CN1304050C - Pharmaceutical composition comprising factor VII polypeptides and factor XI polypeptides - Google Patents

Abstract

This invention relates to compositions containing factor VII or factor VII-related polypeptides and factor XI or factor XI-related polypeptides, and their use in treating bleeding episodes.

Description

Pharmaceutical compositions containing factor VII polypeptides and factor XI polypeptides

technical field

The present invention relates to pharmaceutical compositions comprising Factor VII or Factor VII-related polypeptides and Factor XI or Factor XI-related polypeptides. The present invention also relates to the combination of factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide in the preparation of a medicament for treating a subject suffering from bleeding episodes, or preventing their onset. The invention also relates to methods of treating bleeding episodes in a subject and methods of promoting coagulation in a subject. The invention also relates to kits containing these compounds.

Background technique

Hemostasis is initiated by the formation of a complex between tissue factor (TF) exposed to circulating blood following vessel wall injury and circulating FVIIa present in amounts corresponding to approximately 1% of the total FVII protein mass. This complex anchors to TF-bearing cells and activates FX to FXa and FIX to FIXa on the cell surface. FXa activates prothrombin to thrombin, which activates FVIII, FV, FXI and FXIII. In addition, limited amounts of thrombin formed during the initial step of hemostasis also activate platelets. When thrombin acts on a platelet, it changes shape and exposes the charged phospholipids on its surface. This activated platelet surface forms a template for further activation of FX and sufficient generation of thrombin. FXa is further activated on the surface of activated platelets by forming a FIXa-FVIIIa complex on the surface of activated platelets, and then FXa is still on the surface to convert prothrombin to thrombin. Thrombin then converts fibrinogen to fibrin which is insoluble and stabilizes the initiating platelet embolism. This process is compartmentalized, i.e. localized to sites of TF expression or exposure, thereby minimizing the risk of systemic activation of the coagulation system. Plugs formed by insoluble fibrin are further stabilized by FXIII-catalyzed crosslinking of fibrin fibers.

FVIIa exists mainly as a single-chain zymogen in plasma, which is cleaved by FXa into its double-chain activated form, namely FVIIa. Recombinant activated factor VIIa (rFVIIa) has been developed as a pro-haemostatic agent. Administration of rFVIIa in hemophilic subjects with bleeding who cannot be treated with coagulation factor products due to antibody formation produces a rapid and highly effective prohemostatic response. Bleeding subjects with Factor VII deficiency or subjects with a normal coagulation system but who experience excessive bleeding can also be successfully treated with FVIIa. In these studies, no adverse side effects of rFVIIa (in particular the occurrence of thromboembolism) were found.

Additional exogenous administration of FVIIa increases thrombin formation on the surface of activated platelets. This occurs in hemophiliac subjects who lack FIX or FVIII and thereby lose the most efficient pathway for adequate thrombin formation. In addition, additional FVIIa increases thrombin formation in cases where platelet numbers are reduced or platelets have functional defects.

A commercial preparation of recombinant human FVIIa is sold as NovoSeven(R) (Novo Nordisk A/S, Denmark). Novoseven(R) is indicated for the treatment of bleeding episodes in hemophilia A and B patients. Novoseven(R) is the only recombinant FVIIa available on the market for the effective and reliable treatment of bleeding episodes.

FXI is a component of the intrinsic coagulation pathway. FXI deficiency is associated with mild to moderate bleeding disturbances especially in tissues with high local fibrinolytic activity. Instead, high levels of FXI are believed to be a risk factor for venous thrombosis. FXI is the zymogen of trypsin-like serine proteases activated by FXIIa, thrombin and FXIa. Activated FXI (FXIa) participates in the activation of FIX, which in turn (in conjunction with FVIII) further activates FX and thus leads to thrombin generation.

