EP1411973A1 - Pharmaceutical composition comprising factor vii polypeptides and factor xi polypeptides - Google Patents

Pharmaceutical composition comprising factor vii polypeptides and factor xi polypeptides

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Publication number
EP1411973A1
EP1411973A1 EP02750844A EP02750844A EP1411973A1 EP 1411973 A1 EP1411973 A1 EP 1411973A1 EP 02750844 A EP02750844 A EP 02750844A EP 02750844 A EP02750844 A EP 02750844A EP 1411973 A1 EP1411973 A1 EP 1411973A1
Authority
EP
European Patent Office
Prior art keywords
factor
vll
related polypeptide
factor vll
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02750844A
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German (de)
English (en)
French (fr)
Inventor
Rasmus Rojkjaer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk Health Care AG
Original Assignee
Novo Nordisk Health Care AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk Health Care AG filed Critical Novo Nordisk Health Care AG
Publication of EP1411973A1 publication Critical patent/EP1411973A1/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • 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

Definitions

  • the present invention relates to a pharmaceutical composition comprising factor VII or a factor Vll-related polypeptide and factor XI or a factor Xl-related polypeptide.
  • the invention also relates to the use of a combination of a factor VII or a factor Vll-related polypeptide and a factor XI or a factor Xl-related polypeptide for the manufacture of a medicament for treatment of subjects suffering from bleeding episodes, or prevention hereof.
  • the invention also relates to a method for treatment of bleeding episodes in subjects and to a method for enhancing clot formation in a subject.
  • the present invention also relates to kits comprising these compounds.
  • Haemostasis is initiated by the formation of a complex between tissue factor (TF) being exposed to the circulating blood following an injury to the vessel wall, and FVIIa which is present in the circulation in an amount corresponding to about 1 % of the total FVII protein mass.
  • This complex is anchored to the TF-bearing cell and activates FX into FXa and FIX into FIXa on the cell surface.
  • FXa activates prothrombin to thrombin, which activates FVIII, FV, FXI and FXIII.
  • the limited amount of thrombin formed in this initial step of haemostasis also activates the platelets. Following the action of thrombin on the platelets these change shape and expose charged phospholipids on their surface.
  • This activated platelet surface forms the template for the further FX activation and the full thrombin generation.
  • the further FX activation on the activated platelet surface occurs via a FIXa-FVIIIa complex formed on the surface of the activated platelet, and FXa then converts prothrombin into thrombin while still on the surface.
  • Thrombin then converts fibrinogen into fibrin which is insoluble and which stabilizes the initial platelet plug.
  • This process is compartmentalized, i.e., localised to the site of TF expression or exposure, thereby minimizing the risk of a systemic activation of the coagulation system.
  • the insoluble fibrin forming the plug is furthermore stabilised by FXIII-catalysed cross-linking of the fibrin fibres.
  • FVIIa 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 a factor VII deficiency or subjects having a normal coagulation system but experiencing excessive bleeding can be treated successfully with FVIIa.
  • NovoSeven® Novo Nordisk A/S,Denmark
  • Novoseven® is indicated for treatment of bleeding episodes in haemophilia A and B patients.
  • Novoseven® is the only recombinant FVIIa available on the market for effective and reliable treatment of bleeding episodes.
  • FXI is a component of the intrinsic pathway of coagulation.
  • a deficiency of FXI is associated with a mild to moderate bleeding disorder especially from tissues with a high local fibri- nolytic activity. In contrast, it is believed that high levels of FXI are a risk factor for venous thrombosis.
  • FXI is the zymogen of a trypsin-like serine protease that is activated by FXIIa, thrombin and FXIa.
  • Activated FXI participates in the activation of FIX, which in turn (in combina- tion with FVIII) further activates FX and thus gives rise to generation of thrombin.
  • Japanese patent application No. 59-116213A concerns an aerosol composition for use as a tissue glue containing a blood coagulant as an active component.
  • the blood coagulant may be selected from blood coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, and XIII, prekallikrein, high polymer kininogen and thrombin.
  • a combination of F XIII and thrombin is preferred.
  • European Patent No. 225.160 (Novo Nordisk) concerns compositions of FVIIa and methods for the treatment of bleeding disorders not caused by clotting factor defects or clotting factor inhibitors.
  • European Patent No. 82.182 (Baxter Travenol Lab.) concerns a composition of factor Vila for use in counteracting deficiencies of blood clotting factors or the effects of inhibitors to blood clotting factors in a subject.
  • US Patent No. 5,252,217 concerns a process for preparing a human factor XI concentrate intended for therapeutic use.
  • bleeding episodes including subjects where the bleeding episodes are due to surgery, trauma, or other forms of tissue damage; induced coagulophathy, including coagulopathy in multi-transfused subjects; congenital or acquired coagulation or bleeding disorders, including diminished liver function ("liver disease”); defective platelet function or decreased platelet number; lacking or abnormal essential clotting "compounds” (e.g., platelets or von Willebrand factor protein); in- creased fibrinolysis; anticoagulant therapy or thrombolytic therapy; or stem cell transplantation.
  • induced coagulophathy including coagulopathy in multi-transfused subjects
  • congenital or acquired coagulation or bleeding disorders including diminished liver function ("liver disease”); defective platelet function or decreased platelet number; lacking or abnormal essential clotting "compounds” (e.g., platelets or von Willebrand factor protein); in- creased fibrinolysis; anticoagulant therapy or thrombolytic therapy; or stem cell transplantation.
  • 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 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 provides a pharmaceutical composition comprising factor VII or a factor Vll-related polypeptide, and factor XI or a factor Xl-related polypeptide.
  • the invention provides 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 factor XI or factor Xl-related polypeptide and a pharmaceutically acceptable carrier in a second unit dosage form; and c) Container means for containing said first and second dosage forms.
  • the kit further contains an effective amount of a
  • the invention provides the use of a factor VII or factor Vll-related polypeptide in combination with a factor XI or a factor Xl-related polypeptide for the manufacture of a medicament for treating bleeding episodes in a subject.
  • the invention provides the use of a composition as described in any one of claims 1 to18, for the manufacture of a medicament for treating bleeding episodes in a subject.
  • the medicaments are for reducing clotting time, prolonging the clot lysis time, and increasing clot strength.
  • the medicament is formulated for intravenous administration, preferably injection or infusion, in particular injection.
  • the medicament is formulated in single-unit dosage form; in another it is formulated 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 factor XI or factor Xl-related polypeptide.
  • the medicaments are for treatment of subjects experiencing bleeding episodes due to surgery, trauma, or other forms of tissue damage; coagulophathy, in- eluding coagulopathy in multi-transfused subjects; congenital or acquired coagulation or bleeding disorders, including decreased liver function ("liver disease”); defective platelet function or decreased platelet number; lacking or abnormal essential clotting "compounds” (e.g., platelets or von Willebrand factor protein); increased fibrinolysis; anticoagulant therapy or thrombolytic therapy; stem cell transplantation.
  • the bleedings occur in organs such as the brain, inner ear region, eyes, liver, lung, tumour tissue, gastrointestinal tract; in another series of embodiments, it is diffuse bleeding, such as in haemorrhagic gastritis and profuse uterine bleeding.
  • the bleeding episodes are 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, haematuria (bleeding from the renal tract), cerebral haemorrhage, surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings (e.g., UGI bleeds).
  • the medicament is for treating bleeding episodes due to trauma, or surgery, or lowered count or activity of platelets, in a subject.
  • the invention provides 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 factor XI or factor Xl-related polypeptide, wherein the first and second amount to- gether are effective to treat bleedings.
