or more of the parameters are assayed by means of, e.g., thromboelastography as described by, e.g., Meh et al., Blood Coagulation & Fibrinolysis 2001 ;12:627-637; Vig et al., Hematology, Vol. 6 (3) pp. 205-213 (2001 ); Vig et al., Blood coagulation & fibrinolysis, Vol. 12 (7) pp.
  • thromboelastography as described by, e.g., Meh et al., Blood Coagulation & Fibrinolysis 2001 ;12:627-637; Vig et al., Hematology, Vol. 6 (3) pp. 205-213 (2001 ); Vig et al., Blood
  • the term "prolonging clot lysis time" is intended to encompass the situations when the measured clot lysis time for a test sample containing a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin-activable factor X is prolonged rela- tive to the individual clot lysis time of a control sample containing only the factor VII or factor Vll-related polypeptide or the thrombin-activable factor X, respectively, when tested in the same clot lysis assay.
  • the clot lysis time may be assayed as described above.
  • the term "increasing clot strength" is intended to encompass the situations when the measured clot strength, e.g., mechanical strength, for a test sample containing a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin-activable factor X is increased relative to the individual clot lysis time of a control sample containing only the factor VII or factor Vll-related polypeptide or the thrombin-activable factor X, respectively, when tested in the same clot strength assay.
  • the clot strength may be assayed as described, e.g. in Carr et al, 1991. (Carr ME, Zekert SL. Measurement of platelet-mediated force development during plasma clot formation. AM J MED SCI 1991 ; 302: 13-8), or as described above by means of thromboelastography.
  • enhancing fibrin clot formation is intended to encompass the situations when the measured rate for or degree of fibrin clot formation for a test sample containing a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a preparation of a thrombin-activable factor X is increased relative to the individual rate for or degree of fibrin clot formation of a control sample containing only the factor VII or factor Vll-related polypeptide or the thrombin-activable factor X, respectively, when tested in the same clotting assay.
  • the fibrin clot formation may be assayed as described above.
  • shortening clotting time is intended to encompass the situations when the measured time for clot formation (clotting time) for a test sample containing a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a preparation of a thrombin- activable factor X is increased relative to the individual clotting time of a control sample containing only the factor VII or factor Vll-related polypeptide or the thrombin-activable factor X respectively, when tested in the same clotting assay.
  • the clotting time may be assayed by means of standard PT og aPTT assays, which are known to the general skilled person.
  • bleeding disorder reflects any defect, congenital, acquired or induced, of cellular or molecular origin that is manifested in bleeding episodes.
  • bleeding disorders include, but are not limited to, clotting factor deficiencies (e.g.
  • coagulation factors VIII, IX, Xl or VII defi- ciency of coagulation factors VIII, IX, Xl or VII
  • clotting factor inhibitors e.g., Glanzmann thombasthenia and Bernard-Soulier syndrome
  • thrombocytopenia e.g., von Willebrand's disease
  • coagulophathy e.g., a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to bleedings and/or transfusions (e.g., in multi transfused subjects having been subjected to surgery or trauma).
  • Bleeding refers to extravasation of blood from any component of the circulatory system.
  • the term "bleeding episodes” is meant to include unwanted, uncontrolled and often excessive bleeding in connection with surgery, trauma, or other forms of tissue damage, as well as unwanted bleedings in subjects having bleeding disorders. Bleeding episodes may occur in subjects having a basically normal coagulation system but experiencing a (temporary) coagulopathy, as well as in subjects having congenital or acquired coagulation or bleeding disorders.
  • the bleedings may be likened to bleedings caused by haemophilia because the haemostatic system, as in haemophilia, lacks or has abnormal essential clotting "compounds" (e.g., platelets or von Willebrand factor pro- tein).
  • the normal haemostatic mechanism may be overwhelmed by the demand of immediate haemostasis and they may develop excessive bleeding in spite of a basically (pre-trauma or pre-surgery) normal haemostatic mechanism.
  • Such subjects who further often are multi transfused, develop a (temporary) coagulopathy as a result of the bleeding and/or transfusions (i.e., a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to the bleeding and/or transfusions).