It is well known that subjects with excessive bleeding associated with surgery or severe trauma requiring transfusions develop more complications than subjects who do not experience any bleeding. However, moderate bleeding requiring administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for treatment of coagulation defects, etc.) virus) risk-related complications. Extensive bleeding requiring massive transfusions can lead to multiple organ failure, including impairment of lung and kidney function. Once a subject develops these serious complications, a cascade of events involving many cytokines and an inflammatory response begins, making any treatment very difficult and unfortunately often unsuccessful. Therefore, a major goal in surgery as well as in the treatment of severe tissue injury is to avoid or minimize bleeding. To avoid or minimize such bleeding, it is important to ensure the formation of a stable solid hemostatic plug that is not easily dissolved by fibrinolytic enzymes. Furthermore, it is important to ensure rapid and efficient formation of this embolism or coagulation.

Currently, subjects experiencing bleeding episodes, including trauma victims and subjects with surgery-related bleeding, are usually treated with several injections or infusions of FVIIa, requiring one because of the short half-life of FVIIa (2.5 hours) The above dosing is to maintain a certain level of hemostatic ability. Accelerated arrest of bleeding would have an important benefit to this subject. So was the reduction in the number of doses needed to stop bleeding and maintain hemostasis.

Japanese Patent Application No. 59-116213A relates to an aerosol composition containing a coagulant as an active ingredient for use as a tissue adhesive. The coagulation agent may be selected from coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, and XIII, prekallikrein, high multimeric kininogen, and thrombin. Combinations of FXIII and thrombin are preferred.

European Patent 225.160 (Novo Nordisk) relates to compositions of FVIIa and methods for the treatment of bleeding disorders not caused by coagulation factor deficiencies or coagulation factor inhibitors.

European patent 82.182 (Baxter Travenol Lab.) relates to the use of compositions of factor VIIa in subjects against deficiencies in the coagulation factor or as inhibitors of the effect on the coagulation factor.

International patent application WO 93/06855 (Novo Nordisk) relates to the topical application of FVIIa.

US Patent 5,252,217 relates to a method of preparing human Factor XI concentrates for therapeutic use.

There remains a need in the art for improved treatment of subjects experiencing bleeding episodes, including bleeding episodes caused by: surgery, trauma, or other forms of tissue injury; induced coagulopathy, including during multiple transfusions; Coagulopathy in the subject; congenital or acquired coagulation or bleeding disorders, including reduced liver function ("liver disease"); defective platelet function or reduced number of platelets; major coagulation "compounds" (e.g., platelets or von. Willebrand factor protein) deficiency or abnormality; increased fibrinolysis; anticoagulant therapy or thrombolytic therapy; or stem cell transplantation.

There remains a need in the art for improved, reliable and broadly applicable methods for enhancing coagulation, enhancing or ensuring the formation of stable hemostatic plugs, or improving the convenience of treating a subject, or in a subject, particularly with reduced thrombin production Complete hemostasis was achieved in subjects. There is also a need for methods that reduce the amount of FVIIa required to achieve complete hemostasis and that shorten the time to arrest bleeding.

Summary of the invention

It is an object of the present invention to provide compositions which are effective in the treatment or prevention of bleeding episodes and coagulation disorders.

A second object of the present invention is to provide compositions in single unit dosage form which are effective for the treatment or prevention of bleeding episodes or as procoagulants. Another object of the present invention is to provide compositions, methods of treatment or kits exhibiting a synergistic effect.

Another object of the present invention is to provide compositions, methods of treatment or kits that exhibit no significant side effects, such as high levels of systemic activation of the coagulation system.

Other objects of the present invention will be apparent upon reading this specification.

In a first aspect, the present invention provides a pharmaceutical composition comprising Factor VII or a Factor VII-related polypeptide, and Factor XI or a Factor XI-related polypeptide.

In a second aspect, the invention provides a kit comprising means for treating a bleeding episode, comprising

a) a preparation of an effective amount of Factor VII or a Factor VII-related polypeptide in a first unit dosage form and a pharmaceutically acceptable carrier;

b) a preparation of an effective amount of Factor XI or a Factor XI-related polypeptide in a second unit dosage form and a pharmaceutically acceptable carrier; and

c) container means containing said first and second dosage forms.

In its various embodiments, the kit also contains an effective amount of a TFPI-inhibitor and/or Factor VIII; the TFPI-inhibitor or Factor VIII (or a combination of both) may be presented in separate unit dosage forms Alternatively it may be present in a unit dosage form containing Factor VII or Factor VII-related polypeptide, or Factor XI or Factor XI-related polypeptide.