  • the invention provides 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 factor XI or factor Xl-related polypeptide wherein the first and second amount together are effective to reduce clotting time.
  • the invention provides 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 factor XI or factor Xl-related polypeptide wherein the first and second amount together are effective to enhance haemostasis.
  • the invention provides a 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 factor XI or factor Xl-related polypeptide wherein the first and second amount together are effective to prolong the clot lysis time.
  • the invention provides 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 factor XI or factor Xl-related polypeptide wherein the first and second amount together are effective to increase clot strength.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are administered in single-unit dosage form.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide 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 factor XI or factor Xl-related polypeptide.
  • the first-unit dosage form and the second-unit dosage form are administered with a time separation of no more than 15 minutes.
  • the invention provides a kit containing a treatment for bleeding episodes comprising d) An effective amount of a factor VII or factor Vll-related polypeptide and an effective amount of a factor XI or factor Xl-related polypeptide and a pharmaceutically acceptable carrier in a one-unit dosage form; and e) Container means for containing said one-unit dosage form.
  • the factor VII or factor Vll-related polypeptide is a factor Vll-related polypeptide. In one series of embodiments of the invention the factor Vll-related polypeptide is a factor VII amino acid sequence variant. In one embodiment 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 factor VII.
  • said factor VII is human factor VII.
  • the factor VII is bovine, porcine, canine, equine, murine or salmon factor VII.
  • the factor VII is recombinantly made.
  • the factor VII is derived from plasma.
  • the factor VII is recombinant human factor VII.
  • the factor VII or factor Vll-related polypeptide is in its activated form.
  • the factor VII is recombinant human factor Vila.
  • the factor XI or factor Xl-related polypeptide is a factor Xl-related polypeptide.
  • the factor Xl-related polypeptide is a factor XI amino acid sequence variant.
  • the ratio between the activity of said factor Xl-related polypeptide and the activity of native human plasma factor XI (wild-type FXI) is at least about 1.25 when tested in the "FXI chromogenic assay" as described in the present description.
  • the factor XI or factor Xl-related polypeptide is a factor XI polypeptide. In one em- bodiment the factor XI is human factor XI.
  • the factor XI is bovine, porcine, canine, equine, murine or salmon factor XI. In a preferred embodiment the factor XI is recombinantly made. In another embodiment the factor XI is derived from plasma. In another embodiment the factor XI is platelet-derived factor XI. In a preferred embodiment the factor XI is recombinant human plasma factor XI. In one series of embodiments of the invention the factor XI or factor Xl-related polypeptide is in its activated form. In one embodiment the factor Xl-related polypeptide is a fragment of factor XI.
  • the factor Xl-related polypeptide is a hybrid factor XI polypeptide, e.g., a porcine/human hybrid.
  • the factor XI is human plasma activated factor XI (FXIa).
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor-XI related polypeptide are present in a ratio by mass of between about 100:1 and about 1 :100 (w/w factor Vll:factor XI).
  • 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. 4,784,950), In another embodiment, the factor VII variants have no more than 15 amino acids replaced, deleted or inserted; in other embodiments, the factor VII variants have no more than 10 amino acids, such as 8, 6, 5, or 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, L305l-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 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 Vll-related polypeptides to tissue factor are not impaired and the factor Vll-related polypeptides 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 factor XI or factor Xl-related polypeptide are recombinant human factor Vila and recombinant human plasma factor XI or recombinant human factor Vila and recombinant human plasma factor Xla.
  • 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 mammalian blood is human blood.
  • the mammalian blood is normal human blood; in one embodiment, the blood is blood from a subject having an impaired thrombin generation.
  • the blood is blood from a subject having a deficiency of one or more coagulation factors; in another 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 factor Xl-def icient human blood. In one series of embodiments, the blood is plasma.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide prolong the in vitro clot lysis time in normal human plasma.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide increase the maximal clot strength clot lysis time in normal human plasma in vitro.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide shorten the clotting time in normal human plasma in vitro.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are the sole haemostatic agents contained in the composition. In another embodiment, the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are the sole active haemostatic agents contained in the composition. In another embodiment, the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are the sole coagulation factors administered to the subject. In one embodiment of the invention, the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are the sole active agents administered to the patient. In one embodiment, the composition is substantially free of prothrombin; in another embodiment, the composition is substantially free of FX; in another embodiment, the composition is substantially free of FXa.
  • the pharmaceutical composition is formulated for intravenous administration, preferably injection or infusion, in particular injection.
  • the composition contains at least one pharmaceutical acceptable excipients or carrier.
  • the composition is in single-unit dosage form wherein the single-unit dosage form contains both coagulation factors, ln one embodiment of the invention, the composition is 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 factor XI or factor Xl-related polypeptide as a second-unit dosage form, and comprising container means for containing said first and second unit dosage forms. In one embodiment 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 factor XI or factor Xl-related polypeptide are administered in single-dosage form. In one embodiment of the invention, the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide 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 factor XI or factor Xl-related polypeptide.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are administered simultaneously. In another embodiment, the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl- related polypeptide are administered sequentially. In one embodiment, the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are administered with a time separation of no more than 15 minutes, preferably 10, more preferred 5, more preferred 2 minutes.
  • the factor VII or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide are administered with a time separation of up to 2 hours, preferably from 1 to 2 hours, more preferred up to 1 hour, more preferred from 30 minutes to 1 hour, more preferred up to 30 minutes, more preferred from 15 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).
  • the effective amount of a preparation of a factor XI or factor Xl-related polypeptide is from about 0.01 mg/day to about 500 mg/day (70-kg subject).
  • factor VII or factor Vll-related polypeptide and factor XI or factor Xl-related polypeptide are present in a ratio by mass of between about 100:1 and about 1 :100 (w/w factor Vlhfactor XI)
  • the pharmaceutical composition is in single-dosage form and consists essentially of a preparation of a factor VII or factor Vll-related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide, 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 another 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 factor VIII.
  • the invention concerns a method to enhance haemostasis in a subject suffering from a factor VII responsive syndrome compared to when the subject is treated with factor VII 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 factor XI or factor Xl-related polypeptide, 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 prepara- tion of a factor XI or factor Xl-related polypeptide, 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 factor VII responsive syndrome compared to when the subject is treated with factor VII 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 factor XI or factor Xl-related polypeptide, wherein the first and second amounts together are effective to enhance formation of thrombin.
  • the invention concerns a method for reducing the number of administrations of coagulation factor protein needed to accomplish haemostasis in a subject suffering from a factor VII responsive syndrome compared to the number of administrations needed when factor VII 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 factor XI or factor Xl-related polypeptide, wherein the first and second amounts together are effective to reduce the number of administrations of coagulation factor protein.
  • the invention concerns a method of treating bleedings in a subject suffering from a factor VII 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 factor XI or factor Xl-related polypeptide, 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 pharmaceutical composition is formulated for intravenous administration.
  • the composition further comprises an inhibitor of the fibrino- lytic system, including, without limitation, aprotinin, ⁇ -aminocaproic acid or tranexamic acid.
  • the composition further contains a TFPI inhibitor and/or FVIII.
  • the composition further contains a factor VIII.
  • the factor VIM is an activated factor VIII (factor Villa).
  • the factor VIII is a recombinant factor Villa.
  • the factor VIII is recombinant human factor Villa.
  • the invention relates to the use of a factor Vila in combination with a factor XI for the manufacture of a medicament for enhancing fibrin clot formation in mammalian plasma.