  • Bleedings may also occur in organs such as the brain, inner ear region and eyes; these are areas with limited possibilities for surgical haemostasis and thus problems with achieving satisfactory haemostasis. Similar problems may arise in the process of taking biopsies from various organs (liver, lung, tumour tissue, gastrointestinal tract) as well as in laparoscopic surgery and radical retropubic prostatectomy.
  • Such therapy may include heparin, other forms of proteoglycans, Warfarin or other forms of vitamin K-antagonists as well as aspirin and other platelet aggregation inhibitors, such as, e.g., antibodies or other inhibitors of GP llb/llla activity.
  • Bleeding episodes are also meant to include, without limitation, uncon- trolled and excessive bleeding in connection with surgery or trauma in subjects having acute haemarthroses (bleedings in joints), chronic haemophilic arthropathy, haematomas, (e.g., muscular, retroperitoneal, sublingual and retropharyngeal), bleedings in other tissue, haema- turia (bleeding from the renal tract), cerebral haemorrhage, surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings (e.g., UGI bleeds).
  • acute haemarthroses bleedings in joints
  • chronic haemophilic arthropathy haematomas, (e.g., muscular, retroperitoneal, sublingual and retropharyngeal)
  • haema- turia bleeding from the renal tract
  • cerebral haemorrhage e.g., hepatectomy
  • dental extraction e.g., UGI bleeds
  • the bleeding episodes may be associated with inhibitors against factor VIII; haemophilia A; haemophilia A with inhibitors; haemophilia B; deficiency of factor VII; deficiency of Factor Xl; thrombocytopenia; deficiency of von Willebrand factor (von Willebrand's disease); severe tissue damage; severe trauma; surgery; laparoscopic surgery; haemorrhagic gastritis; taking biopsies; anticoagulant therapy; upper gastroentestinal bleedings (UGI); or stem cell transplantation.
  • the bleeding episodes may be profuse uterine bleeding; occurring in organs with a limited possibility for mechanical haemostasis; occurring in the brain; occurring in the inner ear region; or occurring in the eyes.
  • the terms "bleeding episodes” and “bleedings” may, where appropriate, be used interchangeably.
  • treatment is meant to include both prevention of an ex- pected bleeding, such as, for example, in surgery, and regulation of an already occurring bleeding, such as, for example, in trauma, with the purpose of inhibiting or minimising the bleeding.
  • expected bleeding may be a bleeding expected to occur in a particular tissue or organ, or it may be an unspecified bleeding.
  • Prophylactic administration of a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a throm- bin-activable factor X is thus included in the term "treatment".
  • subject as used herein is intended to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the term “patient”.
  • the factor VII or factor Vll-related polypeptides and thrombin-activable factor Xs as defined in the present specification may be administered simultaneously or sequentially.
  • the factors may be supplied in single-dosage form wherein the single-dosage form contains both coagulation factors, or in the form of a kit-of-parts comprising a preparation of a factor VII or factor Vll-related polypeptide as a first unit dosage form and a preparation of a thrombin- activable factor X as a second unit dosage form.
  • the second unit dosage form may be in the form of a high-, medium- or low-activity thrombin-activable factor X product. High-activity products are preferred. Most preferred are recombinant high-activity products.
  • a preparation of a factor VII or factor Vll-related poly- peptide and a preparation of a thrombin-activable factor X is meant administration of the coagulation factor proteins in single-dosage form, or administration of a first coagulation factor protein followed by administration of a second coagulation factor protein with a time separation of no more than 15 minutes, 10, 5, 2 minutes. Either factor may be administered first.
  • sequential dosing administration of a first coagulation factor protein followed by administration of a second coagulation factor protein with a time separation of up to 2 hours, from 1 to 2 hours, up to 1 hour, from 30 minutes to 1 hour, up to 30 minutes, or from 15 to 30 minutes.
  • Either of the two unit dosage form, or coagulation factor proteins, may be administered first. Both products can be injected through the same intravenous access.
  • factor VII is defined as the amount of factor VII present in 1 ml of normal (pooled) plasma, corresponding to about 0.5 ⁇ g protein. After activation 50 units correspond to about 1 ⁇ g protein.
  • APTT or "aPTT” is meant the activated partial thromboplastin time (described by, e.g., Proctor RR, Rapaport Sl: The partial thromboplastin time with kaolin; a simple screening test for first-stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212, 1961 ).