In a third aspect, the present invention provides the use of a combination of Factor VII or a Factor VII-related polypeptide and Factor XI or a Factor XI-related polypeptide in the preparation of a medicament for treating bleeding episodes in a subject. In another aspect, the present invention provides the use of the composition according to any one of claims 1 to 18 for the preparation of a medicament for treating bleeding episodes in a subject.

In its various embodiments, the medicament is used to shorten coagulation time, prolong coagulation lysis time, and increase coagulation strength.

In another embodiment, the medicament is formulated for intravenous administration, preferably injection or infusion, especially injection.

In one embodiment, the medicament is formulated as a single unit dosage form; in another embodiment it is formulated as a first unit dosage form containing a Factor VII or Factor VII-related polypeptide preparation and a Factor XI or Factor XI-related polypeptide preparation. The form of the second unit dosage form of the preparation.

In various embodiments, the medicament is used to treat subjects experiencing bleeding episodes resulting from: surgery, trauma, or other forms of tissue damage; coagulopathy, including coagulopathy in subjects; congenital or acquired coagulation or bleeding disorders, including reduced liver function ("liver disease"); defective platelet function or decreased platelet numbers; major coagulation "compounds" (e.g., platelets or von Lebrand factor protein) deficiency or abnormality; increased fibrinolysis; anticoagulant therapy or thrombolytic therapy; stem cell transplantation. In one series of embodiments, the hemorrhage occurs in an organ such as the brain, inner ear region, eye, liver, lung, tumor tissue, gastrointestinal tract; in another series of embodiments, it is a diffuse type of hemorrhage, e.g. gastritis and excessive uterine bleeding. In another series of embodiments, the bleeding episode is in a subject with acute hemorrhagic arthropathy (joint bleeding), chronic hemophilic arthropathy, hematoma, (e.g., muscle, retroperitoneal, sublingual and retropharyngeal) Bleeding associated with surgery or trauma, bleeding in other tissues, hematuria (renal bleeding), cerebral hemorrhage, surgery (eg, liver resection), tooth extraction, and gastrointestinal bleeding (eg, UGI bleeding). In one embodiment, the medicament is for treating a bleeding episode in a subject due to trauma, or surgery, or a decrease in platelet count or activity.

In another aspect, the invention provides a method of treating a bleeding episode in a subject, the method comprising administering to a subject in need thereof a first amount of Factor VII or a preparation of a Factor VII-related polypeptide and a second amount of Factor XI or a preparation of a Factor XI-related polypeptide, wherein the first and second amounts together are effective for treating bleeding.

In another aspect, the present invention provides a method of reducing clotting time in a subject, the method comprising administering to a subject in need thereof a first amount of Factor VII or a preparation of Factor VII-related polypeptide and a second amount of Factor XI or a preparation of a Factor XI-related polypeptide, wherein the first and second amounts are together effective to shorten clotting time.

In another aspect, the invention provides a method of enhancing hemostasis in a subject, the method comprising administering to a subject in need thereof a first amount of Factor VII or a preparation of Factor VII-related polypeptide and a second amount of Factor VII A preparation of XI or Factor XI-related polypeptide wherein the first and second amounts are together effective to enhance hemostasis.

In another aspect, the present invention provides a method of prolonging coagulation lysis time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of Factor VII or a Factor VII-related polypeptide and a second amount of A preparation of Factor XI or a Factor XI-related polypeptide, wherein the first and second amounts are together effective to prolong coagulation and lysis time.

In another aspect, the present invention provides a method of increasing coagulation strength in a subject, the method comprising administering to a subject in need thereof a first amount of a Factor VII or a preparation of a Factor VII-related polypeptide and a second amount of A preparation of Factor XI or a Factor XI-related polypeptide wherein the first and second amounts are together effective to increase the strength of coagulation.

In a series of embodiments of the method, the Factor VII or Factor VII-related polypeptide and the Factor XI or Factor XI-related polypeptide are administered in a single unit dosage.