  • the invention in another aspect, relates to a method of enhancing fibrin clot formation in a subject, which method comprises 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 factor XI or factor Xl-related polypeptide, wherein the first and second amounts together are effective in treating bleedings.
  • the pharmaceutical composition (when in single-preparation form) consists essentially of a factor Vila and a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor.
  • the pharmaceutical composition (when in single-preparation form) consists essentially of a factor Vila and a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor, and a TFPI-inhibitor.
  • the pharmaceutical composition (when in single-preparation form) consists essentially of a factor Vila and a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor, and a factor VIII, and, optionally, a TFPI-inhibitor.
  • the pharmaceutical composition (when in form of a kit) consists of a first unit dosage form consisting essentially of a factor Vila and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor; and a second unit dosage form consisting essentially of a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor.
  • the pharmaceutical composition (when in form of a kit) consists of a first unit dosage form consisting essentially of a factor Vila and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor; and a second unit dosage form consisting essentially of a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor; wherein either the first unit dosage form or the second unit dosage form or both dosage forms further contain a factor VIII and/or a TFPI-inhibitor.
  • FIG. 2 In the presence of 10 nM FVIIa, addition of FXI resulted in a further prolongation of the clot lysis time. The effect was dose-dependent and optimal at 30 nM FXI.
  • FIG. 3 Thromboelastography (roTEG) measurements were utilized to analyze the effect of FVIIa and FXI on the Maximal Clot Firmness (MCF), as well as the clots resistance to t-PA mediated lysis.
  • Figure 4 These results demonstrate that FVIIa and FXI when added to plasma in a synergistic fashion shorten the clotting time in NHP.
  • 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 VIM, 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.
  • 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 neuro- logical 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.
  • major objectives in treatment of bleedings are to obtain haemostasis in a minimum of time, thus keeping the blood loss at a minimum.
  • the present invention thus provides beneficial compositions, uses and methods of treatment for treatment of bleeding episodes in subjects in need of such treatment.
  • the compo- sitions, 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, faster arrest of bleedings, reduction in the number of administrations needed to stop bleeding and maintain haemostasis. /
  • a preparation of a factor VII or factor Vll-related polypeptide e.g., factor Vila
  • a preparation of a factor XI or factor Xl-related polypeptide provides a shortened clotting time, a firmer clot and an increased resistance to fibrinolysis com- pared to the clotting time, clot firmness and resistance when either factor Vila or factor XI 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 factor XI or factor Xl-related polypeptide also provides for a reduced time to obtain bleeding arrest and a reduced number of administra- tions to maintain haemostasis compared to the situation when either factor Vila or factor XI is administered alone.
  • the present invention provides a beneficial effect of simultaneous or sequential dosing of a preparation of a factor XI or factor X-related polypeptide and a preparation of a factor VII or factor Vll-related polypeptide.
  • the pharmaceutical composition according to the present invention may be in the form of a single composition or it may be in the form of a multi-component kit (kit-of-parts).
  • kit-of-parts The 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 factor XI or factor Xl-related polypeptide is an advantageous product ensuring short clotting times, rapid formation of haemostatic plugs, and formation of stable haemostatic plugs. It has been found by the present inventor that a combination of a factor VII or factor Vll- related polypeptide and a factor XI or a factor Xl-related polypeptide is an advantageous product ensuring the formation of solid, stable and quickly formed haemostatic plugs. The present inventors have shown that a combination of a factor Vila and a factor XI can reduce the clotting time of normal human plasma more effectively than either factor Vila or factor XI alone.
  • a combination of a factor Vila and a factor XI can increase the firmness of the clot more effectively than either factor Vila or factor XI alone.
  • a factor Vila at a concentration where no further increase in clot firmness was observed
  • a factor XI also at a concentration where no further increase in clot firmness was observed
  • combination of a factor Vila and a factor XI can prolong the in vitro clot lysis time in normal human plasma more effectively than either factor Vila or factor XI alone.
  • combination of a factor Vila and a factor XI can prolong the half-clot lysis time in normal human plasma more effectively than either factor Vila or factor XI alone. It has also been shown that combination of a factor Vila and a factor XI can protect the clot from fibrinolysis, in particular tPA-mediated fibrinolysis, in normal human plasma more effectively than either factor Vila or factor XI alone. Thus, by enhancing coagulation a more effective treatment of bleeding in subjects can be obtained.
  • the full thrombin generation is necessary for a solid, stabile haemostatic plug to be formed, and thereby for the maintenance of haemostasis.
  • the fibrin structure of such a plug is dependent on both the amount of thrombin formed and the rate of the initial thrombin generation.
  • a porous fibrin plug which is highly permeable, is being formed.
  • the fibrinolytic enzymes normally present on the fibrin surface easily dissolve such a fibrin plug.
  • the formation of a stable fibrin plug is also dependent on the presence of factor Xllla, which is being activated by thrombin and therefore also dependent on the full thrombin generation.
  • TAFI thrombin activatable fibrinolytic inhibitor
  • Subjects with thrombocytopenia 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 conse- quence, 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.
  • their lowered fibrinogen level interfere negatively with the activation of factor XIII.
  • These subjects therefore, have a defective, or less efficient, haemostasis leading to the formation of fibrin plugs which are easily and prematurely dissolved by proteolytic enzymes, such enzymes in addition being extensively released in situations characterized by extensive trauma and organ damage.
  • compositions according to the invention are administered.
  • This composition is especially beneficial in subjects with a lowered number of platelets and in subjects with lowered plasma levels of fibrinogen and/or other coagulation proteins.
  • any factor VII polypeptide may be used that is effective in preventing or treating bleeding.
  • 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%, preferably at least about 30%, more preferably at least about 50%, and most preferably at least about 70%, of the specific biological activity of human factor Vila.
  • factor VII polypeptides include polypeptides that exhibit at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least about 140%, and most preferably at least about 160%, of the specific biological activity of human factor Vila.
  • factor VII polypeptides include polypeptides that exhibit at least about 70 %, preferably at least about 80 %, more preferably at least about 90 %, and most preferable at least about 95 %, of identity with the sequence of wild-type factor VII as disclosed in U.S. Patent No. 4,784,950.
  • 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.
  • 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.
  • 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, as well as polypeptides in which the factor Vila biological activity has been substantially modified or reduced relative to the activity of wild-type human factor Vila.
  • polypeptides include, without limitation, factor Vll or factor Vila that has been chemically modified and factor Vll variants into which specific amino acid sequence alterations have been introduced that modify or disrupt 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 Vll, whether exhibiting substantially the same or better bioactivity than wild-type factor Vll, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type factor Vll, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wild- type factor Vll 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
  • Factor Vll-related polypeptides having substantially the same or improved biological activity relative to wild-type factor Vila encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably 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 reduced biological activity relative to wild-type factor Vila are those that exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1 % 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 variants having a substantially modified biological activity relative to wild-type factor Vll include, without limitation, factor Vll variants that exhibit TF- independent factor X proteolytic activity and those that bind TF but do not cleave factor X.
  • the factor Vll polypeptides are factor Vll-related polypeptides, in particular variants, wherein the ratio between the activity of said factor Vll 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 Vll polypeptides are factor Vll-related polypeptides, in particular variants, wherein the ratio between the activity of said factor Vll 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 Vll polypeptide is human factor Vll, as disclosed, e.g., in U.S. Patent No. 4,784,950 (wild-type factor Vll).
  • the factor Vll polypeptide is human factor Vila.
  • the factor Vll polypeptides are factor Vll-related polypeptides that exhibits at least about 10%, preferably at least about 30%, more preferably at least about 50%, and most preferably at least about 70%, of the specific biological activity of human factor Vila.