  • factor Vll-responsive syndrome is meant a syndrome where exogenous factor VII, preferably factor Vila, administered to the subject in need thereof may prevent, cure or ameliorate any symptoms, conditions or diseases, expected or present, caused by the syndrome. Included are, without limitation, syndromes caused by a reduced level of clotting factors VIII, IX, Xl or VII, clotting factor inhibitors, defective platelet function (e.g., Glanzmann thombasthenia and Bernard-Soulier syndrome), thrombocytopenia, von Willebrand's disease, and coagulopathy such as that caused by a dilution of coagulation proteins, increased fibri- nolysis and lowered number of platelets due to bleedings and/or transfusions (e.g., in multi transfused subjects having been subjected to surgery or trauma).
  • clotting factors VIII, IX, Xl or VII clotting factor inhibitors
  • defective platelet function e.g., Glanzmann thombasthenia and Bernard-Soulier syndrome
  • Het-life refers to the time required for the plasma concentration of a factor VII or factor Vll-related polypeptide or a thrombin-activable factor X to decrease from a particular value to half of that value.
  • primary haemostasis is meant the initial generation of thrombin by FXa and
  • TF:factor Vila the subsequent activation of platelets and formation of the initial loose plug of activated, adhered platelets which has not yet been stabilized by fibrin and, finally, by cross- linked fibrin. If not stabilized by the fibrin formed during the second step of the haemostatic process (maintained haemostasis), the plug is easily dissolved by the fibrinolytic system.
  • full haemostasis is meant the formation of a stable and solid fibrin clot or plug at the site of injury which effectively stops the bleeding and which is not readily dissolved by the fibrinolytic system.
  • haemostasis will be used to represent full haemostasis as described above.
  • isolated refers to coagulation factors, e.g., thrombin- activable factor Xs that have been separated from the cell in which they were synthesized. Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like.
  • Separation of polypeptides from the medium in which they naturally occur may be achieved by any method known in the art, including, without limitation, affinity chromatography, such as, e.g., on an anti-Factor VII antibody column, respectively; hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction and the like.
  • affinity chromatography such as, e.g., on an anti-Factor VII antibody column, respectively
  • hydrophobic interaction chromatography e.g., ion-exchange chromatography
  • size exclusion chromatography e.g., electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction and the like.
  • electrophoretic procedures
  • TAFX thrombin-activable factor X in its zymogenic, unactivated form
  • Human purified Factor Vila suitable for use in the present invention is preferably made by DNA recombinant technology, e.g. as described by Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2412-2416, 1986, or as described in European Patent No. 200.421 (ZymoGenetics, Inc.).
  • Factor VII may also be produced by the methods described by Broze and Majerus, J.Biol.Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J.Clin. Invest. 71 : 1836-1841 , 1983. These methods yield Factor VII without detectable amounts of other blood coagulation factors.
  • An even further purified Factor VII preparation may be obtained by including an additional gel filtration as the final purification step, factor VII is then converted into activated factor Vila by known means, e.g. by several different plasma proteins, such as factor XIIa, IX a or Xa.
  • factor VII may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia fine Chemicals) or the like.
  • Factor VII -related polypeptides may produced by modification of wild-type Factor VII or by recombinant technology.
  • Factor VII -related polypeptides with altered amino acid sequence when compared to wild-type Factor VII may be produced by modifying the nucleic acid sequence encoding wild-type factor VII either by altering the amino acid codons or by removal of some of the amino acid codons in the nucleic acid encoding the natural factor VII by known means, e.g. by site-specific mutagenesis.
  • Thrombin-activable factor X suitable for use in the present invention is preferably made by DNA recombinant technology, e.g. as described in J.Biol Chem 280 No50 p41352 (2005) and in WO 03/035861 , where the activation peptide sequence is altered by site-directed mutagenesis.
  • substitutions can be made outside the regions critical to the function of the factor Vila or thrombin-activable factor X-molecule and still result in an active polypeptide.
  • Amino acid residues essential to the activity of the Factor VII or factor Vll-related polypeptide or thrombin-activable factor X, and therefore pref- erably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085).
  • Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et a/., 1992, FEBS Letters 309: 59-64).
  • the introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure that utilizes a super coiled, double stranded DNA vector with an insert of interest and two synthetic primers con- taining the desired mutation.
  • the oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated.