In another series of embodiments, the Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are in a first unit dosage form comprising Factor VII or Factor VII-related polypeptide preparation and comprising Factor XI or Factor XI-related polypeptide. The XI-related polypeptide preparation is administered in a second unit dosage form. In one series of embodiments thereof, the first unit dosage form and the second unit dosage form are administered at a time interval of not more than 15 minutes.

In another aspect, the invention provides a kit for treating bleeding episodes comprising

d) an effective amount of Factor VII or Factor VII-related polypeptide and an effective amount of Factor XI or Factor XI-related polypeptide and a pharmaceutically acceptable carrier in a unit dosage form; and

e) container means containing said one unit dosage form.

In one series of embodiments of the invention, the Factor VII or Factor VII-related polypeptide is a Factor VII-related polypeptide. In one series of embodiments of the invention, the Factor VII-related polypeptide is a Factor VII amino acid sequence variant. In one embodiment, the ratio between the activity of the Factor VII-related polypeptide and the activity of native human Factor VIIa (wild-type FVIIa) when tested in the "in vitro hydrolysis assay" described herein is at least about 1.25.

In one series of embodiments of the invention, the Factor VII or Factor VII-related polypeptide is Factor VII. In one embodiment, the Factor VII is human Factor VII. In one embodiment, Factor VII is bovine, porcine, canine, equine, murine or salmon Factor VII. In another embodiment, Factor VII is produced recombinantly. In another embodiment, the Factor VII is derived from plasma. In a preferred embodiment, Factor VII is recombinant human Factor VII. In one series of embodiments of the invention, the Factor VII or Factor VII-related polypeptide is an activated form thereof. In a preferred embodiment of the invention, Factor VII is recombinant human Factor VIIa.

In one series of embodiments, the Factor XI or Factor XI-related polypeptide is a Factor XI-related polypeptide. In one embodiment, the Factor XI-related polypeptide is a Factor XI amino acid sequence variant. In one embodiment, the ratio between the activity of said factor XI-related polypeptide and the activity of native human plasma factor XI (wild-type FXI) when detected in the "FXI chromogenic assay" described in the specification is At least about 1.25. In one embodiment, the Factor XI or Factor XI-related polypeptide is a Factor XI polypeptide. In one embodiment, Factor XI is human Factor XI. In one embodiment, the factor XI is bovine, porcine, canine, equine, murine or salmon factor XI. In a preferred embodiment, Factor XI is produced recombinantly. 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, Factor XI is recombinant human plasma Factor XI. In one series of embodiments of the invention, Factor XI or Factor XI-related polypeptide is an activated form thereof. In one embodiment, the Factor XI-related polypeptide is a fragment of Factor XI. In one embodiment, the Factor XI-related polypeptide is a hybrid Factor XI polypeptide, eg, a porcine/human hybrid. In one embodiment, Factor XI is human plasma activated Factor XI (FXIa).

In one embodiment, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are present in a mass ratio of between about 100:1 to about 1:100 (w/w Factor VII:Factor XI).

In one embodiment, the Factor VII-related polypeptide is an amino acid sequence variant having no more than 20 amino acid substitutions, deletions or insertions 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 variant has no more than 15 amino acid substitutions, deletions or insertions; in another embodiment, the Factor VII variant has no more than 10 amino acids compared to wild-type Factor VII (e.g. 8, 6, 5, or 3 amino acids) substitutions, deletions or insertions. In one embodiment, the Factor VII variant is selected from L305V-FVIIa, L305V/M306D/D309S-FVIIa, L305I-FVIIa, L305T-FVIIa, F374P-FVIIa, V158T/M298Q-FVIIa, V158D/E296W/M298Q-FVIIa, K337A-FVIIa, M298Q-FVIIa, V158D/M298Q-FVIIa, L305V/K337A-FVIIa, V158D/E296V/M298Q/L305V-FVIIa, V158D/E296V/M298Q/K337A-FVIIa, V158D/E2986V/LK FVIIa, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII.