  • the factor Vll polypeptides have an amino acid sequence that differs from the sequence of wild-type factor Vll by insertion, deletion, or substitution of one or more amino acids.
  • Non-limiting examples of factor Vll variants having substantially the same or better biological activity compared to wild-type factor Vila include, but are not limited to, those described in Danish Patent Applications Nos. PA 2000 00734 and PA 2000 01360 (corresponding to WO 01/83725), and PA 2000 01361 (corresponding to WO 02/22776).
  • Non-limiting examples of factor Vll variants having substantially the same or improved biological activity as wild-type factor Vll include S52A-FVII, S60A-FVM (lino et al., Arch. Biochem. Biophys.
  • 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. 48:501-505, 1995); and oxidized forms of factor Vila (Kornfelt et al.. Arch. Biochem. Biophys. 363:43-54, 1999).
  • Non-limiting examples of factor Vll variants having substantially reduced or modified biological activity relative to wild-type factor Vll include R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990), S344A-FVIIa (Kazama et al., J.
  • factor Vila in blood clotting derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of factor IX or factor X to produce activated factor IX or X (factor IXa or Xa, respectively).
  • factor Vll biological activity biological activity of factor Vll polypeptides
  • biological activity may be quantified by measuring the ability of a preparation to promote blood clotting using factor Vll-def icient 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 Vll units" by comparison with a pooled human serum standard containing 1 unit/ml factor Vll activity.
  • factor Vila biological activity may be quantified by (i) Measuring the ability of factor Vila or a factor Vila -related polypeptide to produce activated factor X (factor Xa) in a system comprising TF embedded in a lipid membrane and factor X. (Persson et al., J. Biol. Chem. 272:19919-19924, 1997); (ii) Measuring factor X hydrolysis in an aqueous system ("In Vitro Proteolysis Assay", see below); (iii) Measuring the physical binding of factor Vila or a factor Vila -related polypeptide to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts.
  • factor Vll biological activity or "factor Vll 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 limita- tion, NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark).
  • any factor XI polypeptide may be used that is effective in preventing or treating bleeding.
  • This includes factor XI polypeptides derived from blood or plasma, or produced by recombinant means.
  • platelets may contain a structurally different form of FXI (possible due to alternative splicing of the FXI gene). Platelet factor XI is described in Lipscomb, M.S. & Walsh, P.N. (1979), Journal of Clinical Investigation, 63, 1006-1014.
  • factor XI polypeptide encompasses, without limitation, factor XI, as well as factor Xl-related polypeptides.
  • factor XI is intended to encompass, without limitation, polypeptides having the amino acid sequence as described in Sun, Y. & Gailani, D. (1996), J. Biol. Chem. 271 : 29023-29028 (wild-type human factor XI, plasma), as well as wild-type factor XI derived from other species, such as, e.g., bovine, porcine, canine, murine, and salmon factor XI.
  • the factor XI polypeptide is wild-type human factor XI, as disclosed, e.g., in Sun, Y. & Gailani, D. (1996), J. Biol. Chem. 271 : 29023-29028.
  • factor XI is also intended to encompass factor XI 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 Xla.
  • Factor Xl-related polypeptides include, without limitation, factor XI polypeptides that have either been chemically modified relative to human factor XI and/or contain one or more amino acid sequence alterations relative to human factor XI (i.e., factor XI variants), and/or contain truncated amino acid sequences relative to human factor XI (i.e., factor XI fragments). Such factor Xl-related polypeptides may exhibit different properties relative to human factor XI, including stability, phospholipid binding, altered specific activity, and the like.
  • factor Xl-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 Xla-related polypeptides” or "activated factor Xl-related polypeptides".
  • factor Xl-related polypeptides encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild- type human factor XI, as well as polypeptides, in which the factor XI biological activity has been substantially modified or reduced relative to the activity of wild-type human factor XI.
  • polypeptides include, without limitation, factor XI or factor Xla that has been chemically modified and factor XI variants into which specific amino acid sequence alterations have been introduced that modify or disrupt 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 XI.
  • Factor Xl-related polypeptides including variants of factor XI, whether exhibiting substantially the same or better bioactivity than wild-type factor XI, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type factor XI, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wild- type factor XI by insertion, deletion, or substitution of one or more amino acids.
  • Factor Xl-related polypeptides encompass those that exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, and at least about 130%, of the specific activity of wild-type factor XI that has been produced in the same cell type, when tested in the factor XI activity assay as described in the present specification.
  • Factor Xl-related polypeptides including variants, having substantially the same or improved biological activity relative to wild-type factor XI encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130% of the specific biological activity of wild-type human factor XI that has been produced in the same cell type when tested in one or more of the specific factor XI activity assay as described.
  • factor XI biological activity may be quantified as described later in the present description ("assay part").
  • Factor Xl-related polypeptides including variants, having substantially reduced biological activity relative to wild-type factor XI are those that exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1 % of the specific activity of wild-type factor XI that has been produced in the same cell type when tested in one or more of the specific factor XI activity assays as described above.
  • Non-limiting examples of factor XI polypeptides include plasma-derived human factor XI as described, e.g., in Gailani & Broze (1993), Blood Coagul. Fibrinolysis, 4:15-20, or Kerbiriou & Griffin (1979), J. Biol. Chem., 254:12020-12207, or ,
  • the factor XI are factor Xl-reiated polypeptides wherein the ratio between the activity of said factor XI polypeptide and the activity of native human factor XI (wild-type factor XI) is at least about 1.25 when tested in the " FXI chromogenic assay" (see below); in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0.
  • Factor Xl-related polypeptides also include fragments of factor XI or factor Xl-related polypeptides retaining their characteristic haemostasis-related activity.
  • the haemostasis-related activity of a factor XI polypeptide may, for example, be measured using the factor Xl-activity assay described in the present specification.
  • the factor XI is human plasma factor XI or activated human plasma factor Xla.
  • the FXI is platelet factor XI. In another embodiment, the FXI is recombinantly made.
  • amino acids 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 Vll, and that, for example, [K337A]-FVIIa designates the FVM-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.
  • Table 1 Abbreviations for amino acids:
  • factor Vila or “FVIIa” may be used interchangeably.
  • factor Vila includes zymogen factor Vll (single-chain factor Vll).
  • factor XI or “FXI” may be used interchangeably.
  • factor VIII or “FVIII” may be used interchangeably.
  • factor VIII or “FVIII” included activated factor VIII (FVIIIa), variants and truncated forms retaining the characteristic FVMI-related haemostatic activity; the term includes recombinantly made FVIII and plasma-derived FVIII. Human FVIII and human recombinant FVIII are preferred
  • 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 (e.g., as in pooled, normal human plasma), and includes, without limitations, subjects lacking factor VIII; 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 or FVII; subjects having a lowered level of several coagulation factors (e.g., due to exessive bleeding as a consequence of trauma or extensive surgery); and subjects with lowered plasma concentrations of fibrinogen (e.g., multitransfused subjects).
  • level of thrombin generation or "normal thrombin generation” is meant the level of the patient's level of thrombin generation compared to the level in healthy subjects. The level is designated as a percentage of the normal level. The terms may, where appropriate, be used interchangeably.
  • 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 Vll or factor Vll-related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide is prolonged relative to the individual thrombin generation of a control sample containing only the factor Vll or factor Vll-related polypeptide or the factor XI or factor Xl-related polypeptide, 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”).