  • Dpn ⁇ is specific for methylated and hemi-methylated DNA to digest the parental DNA template and to select for muta- tion-containing synthesized DNA.
  • Other procedures known in the art for creating, identifying and isolating variants may also be used, such as, for example, gene shuffling or phage display techniques.
  • Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove nonadherent cells; and the like.
  • Factor VII or factor Vll-related polypeptides may be further purified. Purification may be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, e.g., on an anti-Factor VII antibody column (see, e.g., Waka- bayashi et al., J. Biol. Chem. 261 :11097, 1986; and Thim et al., Biochem.
  • affinity chromatography such as, e.g., on an anti-Factor VII antibody column (see, e.g., Waka- bayashi et al., J. Biol. Chem. 261 :11097, 1986; and Thim et al., Biochem.
  • the preparation preferably contains less than about 10% by weight, more preferably less than about 5% and most preferably less than about 1 %, of non-Factor VII or factor Vll-related polypeptides derived from the host cell.
  • Factor VII or factor Vll-related polypeptides may be activated by proteolytic cleav- age, using Factor XIIa or other proteases having trypsin-like specificity, such as, e.g., factor IXa, kallikrein, Factor Xa, and thrombin.
  • Factor XIIa or other proteases having trypsin-like specificity, such as, e.g., factor IXa, kallikrein, Factor Xa, and thrombin.
  • Factor VII or factor Vll-related polypeptides may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia) or the like. The resulting activated Factor VII or factor Vll-related polypeptide may then be formulated and administered as described below.
  • Thrombin-activable factor X for use within the present invention may prepared by DNA recombinant technology as described in J.Biol Chem 280 No50 p41352 (2005) and in WO 03/035861 As will be appreciated by those skilled in the art, it is preferred to use thrombin- activable factor X polypeptides and factor VII polypeptides syngeneic with the subject in order to reduce the risk of inducing an immune response.
  • thrombin-activable factor X Preparation and characterization of non-human thrombin-activable factor X has been disclosed by, e.g., Fujikawa et al., Biochemistry 1973, 12:4938 (bovine FIX)
  • the present invention also encompasses the use of such thrombin-activable factor X polypeptides and factor VII polypeptides within veterinary procedures.
  • the preparations of the present invention may be used to treat bleeding disorders, including, without limitation, those caused by clotting factor deficiencies (e.g., haemophilia A or B).
  • clotting factor deficiencies e.g., haemophilia A or B.
  • the preparations of the present invention may be used to treat any factor VII responsive syndrome, such as, e.g., bleeding disorders, including, without limitation, syndromes caused by a reduced level of clotting factors VIII, IX, Xl or VII, clotting factor inhibitors, defective platelet function (e.g., Glanzmann thombasthenia and Bernard-Soulier syn- drome), thrombocytopenia, von Willebrand's disease, and coagulophathy such as that caused by a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to bleedings and/or transfusions (e.g., in multi transfused subjects having been subjected to surgery or trauma).
  • factor VII responsive syndrome such as, e.g., bleeding disorders, including, without limitation, syndromes caused by a reduced level of clotting factors VIII, IX, Xl or VII, clotting factor inhibitors, defective platelet function (e.g., Glanzmann thomba
  • compositions comprising a preparation of a factor VII or factor VII- related polypeptide and a preparation of a thrombin-activable factor X according to the present invention are primarily intended for parenteral administration for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly; intravenously being most preferred. They may also be administered by continuous or pulsatile infusion.
  • compositions or formulations according to the invention comprise a preparation of a preparation of a factor VII or factor Vll-related polypeptide, or a preparation of a preparation of a thrombin-activable factor X, or a preparation of a preparation of a factor VII or factor Vll-related polypeptide in combination with a preparation of a preparation of a thrombin-activable factor X in combination with, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier or diluent.
  • aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like.
  • the preparations of the invention can also be formulated using non-aqueous carriers, such as, e.g., in the form of a gel or as liposome preparations for delivery or targeting to the sites of injury.
  • non-aqueous carriers such as, e.g., in the form of a gel or as liposome preparations for delivery or targeting to the sites of injury.
  • Liposome preparations are generally described in, e.g., U.S. Patents Nos. 4,837,028, 4,501 ,728, and 4,975,282.