In another embodiment, the Factor VII-related polypeptide has increased tissue factor-independent activity compared to native human Factor Vila. In another embodiment, the increase in activity is not accompanied by a change in substrate specificity. In another embodiment of the present invention, the binding of the factor VII-related polypeptide to tissue factor is not reduced and the factor VII-related polypeptide has at least the activity of wild-type factor VIIa when binding to tissue factor.

In a preferred embodiment, the factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide are recombinant human factor VIIa and recombinant human plasma factor XI or recombinant human factor VIIa and recombinant human plasma factor XIa.

In one embodiment, the clotting time in mammalian blood is reduced. In another embodiment, hemostasis in mammalian blood is enhanced. In another embodiment, the coagulation lysis time is prolonged in mammalian blood. In another embodiment, the strength of coagulation in mammalian blood is increased. In one embodiment, the mammalian blood is human blood. In another embodiment, the mammalian blood is normal human blood; in one embodiment, the blood is blood from a subject with reduced thrombin production. In one embodiment, the blood is from a subject deficient in one or more clotting factors; in another embodiment, the blood is from a subject with an inhibitor of one or more clotting factors In one embodiment, the blood is blood from a subject having a reduced concentration of fibrinogen; in one embodiment, the blood is human blood deficient in Factor XI. In one series of embodiments, the blood is plasma.

In one embodiment, the Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide prolong the lysis time of in vitro coagulation in normal human plasma. In another embodiment, the Factor VII or Factor VII-related polypeptide and the Factor XI or Factor XI-related polypeptide increase in vitro maximal coagulation intensity coagulation lysis time in normal human plasma. In another embodiment, the Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide shorten the in vitro clotting time of normal human plasma.

In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are the only hemostatic agents comprised in the composition. In another embodiment, the Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are the only active hemostatic agents comprised in the composition. In another embodiment, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are the only coagulation factors administered to the subject. In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are the only 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.

In another embodiment, the pharmaceutical composition is formulated for intravenous administration, preferably injection or infusion, especially injection. In one embodiment, the composition contains at least one pharmaceutically acceptable excipient or carrier.

In one embodiment of the invention, the composition is in a single unit dosage form, wherein the single unit dosage form contains two coagulation factors. In one embodiment of the invention, the composition is in the form of a kit of parts comprising a Factor VII or Factor VII-related polypeptide preparation as a first unit dosage form and a Factor XI or Factor XI-related polypeptide preparation 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 may further contain instructions for separately administering the composition or the separate components, as desired.

In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are administered in a single dosage form. In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are in the form of a first unit dosage of a preparation comprising Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide. The preparation of Factor XI-related polypeptide is administered in a second unit dosage form.

In one embodiment of the present invention, factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide are administered simultaneously. In another embodiment, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are administered sequentially. In one embodiment, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are administered at a time interval of no more than 15 minutes, preferably 10, more preferably 5, more preferably 2 minutes. In one embodiment, factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide are separated for up to 2 hours, preferably 1 to 2 hours, more preferably up to 1 hour, more preferably 30 minutes to 1 hour, more preferably Administration is at intervals of up to 30 minutes, more preferably 15 to 30 minutes.

In one embodiment, the effective amount of Factor VII or Factor VII-related polypeptide is an amount from about 0.05 mg/day to about 500 mg/day (70-kg subject). In one embodiment, the effective amount of a Factor XI or Factor XI-related polypeptide preparation is from about 0.01 mg/day to about 500 mg/day (70-kg subject).

In one embodiment, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are present in a mass ratio of between about 100:1 to about 1:100 (w/w Factor VII:Factor XI).

In one embodiment of the invention, the pharmaceutical composition is in single dosage form and consists essentially of a Factor VII or Factor VII-related polypeptide preparation and a Factor XI or Factor XI-related polypeptide preparation, and selected from pharmaceutically acceptable Excipients or carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and one or more components in protease inhibitors.

In another embodiment, the subject is a human; in another embodiment, the subject has impaired thrombin generation; in one embodiment, the subject has reduced plasma concentrations of fibrinogen (e.g., multiple transfused subjects); in one embodiment, the subject has a decreased plasma concentration of Factor VII.