  • “Sole” agents or factors as used herein refers to situations in which the factor Vll or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide, taken together, are the only haemostatic agents, or active haemostatic agents, or coagulation factors contained in the pharmaceutical composition or kit, or are the only haemostatic agents,or active haemostatic agents, or coagulation factors 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.
  • 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., thromboelastograpy 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.
  • thromboelastograpy as described by, e.g., Meh et al.,Blood Coagulation & Fibrinolysis 2001;12:627- 637; Vig et al., Hematology, Vol. 6 (3) pp. 205-2
  • 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 Vll or factor Vll- related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide is prolonged relative to the individual clot lysis time of a control sample containing only the factor VI I or factor Vll-related polypeptide or the factor XI or factor Xl-related polypeptide, 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 Vll or factor Vll-related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide is increased relative to the individual clot lysis time of a control sample containing only the factor Vll or factor Vll-related polypeptide or the factor XI or factor Xl-related polypeptide, 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 Vll or factor Vll-related polypeptide and a preparation of a preparation of a factor XI or factor Xl-related polypeptide is increased relative to the individual rate for or degree of fibrin clot formation of a control sample containing only the factor Vll or factor Vll-related polypeptide or the factor XI or factor Xl-related polypeptide, 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 Vll or factor Vll-related polypeptide and a preparation of a preparation of a factor XI or factor Xl-related polypeptide is increased relative to the individual clotting time of a control sample containing only the factor VI I or factor Vll-related polypeptide or the factor XI or factor Xl-related polypeptide 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.
  • lowered count or activity of platelets refers to the number of platelets (throm- bocytes) present in the subject's plasma and to the biological, coagulation-related activity of such platelets. Lowered counts may be due, e.g., to increased platelet destruction, decreased platelet production, and pooling of a larger than normal fraction of platelets in the spleen.
  • Thrombocytopenia for example, is defined as a platelet count less than 150,000 platelets per microliter; the upper limit of the normal platelet count is generally considered to be between 350,000 and 450,000 platelets per microliter. Platelet count may be measured by automated platelet counters; this is a well known method to the skilled worker.
  • Syndromes due to lowered platelet count include, without limitation, thrombocytopenia, coagulophathy.
  • Activity includes, without limitation, aggregation, adhesion, and coagulant activity of the platelets. Decreased activity may be due, e.g., to glyco- protein abnormalities, abnormal membrane-cytoskeleton interaction, abnormalities of platelet granules, abnormalities of platelet coagulant activity, abnormalities of signal transduction and se- cretion. Platelet activity, including aggregation, adhesion, and coagulant activity, are measured by standard methods known to the skilled worker, see e.g.,Platelets. A Practical Approach, Ed. S.P. Watson & K.S.
  • 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, XI or Vll clotting factor inhibitors
  • defective platelet function e.g., Glanzmann thombasthenia and Bernard-Soulier syndrome
  • thrombocytopenia e.g., von Willebrand's disease
  • coagulophathy 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) coagulophathy, 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 protein).
  • compounds e.g., platelets or von Willebrand factor protein.
  • 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 achiev- ing 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.
  • the bleeding may also be due to so-called thrombolytic therapy which comprises combined treatment with an antiplatelet agent (e.g., acetylsalicylic acid), an anticoagulant (e.g., heparin), and a fibrinolytic agent (e.g., tissue plasminogen activator, tPA).
  • an antiplatelet agent e.g., acetylsalicylic acid
  • an anticoagulant e.g., heparin
  • a fibrinolytic agent e.g., tissue plasminogen activator, tPA
  • Bleeding episodes are also meant to include, without limitation, uncontrolled and excessive bleeding in connection with surgery or trauma in subjects having acute haemarthroses (bleedings in joints), chronic haemophilic ar- thropathy, haematomas, (e.g., muscular, retroperitoneal, sublingual and retropharyngeal), bleedings in other tissue, haematuria (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 ar- thropathy e.g., ar- thropathy
  • haematomas e.g., muscular, retroperitoneal, sublingual and retropharyngeal
  • bleedings in other tissue e.g., haematuria (bleeding from the renal tract), cerebral haemorrhage, surgery (e.g., hepat
  • the bleeding episodes may be associated with inhibitors against factor VIII; haemophilia A; haemophilia A with inhibitors; haemophilia B; deficiency of factor Vll; deficiency of factor XI; 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 expected 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 Vll or factor Vll-related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide 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 Vll or factor Vll-related polypeptides and factor XI or factor Xl-related polypeptides as defined in the present specification may be administered simultaneously or sequen- tially.
  • 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 Vll or factor Vll-related polypeptide as a first unit dosage form and a preparation of a factor XI or factor Xl-related polypeptide as a second unit dosage form.
  • a first or second or third, etc., unit dose is mentioned throughout this specification this does not indicate the prefer- red order of administration, but is merely done for convenience purposes
  • a preparation of a factor Vll or factor Vll-related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide 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 se- paration of no more than 15 minutes, preferably 10, more preferred 5, more preferred 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, preferably from 1 to 2 hours, more preferred up to 1 hour, more preferred from 30 mi- nutes to 1 hour, more preferred up to 30 minutes, more preferred from 15 to 30 minutes.
  • Either of the two unit dosage form, or coagulation factor proteins may be administered first.
  • both products are injected through the same intravenous access.
  • level of factor XI or "factor XI level” is meant the level of the patient's clotting factor XI activity compared to the level in healthy subjects. The level is designated as a percenta- ge of the normal level. The terms may, where appropriate, be used interchangeably.
  • reduced level of factor XI or “reduced factor XI level” is meant a decrease in the presence or activity of factor XI in the blood stream compared to the mean factor XI level in a population of subjects having no coagulation factor XI deficiency or inhibitors to coagulation factor XI.
  • the level of circulating factor XI can be measured by either a coagulant or an immuno- logic assay, factor XI procoagulant activity is determined by the ability of the patient's plasma to correct the clotting time of factor Xl-def icient plasma (e.g., an APTT assay, see below; see also "assay part” of the present description).
  • One unit of factor XI has been defined as the amount of factor XI present in one milli- litre of normal (pooled) human plasma (corresponding to a factor XI level of 100 %).
  • factor Vll is defined as the amount of factor Vll present in 1 ml of normal plasma, corresponding to about 0.5 ⁇ g protein. After activation 50 units correspond to about 1 ⁇ g protein.
  • defect is meant a decrease in the presence or activity of, e.g., factor XI in plasma compared to that of normal healthy individuals.
  • the term may, where appropriate, be used interchangeably with “reduced factor XI level”.
  • APTT or "aPTT” is meant the activated partial thromboplastin time (described by, e.g.. Proctor RR, Rapaport SI: 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 Xl-responsive syndrome is meant a syndrome where exogenous factor XI 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 limita- tion, syndromes caused by a reduced level of factor XI, e.g., bleeding disorders caused by inhibitors to factor XI. A factor Xl-responsive syndrome may also be treated with a composition according to the present invention.
  • factor Vll-responsive syndrome is meant a syndrome where exogenous factor Vll, preferably factor Vila, administered to the subject in need thereof may prevent, cure or amelio- rate 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, XI or Vll, clotting factor inhibitors, defective platelet function (e.g., Glanzmann thombasthenia and Bernard-Soulier syndrome), 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).
  • clotting factors VIII, IX, XI or Vll clotting factor inhibitors
  • defective platelet function e.g., Glanzmann thomba
  • Het-life refers to the time required for the plasma concentration of a factor Vll or factor Vll-related polypeptide or a factor XI or factor Xl-related polypeptide 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.