  • the compositions may be sterilised by conventional, well-known sterilisation techniques.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilised, the lyophilised preparation being combined with a sterile aqueous solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances or adjuvants, including, without limitation, pH adjusting and buffering agents and/or tonicity adjusting agents, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • Formulations may further include one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, controlled release, etc.
  • a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution and 10 mg of the preparation.
  • compositions containing the preparations of the present invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject already suffering from a disease, as described above, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disease and its complications.
  • An amount adequate to accomplish this is defined as "therapeutically effective amount”.
  • Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix.
  • Local delivery of the preparations of the present invention may be carried out, e.g., by means of a spray, perfusion, double balloon catheters, stent, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods.
  • the pharmaceutical compositions should provide a quantity of the preparation sufficient to effectively treat the condition.
  • the concentration of factor VII or factor Vl l-related polypeptide, thrombin-activable factor X, or factor VII or factor Vl l-related polypeptide in combination with thrombin-activable factor X in these formulations can vary widely, i.e., from less than about 0.5% by weight, usually at or at least about 1 % by weight to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Administration by injection or infusion, in particular injection, is preferred.
  • the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X are prepared in a form suitable for intravenous administration, such as a preparation that is either a dissolved lyophilized powder or a liquid formulation containing both the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X in one dosage form, or a dissolved lyophilized powder or a liquid formulation containing the factor VII or factor Vll- related polypeptide in one dosage form and dissolved lyophilized powder or a liquid formulation containing the thrombin-activable factor X in another dosage form.
  • the amount of factor VII or factor Vll-related polypeptide and the amount of thrombin-activable factor X together comprise an aggregate effective amount for treating the bleeding episode.
  • the materials of the present invention may generally be employed in serious disease or injury states, that is, life threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and general lack of immunogenicity of factor Vila and thrombin-activable factor X in humans, it is possible and may be felt desirable by the treating physician to administer a substantial excess of these compositions.
  • compositions containing a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a thrombin-activable factor X are administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulative capability. Such an amount is defined to be a "prophylactically effective dose.” It is to be understood that the amount of factor VII or factor Vll-related polypeptide and the amount of thrombin-activable factor X together comprise an aggregate effective amount for preventing a bleeding episode. Single or multiple administrations of the compositions can be carried out with dose levels and patterns being selected by the treating physician. The compositions may be administered one or more times per day or week. An effective amount of such a pharmaceutical composition is the amount that provides a clinically significant effect against bleeding episodes. Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health of the subject, and other factors evident to those skilled in the art.
  • composition of the invention is generally administered in a single dose before the expected bleeding or at the start of the bleeding. It may however also be given repeatedly (in multiple doses) for example with intervals of 2-4-6-12 hour, depending on the dose given and the condition of the subject.
  • the factor VII or factor Vll- related polypeptide and the thrombin-activable factor X will typically be administered within about 24 hours prior to performing the intervention, and for as much as 7 days or more thereafter.
  • Administration as a coagulant can be by a variety of routes as described herein.
  • the composition may be in the form of a single preparation (single-dosage form) comprising both a preparation of a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a preparation of a thrombin-activable factor X in suitable concentrations.
  • the composition may also be in the form of a kit-of-parts consisting of a first unit dosage form comprising a preparation of a preparation of a factor VII or factor Vll-related polypeptide and a second unit dosage form comprising a preparation of a preparation of a thrombin-activable factor X.
  • the factor VII or factor Vll-related polypeptide and the thrombin-activable factor X should be administered one after the other, for example within about 15 minutes of each other, for example within 10 minutes of each other or, within 5 minutes or, within 2 minutes of each other. Either of the two unit dosage forms can be administered first.
  • the kit includes at least two separate pharmaceutical compositions.
  • the kit includes container means for containing the separate compositions such as a divided bottle or a divided foil packet.
  • the kit includes directions for the administration of the separate components.
  • the kit form is particularly advantageous when the separate components are preferably administered in different dosage forms, are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.
  • the amount of factor VII or factor Vl I -related polypeptide and the amount of thrombin- activable factor X administered according to the present invention may vary from a ratio of between about 1 :100 to about 100:1 (w/w).