In another aspect, the invention relates to a method of enhancing hemostasis in a subject with factor VII response syndrome as compared to treatment of the subject with factor VII as the sole coagulation protein, the method comprising administering to the subject in need thereof The subject is administered a first amount of Factor VII or a preparation of Factor VII-related polypeptide and a second amount of Factor XI or a preparation of Factor XI-related polypeptide, wherein the first and second amounts together are effective to enhance hemostasis.

In another aspect, the invention relates to a method of enhancing thrombin formation in a subject, the method comprising administering to a subject in need thereof a first amount of Factor VII or a preparation of Factor VII-related polypeptide and a second amount of Factor VII A preparation of XI or Factor XI-related polypeptide wherein the first and second amounts are together effective to enhance thrombin formation.

In another aspect, the invention relates to a method of enhancing thrombin formation in a subject with factor VII response syndrome as compared to treatment of the subject with factor VII as the sole coagulation protein, the method comprising administering The subject is administered a first amount of Factor VII or a preparation of Factor VII-related polypeptide and a second amount of Factor XI or a preparation of Factor XI-related polypeptide, wherein the first and second amounts together are effective to enhance thrombin formation.

In another aspect, the present invention relates to reducing the time required to achieve hemostasis in a subject with Factor VII Response Syndrome compared to the number of doses required for the subject to administer Factor VII as the only coagulation factor protein. A method for administering the number of coagulation factor proteins, the method comprising administering to a subject in need thereof a first amount of a factor VII or a preparation of a factor VII-related polypeptide and a second amount of a preparation of factor XI or a factor XI-related polypeptide, wherein Together, the first and second amounts are effective to reduce the number of administrations of the coagulation factor protein.

In another aspect, the invention relates to a method of treating bleeding in a subject suffering from factor VII response syndrome, the method comprising administering to the subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of Factor XI or a Factor XI-related polypeptide, wherein the first and second amounts together are effective for treating bleeding.

In one embodiment, Factor VII is human recombinant Factor VIIa (rFVIIa). In another embodiment, rFVIIa is NovoSeven(R) (Novo Nordisk A/S, Bagsvaerd, Denmark).

In one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the composition also contains an inhibitor of the fibrinolytic system, including, but not limited to, aprotinin, ε-aminocaproic acid, or tranexamic acid. In one embodiment, the composition also contains a TFPI inhibitor and/or FVIII.

In one embodiment, the composition also contains Factor VII. In one embodiment, Factor VIII is activated Factor VIII (Factor VIIIa). In another embodiment, Factor VIII is recombinant Factor VIIIa. In another embodiment, Factor VIII is recombinant human Factor VIIIa.

In another embodiment, the present invention relates to the use of Factor Vila in combination with Factor XI for the manufacture of a medicament for enhancing the formation of fibrin coagulation in mammalian plasma.

In another aspect, the present invention relates to a method of enhancing fibrin coagulation formation in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of Factor VII or a Factor VII-related polypeptide and a second amount of A preparation of Factor XI or a Factor XI-related polypeptide wherein the first and second amounts together are effective in treating bleeding.

In one embodiment of the invention, the pharmaceutical composition (when in the form of a single preparation) consists essentially of Factor VIIa and Factor XI, and optionally a pharmaceutically acceptable excipient or carrier, and optionally A stabilizer, and optionally a detergent, and optionally a neutral salt, and optionally an antioxidant, and optionally a preservative, and optionally a protease inhibitor.

In another embodiment of the invention, the pharmaceutical composition (when in the form of a single preparation) consists essentially of Factor VIIa and Factor XI, and optionally a pharmaceutically acceptable excipient or carrier, and any optionally a stabilizer, and optionally a detergent, and optionally a neutral salt, and optionally an antioxidant, and optionally a preservative, and optionally a protease inhibitor, and TFPI inhibitor composition.

In another embodiment of the invention, the pharmaceutical composition (when in the form of a single preparation) consists essentially of Factor VIIa and Factor XI, and optionally a pharmaceutically acceptable excipient or carrier, and any optionally a stabilizer, and optionally a detergent, and optionally a neutral salt, and optionally an antioxidant, and optionally a preservative, and optionally a protease inhibitor, and Factor VIII, and optionally a TFPI inhibitor.