  • secondary haemostasis or “maintained haemostasis” is meant the secondary, full, and major, burst or generation of thrombin taking place on the surface of activated platelets and catalysed by factor Villa and factor Villa, the subsequent formation of fibrin and the stabilization of the initial platelet plug.
  • 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.
  • the total amount of protein in a preparation may be measured by generally known methods, e.g, by measuring optical density.
  • Amounts of factor XI coagulation or factor Vll protein ("antigen”) may be measured by generally known methods such as standard Elisa immuno assays.
  • such assay is conducted by contacting, e.g., a solution of the factor XI protein- containing preparation with an anti-FXI antibody immobilised onto the elisa plate, sub- sequently contacting the immobilised antibody-factor XI complex with a second anti FXI antibody carrying a marker, the amounts of which, in a third step, are measured.
  • the amounts of each coagulation factor may be measured in a similar way using appropriate antibodies.
  • the total amount of coagulation factor protein present in a preparation is determined by adding the amounts of the individual coagulation factor proteins.
  • the preparation comprises isolated coagulation factor, ln another embodiment the preparation is free of coagulation factor II and coagulation factor Ma (prothrombin and thrombin) and/or factor X or Xa.
  • isolated refers to coagulation factors, e.g., factor XI or factor Xl-related polypeptides that have been separated from the cell in which they were synthesized or the medium in which they are found in nature (e.g., plasma or blood). Separation of po- lypeptides 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 Vll or anti-factor XI 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 Vll or anti-factor XI 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
  • TFPI inhibitor means compounds inhibiting the anti-coagulative activity of TFPI (tissue factor pathway inhibitor).
  • the term includes compounds such as those disclosed in European Patent No. 558 529, WO 96/28153 and US 5,622,988.
  • TFPI and "EPI” (extrinsic pathway inhibitor) may be used interchangeably.
  • an "effective amount" of a factor Vll polypeptide and a factor XI polypeptide is defined as the amount of a factor Vll polypeptide, e.g., FVIIa, and a fac- tor XI polypeptide that together suffices to prevent or reduce bleeding or blood loss, so as to cure, alleviate or partially arrest the disease and its complications.
  • the term "activity of factor Vila” or "factor Vila-activity” includes 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.
  • composition according to the invention may further comprise a TFPI-inhibitor.
  • composition according to the invention may further comprise a factor Vlll.
  • Such a composition should preferably be administered to subjects who do not have inhibitors to factor Vlll.
  • 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 Vll 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.CIin.lnvest. 71: 1836-1841, 1983. These methods yield factor Vll without detectable amounts of other blood coagulation factors.
  • An even further purified factor Vll 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 Xlla, IX a or Xa.
  • factor Vll may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia fine Chemicals) or the like.
  • Factor Vll -related polypeptides may produced by modification of wild-type factor Vll or by recombinant technology, factor Vll -related polypeptides with altered amino acid sequence when compared to wild-type factor Vll may be produced by modifying the nucleic acid sequence encoding wild-type factor Vll 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 Vll by known means, e.g. by site-specific mutagenesis.
  • substitutions can be made outside the regions critical to the function of the factor Vila or factor Xl-molecule and still result in an active polypeptide.
  • Amino acid residues essential to the activity of the factor Vll or factor Vll-related polypeptide or factor XI or factor Xl-related polypeptide, and therefore preferably 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;
  • 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 containing 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.
  • the product is treated with Dpnl, which is specific for methylated and hemi- methylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA.
  • Dpnl is specific for methylated and hemi- methylated DNA to digest the parental DNA template and to select for mutation-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 non- adherent cells; and the like.
  • factor Vll or factor Vll-related polypeptides may be further purified. Purification may be achieved using any method known in the art, including, without limitation, aff in- ity chromatography, such as, e.g., on an anti-factor Vll antibody column (see, e.g., Wakabayashi 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 Vll or factor Vll-related polypeptides derived from the host cell.
  • Factor Vll or factor Vll-related polypeptides may be activated by proteolytic cleavage, using factor Xlla or other proteases having trypsin-like specificity, such as, e.g., factor IXa, kallik- rein, factor Xa, and thrombin. See, e.g., Osterud et al., Biochem. 11 :2853 (1972); Thomas, U.S. Patent No. 4,456,591; and Hedner et al., J. Clin. Invest. 71 :1836 (1983).
  • factor Vll 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 Vll or factor Vll-related polypeptide may then be formulated and administered as described below.
  • Factor XI for use within the present invention may be prepared from plasma according to known methods, such as those disclosed by Koide et al. (Biochemistry 16: 2279-2286, 1977) and Bouma et al. (J. Biol. Chem. 252: 6432-6437, 1977), incorporated herein by reference. It is preferred, however, to use recombinant factor XI so as to avoid to the use of blood- or tissue- derived products that carry a risk of disease transmission. Methods for preparing recombinant factor XI are known in the art. See, for example, Kemball-Cook et al. (Gene 139(2): 275-279, 1994), Fujikawa et al. (Biochemistry 25: 2417-2424, 1986), Meijers et al. (Blood 79(6): 1435-1440, 1992), which are incorporated herein by reference in their entirety.
  • Factor XI -related polypeptides may produced by modification of wild-type factor XI or by recombinant technology, factor XI -related polypeptides with altered amino acid sequence when compared to wild-type factor XI may be produced by modifying the nucleic acid sequence encoding wild-type factor XI 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 XI by known means, e.g. by site-specific mutagenesis, as described in more detail above.
  • 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.
  • factor XI or factor Xl-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 XI antibody column; 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, as described in more detail above.
  • 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 XI or factor Xl-related polypeptides derived from the host cell.
  • the re- suiting activated factor XI or factor Xl-related polypeptide may then be formulated and administered as described below.
  • factor XI and factor Vila proteins syngeneic with the subject in order to reduce the risk of inducing an immune response.
  • Preparation and characterization of non-human factor XI has been disclosed by , for example, Gailani (Blood 90(3): 1055-1064, 1997).
  • the present invention also encompasses the use of such factor XI and factor Vila proteins within veterinary procedures.
  • compositions and Methods of Use may be used to treat any factor Vll responsive syndrome, such as, e.g., bleeding disorders, including, without limitation, syndromes caused by a reduced level of clotting factors Vlll, IX, XI or Vll, clotting factor inhibitors, defective platelet function (e.g., Glanzmann thombasthenia and Bernard-Soulier syndrome), 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 Vll responsive syndrome such as, e.g., bleeding disorders, including, without limitation, syndromes caused by a reduced level of clotting factors Vlll, IX, XI or Vll, clotting factor inhibitors, defective platelet function (e.g., Glanzmann
  • compositions comprising a preparation of a factor Vll or factor Vll-related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide 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 factor Vll or a factor VM-related polypeptide, and factor XI or a factor Xl-related polypeptide, either formulated in a single-unit dosage form or in the form of a kit-of parts, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier or diluent.
  • pharmaceutical compositions suitable for use according to the present invention is made by mixing a factor Vila, or a factor XI, or a factor Vila in combination with a factor XI, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent.
  • aqueous carriers 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. 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.
  • 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.
  • diluents such as those disclosed in Remington's Pharmaceutical Sciences. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990.
  • 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.
  • the 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 Vll or factor Vll-related polypeptide, factor XI or factor XI- related polypeptide, or factor Vll or factor Vll-related polypeptide in combination with factor XI or factor Xl-related polypeptide 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 Vll or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide 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 Vll or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide in one dosage form, or a dissolved lyophilized powder or a liquid formulation containing the factor Vll or factor Vll-related polypeptide in one dosage form and dissolved lyophilized powder or a liquid formulation containing the factor XI or factor Xl-related polypeptide in another dosage form.