  • the ratio of factor VII to thrombin-activable factor X may thus be, e.g., about 1 :100, or 1 :90, or 1 :80, or 1 :70 or 1 :60, or 1 :50, or 1 :40, or 1 :30, or 1 :20, or 1 :10, or 1 :5, or 1 :2, or 1 :1 , or 2:1 , or 5:1 , or 10:1 , or 20:1 , or 30.1 , or 40:1 , or 50:1 , or 60: 1 , or 70: 1 , or 80: 1 , or 90: 1 , or 100: 1 ; or between about 1 :90 to about 1 : 1 , or between about 1 :80
  • the dose of the Factor VII or factor Vll-related polypeptide ranges from what corresponds to about 0.05 mg to about 500 mg/day of wild-type Factor VII, e.g., from about 1 mg to about 200 mg/day, or, e.g., from about 5 mg to about 175 mg/day for a 70-kg subject as loading and maintenance doses, depending on the weight of the subject, the condition and the severity of the condition.
  • the dose of the thrombin-activable factor X ranges from what corresponds to about 0.01 mg to about 500 mg/day of wild-type factor X, e.g., from about 1 mg to about 200 mg/day, or, e.g., from about 5 mg to about 175 mg/day for a 70-kg subject as loading and maintenance doses, depending on the weight of the subject, the condition and the severity of the condition.
  • a suitable assay for testing for factor Vila activity and thereby selecting suitable factor Vila variants can be performed as a simple preliminary in vitro test: In Vitro Hydrolysis Assay Native (wild-type) factor Vila and factor Vila variant (both hereafter referred to as "factor Vila") may be assayed for specific activities. They may also be assayed in parallel to directly compare their specific activities. The assay is carried out in a microtiter plate (Max- iSorp, Nunc, Denmark).
  • the chromogenic substrate D-lle-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mM, is added to factor Vila (final concentration 100 nM) in 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI 2 and 1 mg/ml bovine serum albumin.
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA).
  • Ratio (A405 nm factor Vila variant)/(A405 nm factor Vila wild-type).
  • factor Vila variants with an activity comparable to or higher than native factor Vila may be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is around, versus above 1.0.
  • the activity of factor Vila or factor Vila variants may also be measured using a physiological substrate such as factor X, suitably at a concentration of 100-1000 nM, where the factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. S-2765).
  • the activity assay may be run at physiological temperature.
  • Factor Vila Native (wild-type) Factor Vila and Factor Vila variant (both hereafter referred to as "Factor Vila") are assayed in parallel to directly compare their specific activities.
  • the assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • Factor X cleavage is then stopped by the addition of 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1 mg/ml bovine serum albumin.
  • the amount of Factor Xa generated is measured by addition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chro- mogenix, Sweden), final concentration 0.5 mM.
  • the absorbance at 405 nm is measured con- tinuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA).
  • the absorbance developed during 10 minutes, after subtraction of the absorbance in a blank well containing no FVIIa, is used to calculate the ratio between the proteolytic activities of variant and wild- type Factor Vila:
  • Ratio (A405 nm Factor Vila variant)/(A405 nm Factor Vila wild-type). Based thereon, factor Vila variants with an activity comparable to or higher than native factor Vila may be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is around, versus above 1.0.
  • Thrombin generation assay The ability of factor VII or factor Vl l-related polypeptides (e.g., variants) or thrombin- activable factor Xs (e.g., variants) or a combination of a factor VII and a thrombin-activable factor X to generate thrombin can be measured in an assay comprising all relevant coagulation factors and inhibitors at physiological concentrations and activated platelets (as de- scribed on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 which is hereby incorporated as reference).
  • a pharmaceutical composition comprising a preparation of a factor VII or a factor Vll-related polypeptide, and a preparation of a thrombin-activable factor X.
  • composition according to clause 1 wherein said factor VII or factor Vll-related polypeptide is a factor Vll-related polypeptide.
  • said factor Vll-related polypeptide is a factor VII amino acid sequence variant.
  • composition according to clauses 2 or clause 3, wherein the ratio between the activity of said factor Vll-related polypeptide and the activity of native human factor Vila (wild- type FVIIa) is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay" as described in the present description.
  • composition according to clause 1 wherein said factor VII or factor Vll-related polypeptide is a factor VII polypeptide.