In another embodiment, the pharmaceutical composition (when in kit form) consists essentially of Factor VIIa and optionally one pharmaceutically acceptable excipient or carrier, and optionally one stabilizer , and optionally a detergent, and optionally a neutral salt, and optionally an antioxidant, and optionally a preservative, and optionally a first unit dosage form consisting of a protease inhibitor , and consist essentially of Factor XI and optionally a pharmaceutically acceptable excipient or carrier, and optionally a stabilizer, and optionally a detergent, and optionally a neutral salt, and a second unit dosage form of optionally one antioxidant, and optionally one preservative, and optionally one protease inhibitor.

In another embodiment, the pharmaceutical composition (when in kit form) consists essentially of Factor VIIa and optionally one pharmaceutically acceptable excipient or carrier, and optionally one stabilizer , and optionally a detergent, and optionally a neutral salt, and optionally an antioxidant, and optionally a preservative, and optionally a first unit dosage form consisting of a protease inhibitor , and consist essentially of Factor XI and optionally a pharmaceutically acceptable excipient or carrier, and optionally a stabilizer, and optionally a detergent, and optionally a neutral salt, and optionally an antioxidant, and optionally a preservative, and optionally a second unit dosage form consisting of a protease inhibitor; wherein the first unit dosage form or the second unit dosage form or both dosage forms All further contain Factor VIII and/or TFPI inhibitors.

Description of drawings

Figure 1: Addition of FVIIa results in a dose-dependent prolongation of coagulation lysis time. This effect is optimal at 1OnM FVIIa.

Figure 2: In the presence of 10 nM FVIIa, addition of FXI leads to further prolongation of coagulation and lysis times. This effect is dose dependent and optimal at 30 nM FXI.

Figure 3: Analysis of the effect of FVIIa and FXI on maximum coagulation firmness (MCF), and coagulation resistance to t-PA-mediated lysis, as measured by thromboelastography (roTEG).

Figure 4: These results demonstrate that FVIIa and FXI, when added to plasma, synergistically shorten coagulation time in NHP.

Detailed description of the invention

Subjects who had excessive bleeding associated with surgery or severe trauma and thus required blood transfusions developed more complications than those who did not experience any bleeding. However, moderate bleeding can also lead to complications where administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for treatment of coagulation defects, etc.) Hepatitis, HIV, parvovirus, or other as yet unknown viruses) as well as non-viral pathogens. Extensive bleeding requiring massive transfusions can lead to multiple organ failure, including impairment of lung and kidney function. Once a subject develops these serious complications, a cascade of events involving many cytokines and an inflammatory response begins, making any treatment very difficult and unfortunately often unsuccessful. Patients who experience severe blood loss are clinically unstable. The patient is at risk of experiencing atrial fibrillation, which can cause fatal cardiac arrest; impaired renal function; or pulmonary fluid extravasation (also known as "wet lung" or ARDS). Therefore, a major goal in surgery as well as in the treatment of severe tissue injury is to avoid or minimize bleeding. To avoid or minimize this unwanted bleeding, it is important to ensure the formation of a stable solid hemostatic plug that is not easily dissolved by fibrinolytic enzymes. Furthermore, it is important to ensure rapid and efficient formation of this embolism or coagulation.

Subjects with thrombocytopenia (decreased platelet count or activity) also have impaired thrombin generation and a defect in the stabilization of the fibrin plug, resulting in a predisposed premature dissolution of the hemostatic plug. Furthermore, subjects who have suffered severe trauma or organ damage and thus frequently receive blood transfusions often have reduced platelet counts as well as reduced levels of fibrinogen, Factor VIII, and other clotting proteins. These subjects experience impaired (or reduced) thrombin generation. Thus, these subjects have defective, or less efficient, hemostasis leading to the formation of fibrin thrombi that are readily and prematurely lysed by proteolytic enzymes that are critical in conditions characterized by extensive trauma and organ damage. The following further broad release.

Bleeding into tissue can also lead to the formation of a hematoma. The size of the hematoma (especially intercranial and spinal) is closely related to the degree of neurological damage, difficulty of rehabilitation, and/or the severity and extent of permanent neurological damage after rehabilitation. The most serious consequences of hematomas occur when they are located in the brain and they can even lead to the death of the patient.