  • the amount of factor Vll or factor Vll-related polypeptide and the amount of factor XI or factor Xl-related polypeptide together comprise an aggregate effective amount for treating the bleeding episode.
  • compositions containing a preparation of a factor Vll or factor Vll-related polypeptide and a preparation of a factor XI or factor Xl-related polypeptide 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 Vll or factor Vll-related polypeptide and the amount of factor XI or factor Xl-related polypeptide together comprise an aggregate effective amount for preventing a bleeding episode.
  • 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) preferably with intervals of 2-4-6-12 hour, depending on the dose given and the condition of the subject.
  • the factor Vll or factor Vll- related polypeptide and the factor XI or factor Xl-related polypeptide 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 Vll or factor Vll-related polypeptide and a preparation of a preparation of a factor XI or factor Xl-related polypeptide 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 Vll or factor Vll-related polypeptide and a second unit dosage form comprising a preparation of a preparation of a factor XI or factor Xl-related polypeptide.
  • the factor Vll or factor Vll-related polypeptide and the factor XI or factor Xl-related polypeptide should be administered one after the other, preferably within about 15 minutes of each other, for example within 10 minutes of each other or, preferably. within 5 minutes or, more preferred, 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 Vll or factor Vll-related polypeptide and the amount of factor XI or factor Xl-related polypeptide 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 Vll to factor XI 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 to about 1:2, or between about 1 :70 to about 1 :5, or between about 1 :60 to about 1 :10, or between about 1:50 to about 1:25, or between about 1 :
  • the dose of the factor Vll or factor Vll-related polypeptide ranges from what corresponds to about 0.05 mg to about 500 mg/day of wild-type factor Vll, 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 factor XI or factor Xl-related polypeptide ranges from what corre- sponds to about 0.05 mg to about 500 mg/day of wild-type factor XI, e.g., from about 1 mg to about 200 mg/day, or, e.g., from about 1 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 combination of a factor Vila and a factor XI shows a synergistic effect in an in vitro clot firmness- and fibrinolysis time-assay. Moreover, the combination of a factor Vila and a factor XI shows a synergistic effect in forming stable fibrin clots, increasing the half-clot lysis time, increasing clot strength and increasing resistance to fibrinolysis.
  • the composition may be in the form of a single preparation comprising both a factor Vila and a factor XI in suitable concentrations.
  • the composition may also be in the form of a kit consisting of a first unit dosage form comprising a factor Vila and a second unit dosage form comprising a factor XI and, optionally, one or more further unit dosage forms comprising a factor Vlll and/or an TFPI inhibitor.
  • the factor Vila and the factor XI should be administered sequentially, preferably within about 1-2 hours of each other, for example within 30 minutes of each other or, preferably, within 10 minutes or, more preferred, within 5 minutes of each other. Either of the two unit dosage forms can be administered first.
  • the present invention relates to the prevention or treatment of bleeding episodes or for coagulative treatment by treatment with a combination of active ingredients that may be administered separately
  • the invention also relates to combining separate pharmaceutical compositions in kit form.
  • 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
  • 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 (MaxiSorp, 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 NaCl, 5 mM CaCl 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 (A 405 nm factor Vila variant)/(A 05 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 Vll (wild-type FVII) is around, versus above 1.0.
  • 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).
  • 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 Vila (10 nM) and factor X (0.8 mi- croM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCI2 and 1 mg/ml bovine serum albumin, are incubated for 15 min.
  • factor X cleavage is then stopped by the addition of 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 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, Chromogenix, Sweden), final concentration 0.5 mM.
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader
  • 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 Vll (wild-type FVII) is around, versus above 1.0.
  • Thrombin generation assay The ability of factor Vll or factor Vll-related polypeptides or factor XI or factor Xl-related polypeptides (e.g., variants) to generate thrombin can be measured in an assay comprising all relevant coagulation factors and inhibitors at physiological concentrations and activated platelets (as described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 which is hereby incorporated as reference).
  • a suitable assay for testing for factor XI amidolytic activity and thereby selecting suitable factor XI variants can be performed as a simple in vitro test using a chromogenic substrate as described, for example, in Gailani et al. (Blood 97(10): 3117-3122, 2001) ("the FXI chromogenic assay").
  • Factor XI biological activity may also be performed as a simple in vitro test measuring the activation of factor IX to IXa as described for example, in Gailani et al. (Blood 97(10): 3117- 3122, 2001).
  • Suitable assays for testing for factor Vlll activity can be performed as simple in vitro tests as described, for example, in Kirkwood TBL, Rizza CR, Snape TJ, Rhymes IL, Austen DEG. Identification of sources of interlaboratory variation in factor Vlll assay. B J Haematol 1981; 37; 559-68.; or Kessels et al., British Journal of Haematology, Vol. 76 (Suppl.1) pp. 16 (1990)).
  • factor Vlll activity may also be measured by a two-step chromogenic assay based on the amidolytic activity of generated FXa (Wagenvoord et al, 1989, Haemostasis, 19(4):196-204).
  • factor Vlll biological activity may also be quantified by measuring the ability of a preparation to correct the clotting time of factor Vlll-def icient plasma, e.g., as described in Nilsson et al., 1959. (Nilsson IM, Blombaeck M, Thilen A, von Francken I., Carriers of haemophilia A - A laboratory study, Acta Med Scan 1959; 165:357).
  • biological activity is expressed as units/ml plasma (1 unit corresponds to the amount of FVIII present in normal pooled plasma.
  • a pharmaceutical composition comprising a factor Vll or a factor Vll-related polypeptide, and a factor XI or factor Xl-related polypeptide.
  • Embodiment 2 The composition as in aspect 1, wherein said factor Vll or factor Vll- related polypeptide is a factor Vll-related polypeptide.
  • Embodiment 3 The composition as in aspect 1, wherein the factor Vila is human factor Vila
  • Embodiment 4 The composition as in aspect 1 or aspect 3, wherein the factor Vila and the factor XI is recombinant human factor Vila and recombinant human factor XI.
  • Embodiment 5 The composition as in any one of aspects 1-4, wherein the factor XI is platelet factor XI.
  • Embodiment 6 The composition as in any one of aspects 1-5, wherein the factor XI is activated factor XI.
  • Embodiment 7 The composition as in any one of aspects 1-6, wherein the composition further contains a TFPI inhibitor.
  • Embodiment 8 The composition as in any one of aspects 1-7, wherein the composition further contains a factor Vlll.
  • a kit containing a treatment for bleeding episodes comprising a) an effective amount of a factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms.
  • Embodiment 10 The composition as in aspect 2, comprising a) an effective amount of a factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; c) an effective amount of a TFPI inhibitor and a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing said first, second and third dosage forms.
  • a kit containing a treatment for bleeding episodes comprising a) an effective amount of a factor Vila and a TFPI inhibitor and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms.
  • a kit containing a treatment for bleeding episodes comprising a) an effective amount of a factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XI and a TFPI inhibitor and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms.
  • Embodiment 9 A kit as in any one of embodiments 2-4 further containing a factor Vlll, either formulated in a separate unit dosage form, or contained within a unit dosage form also containing one or more of the compounds selected from the list of a factor
  • Aspect 6 Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for treating bleeding episodes in a subject.
  • Aspect 7 Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for reducing clotting time in a subject.
  • Aspect 8 Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for prolonging the clot lysis time in normal mammalian plasma.
  • Aspect 9 Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for increasing clot strength in normal mammalian plasma.