  • composition according to clause 8 wherein said factor VII polypeptide is recombinant human factor Vila. 10. A composition according to any one of clauses 1 to 9, wherein said factor VII or factor Vll-related polypeptide and said thrombin-activable factor X are present in a ratio by mass of between about 100.1 and about 1 :100 factor Vll:thrombin-activable factor X
  • a kit of parts containing a treatment for bleeding episodes comprising a) An effective amount of a preparation of a factor VII or factor Vll-related polypeptide and a pharmaceutically acceptable carrier in a first unit dosage form; b) An effective amount of a preparation of a thrombin-activable factor X and a pharmaceutically acceptable carrier in a second unit dosage form; and c) Container means for containing said first and second dosage forms.
  • kit according to clause 11 wherein said factor VII or factor Vll-related polypeptide is a factor VII polypeptide. 16. A kit according to clause 15, wherein said factor VII polypeptide is human factor
  • kit according to clause 16 wherein said factor VII polypeptide is recombinant human factor VII.
  • a method for treating bleeding episodes in a subject comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vl l-related polypeptide and a second amount of a preparation of a thrombin-activable factor X, wherein the first and second amount together are effective to treat bleedings.
  • 36. A method for reducing clotting time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor VII- related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to reduce clotting time.
  • a method to enhance haemostasis in a subject comprising admin- istering to a subject in need thereof a first amount of a preparation of a factor VII or factor VII- related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to enhance haemostasis.
  • a method for reducing the number of administrations of coagulation factor protein needed to arrest bleeding and maintain haemostasis in a subject comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to arrest bleeding and maintain haemostasis.
  • a method for reducing the amount of administered coagulation factor protein needed to arrest bleeding and maintain haemostasis in a subject comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to arrest bleeding and maintain haemostasis.
  • a method for prolonging the clot lysis time in a subject comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to prolong the clot lysis time.
  • 41. A method for increasing clot strength in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to increase clot strength.
  • a method for enhancing fibrin clot formation in a subject comprising administering to a subject in need thereof a first amount of a preparation of a factor VII or factor Vll-related polypeptide and a second amount of a preparation of a thrombin-activable factor X wherein the first and second amount together are effective to enhance fibrin clot formation.
  • kits containing a treatment for bleeding episodes comprising a) an effective amount of a factor VII or factor Vll-related polypeptide and an effective amount of a thrombin-activable factor X and a pharmaceutically acceptable carrier in a one-unit dosage form; and b) container means for containing said one-unit dosage form.
  • a composition according to clause 1 wherein said factor VII or factor VII related polypeptide is an isolated factor VII or factor Vll-related polypeptide.
  • a composition according to clause 1 wherein said thrombin-activable factor X is an isolated thrombin-activable factor X.

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Abstract

L'invention concerne une préparation pharmaceutique comprenant un facteur VII ou un polypeptide lié au facteur VII, et un facteur X activable par la thrombine. L'invention concerne également l'utilisation d'un facteur VII ou d'un polypeptide lié au facteur VII et d'un facteur X activable par la thrombine pour la fabrication d'un médicament à usage pharmaceutique, ainsi que les techniques de prévention et de traitement des épisodes hémorragiques.
PCT/EP2007/058797 2006-08-24 2007-08-24 Combinaison d'un fvii et d'un facteur x activable par la thrombine WO2008023052A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1240901A1 (fr) * 1999-12-24 2002-09-18 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Compositions medicinales de traitement et de prevention de maladies basees sur une coagulation sanguine anormale
WO2005058283A2 (fr) * 2003-12-19 2005-06-30 Novo Nordisk Health Care Ag Compositions stabilisees de polypeptides de facteur vii
WO2005107795A1 (fr) * 2004-05-11 2005-11-17 Novo Nordisk Health Care Ag Utilisation du facteur viia dans le traitement de traumas par brulures

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1240901A1 (fr) * 1999-12-24 2002-09-18 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Compositions medicinales de traitement et de prevention de maladies basees sur une coagulation sanguine anormale
WO2005058283A2 (fr) * 2003-12-19 2005-06-30 Novo Nordisk Health Care Ag Compositions stabilisees de polypeptides de facteur vii
WO2005107795A1 (fr) * 2004-05-11 2005-11-17 Novo Nordisk Health Care Ag Utilisation du facteur viia dans le traitement de traumas par brulures

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