  • Aspect 10 Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for enhancing fibrin clot formation in normal human plasma.
  • a method of enhancing fibrin clot formation in a subject comprises administering to a subject an effective amount of a factor Vila in combination with an effective amount of a factor XI.
  • Aspect 12 A method for treating bleeding episodes in a subject comprising administering to a subject an effective amount of a factor Vila in combination with an effective amount of a factor XI.
  • Embodiment 10 Method as in aspect 19 or 20, wherein the factor Vila and the factor XI are administered in one-dosage form.
  • Embodiment 11 Method as in aspect 19 or 20, wherein the factor Vila and the factor XI is administered sequentially.
  • Clot lysis assay Normal human plasma diluted 10-fold with buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCI, pH 7.4) containing Innovin (Dade Behring, 2000-fold dilution), rFVIIa (Novo Nordisk A S, Bagsvaerd, Denmark; various concentrations) and t-PA (American Diagnostics, 8 nM) was added to 96-well ELISA plates and turbidity at 650 nm was measured over time at room temperature. Where indicated, purified human FXI (Haematologic Technologies, various concentrations) was included.
  • Rotatonal thromboelastography Measurements was conducted on citrated normal human plasma added 5 nM t-PA and the effect of addition of 1 nM FVIIa alone or in combination with 30 nM FXI (Haematologic Technologies) was analyzed. Clotting was initiated by addition of Innovin (final concentration 2000-fold diluted, Dade Behring # 526945) and calcium (final concentration 15 mM) in a 20 mM HEPES, 150 mM NaCl, pH 7.4 buffer.
  • Clot lysis assay Addition of FVIIa results in a dose-dependent prolongation of the clot lysis time (Fig. 1). This effect was optimal at 10 nM FVIIa. In the presence of 10 nM FVIIa, addition of FXI resulted in a further prolongation of the clot lysis time (Fig. 2). The effect was dose-dependent and optimal at 30 nM FXI.
  • Thromboelastography roTEG measurements were utilized to analyze the effect of FVIIa and FXI on the Maximal Clot Firmness (MCF), as well as the clots resistance to t-PA mediated lysis.
  • MCF Maximal Clot Firmness
  • FVIIa/FXI the MCF was 25 mm and the time required for half clot lysis was 12.3 minutes (Fig. 3).
  • Addition of FXI (30 nM) did not alter MCF but prolonged the half-clot lysis time to 16.1 min (Fig. 3).
  • addition of FVIIa (1 nM) resulted in clot protection from t-PA- mediated fibrinolysis (half-clot lysis time; 16.7 min) without any effect on MCF (Fig. 3).
  • FVIIa (1 nM) together with FXI (30 nM) increases the MCF (29 mm), as well as the half-clot lysis time (24.2 min, Fig. 3).
  • Clot assay Aliquots (55 ⁇ l) of rFVIIa (1 ⁇ g/ml) alone, FXI (25 nM) alone, or rFVIIa and FXI in 50 mM Pipes, 100 mM NaCl, 2 mM EDTA, 1 % BSA, pH 7.2, were incubated for 5 min with a 55 ⁇ l aliquot containing 100 ⁇ M PC/PS vesicles and 50 mM CaCl2 in the same buffer. A 55 ⁇ l aliquot of normal human plasma (NHP) was then added and clotting followed for 400 seconds in an ACL clotting machine using the standard APTT program.
  • NHS normal human plasma
  • Clot assay Prior to addition of rFVIIa or FXI, NHP did not clot within the 400 seconds monitoring time. Following addition of FXI (25 nM) the clotting time was still longer than 400 s. Addition of FVIIa (1 ⁇ g/ml) reduced the clot time to 159.4 ⁇ 1.4 seconds (Fig 4). Addition of both FVIIa (1 ⁇ g/ml) and FXI (25 nM) reduced the clot time to 95.0 ⁇ 1.4 seconds (Fig 4).

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EP02750844A 2001-07-20 2002-07-19 Pharmaceutical composition comprising factor vii polypeptides and factor xi polypeptides Withdrawn EP1411973A1 (en)

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AU5807500A (en) 1999-07-14 2001-02-05 Novo Nordisk A/S Use of fviia or a tissue factor antagonist for regulating gene expression and cell migration or chemotaxis
ES2381110T3 (es) 2003-09-09 2012-05-23 Novo Nordisk Health Care Ag Polipéptidos de factor VII de coagulación
BRPI0416461A (pt) * 2003-11-20 2007-03-06 Novo Nordisk Healthcare Ag método para o tratamento de episódios de sangramento, formulação farmacêutica, e, uso de um polipeptìdeo fxi
EP1713497B1 (en) * 2004-02-05 2016-04-13 Novo Nordisk Health Care AG Use of factor viia for treating late complications of trauma
WO2006128497A1 (en) * 2005-06-01 2006-12-07 Novo Nordisk A/S Pharmaceutical formulation of factor xi
ES2773766T3 (es) 2008-12-19 2020-07-14 Baxalta GmbH Inhibidores de TFPI y métodos de uso
AU2011227714B2 (en) 2010-03-19 2014-09-04 Takeda Pharmaceutical Company Limited TFPI inhibitors and methods of use
EP2624859B1 (en) 2010-10-06 2017-03-01 Medimmune Limited Factor ii alone or in combination with further factors for treatment of impaired haemostasis associated with dilutional coagulopathy
CA2867363C (en) 2012-03-21 2023-02-28 Baxter International Inc. Tfpi inhibitors and methods of use
KR20160111520A (ko) * 2014-01-31 2016-09-26 다나-파버 캔서 인스티튜트 인크. 디히드로프테리디논 유도체 및 그의 용도
EP3307728A4 (en) 2015-06-12 2019-07-17 Dana Farber Cancer Institute, Inc. ASSOCIATION THERAPY USING TRANSCRIPTION INHIBITORS AND KINASE INHIBITORS
MX2018006499A (es) 2015-11-25 2018-08-01 Dana Farber Cancer Inst Inc Inhibidores de bromodominio bivalentes y usos de los mismos.
US11958911B2 (en) 2017-02-10 2024-04-16 Shanghai Benemae Pharmaceutical Anti-coagulation factor XI antibody

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JPS59116213A (ja) * 1982-12-24 1984-07-05 Unitika Ltd エアゾ−ル組成物
ATE66374T1 (de) * 1985-11-26 1991-09-15 Novo Nordisk As Zubereitungen und verfahren zur behandlung von blutungsstoerungen.
US5580560A (en) * 1989-11-13 1996-12-03 Novo Nordisk A/S Modified factor VII/VIIa
FR2676231A1 (fr) * 1991-05-07 1992-11-13 Aetsrn Concentre de facteur xi de la coagulation sanguine a haute activite specifique, approprie a usage therapeutique et son procede de preparation.
JP4361728B2 (ja) * 2000-09-13 2009-11-11 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト ヒト凝固因子ｖｉｉ変異型

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CN1304050C (zh) 2007-03-14
HUP0400976A3 (en) 2006-01-30
BR0211256A (pt) 2004-07-27
ZA200400012B (en) 2004-08-17
NO20040235L (no) 2004-03-17
JP2004534855A (ja) 2004-11-18
MXPA04000415A (es) 2004-11-22
RU2004105031A (ru) 2005-04-20
CZ200439A3 (cs) 2005-03-16
RU2298416C2 (ru) 2007-05-10
PL369077A1 (en) 2005-04-18
CA2452677A1 (en) 2003-01-30
KR20040015821A (ko) 2004-02-19
WO2003007983A1 (en) 2003-01-30

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