Therefore, the main goal in the treatment of bleeding is to stop the bleeding in the shortest possible time so that blood loss is kept to a minimum.

The present invention thus provides beneficial compositions, uses thereof and methods of treatment for treating bleeding episodes in a subject in need thereof. The compositions, uses and methods are associated with beneficial effects such as less blood loss before hemostasis, less blood need during surgery, maintenance of blood pressure at acceptable levels before hemostasis, faster stabilization of blood pressure, recovery of treated patients Shorter time to recovery in treated patients, including reduced hematoma formation or smaller hematoma formation in the brain, faster termination of bleeding, and fewer doses needed to stop bleeding and maintain hemostasis.

Factor VII or factor VII-related polypeptide preparations, such as factor VIIa administered in combination with factor XI or factor XI-related polypeptide preparations provide improved clotting time, coagulation hardness and resistance compared to administration of factor VIIa or factor XI alone. Shortened clotting time, greater coagulation firmness and increased resistance to fibrinolysis.

Factor VII or a factor VII-related polypeptide preparation (e.g., factor VIIa) in combination with a factor XI or factor XI-related polypeptide preparation reduces the time required to arrest bleeding compared with factor VIIa or factor XI alone , the number of doses required to maintain hemostasis is reduced. The present invention provides the beneficial effect of administering Factor XI or a preparation of Factor XI-related polypeptide and Factor VII or a preparation of Factor VII-related polypeptide simultaneously or sequentially. The pharmaceutical compositions according to the invention may be in the form of a single composition or may be in the form of a kit of parts (kit of parts). The compositions according to the invention are used as therapeutic and prophylactic procoagulants in mammals including primates such as humans. The invention also provides methods of treating (including prophylactically treating or preventing) a bleeding episode in a subject, including a human.

Whenever a first or second or third, etc., unit dose is mentioned in this specification, it does not imply a preferred order of administration, but is merely for descriptive convenience.

The combination of factor VII or factor VII-related polypeptide preparations and factor XI or factor XI-related polypeptide preparations is a beneficial product that ensures shortened coagulation time, rapid formation of hemostatic plugs, and formation of stable hemostatic plugs. The present inventors have found that the combination of Factor VII or Factor VII-related polypeptides with Factor XI or Factor XI-related polypeptides is a beneficial product that ensures the formation of firm, stable and rapidly forming hemostatic plugs.

The present inventors have shown that the combination of Factor Vila and Factor XI is more effective in shortening the clotting time of normal human plasma than Factor Vila or Factor XI alone. The combination of Factor Vila and Factor XI has also been shown to enhance coagulation firmness more effectively than Factor Vila or Factor XI alone. By combining Factor Vila at a concentration at which no further increase in coagulation firmness was observed, a further increase in coagulation firmness was unexpectedly found. It has also been shown that the combination of Factor Vila and Factor XI is more effective in prolonging in vitro coagulation and lysis times in normal human plasma than Factor Vila or Factor XI alone. It has also been shown that the combination of Factor Vila and Factor XI is more effective in prolonging the hemiclotting time in normal human plasma than Factor Vila or Factor XI alone. It has also been shown that the combination of Factor Vila and Factor XI is more effective than Factor Vila or Factor XI alone in preventing coagulated fibrinolysis, particularly tPA-mediated fibrinolysis, in normal human plasma.

Thus, by enhancing coagulation, a more effective treatment of bleeding in a subject can be achieved.

Without wishing to be bound by theory, it is believed that sufficient thrombin production is necessary to form a firm, stable hemostatic plug, and thus maintain hemostasis. The fibrin structure of the thrombus depends on the amount of thrombin formed and the rate at which thrombin generation begins. Under conditions of impaired thrombin production, a highly permeable porous fibrin thrombus forms. Fibrinolytic enzymes generally present on the surface of fibrin readily dissolve the fibrin thrombus. Formation of a stable fibrin thrombus also depends on the presence of Factor XIIIa, which is activated by thrombin and thus also depends on thrombin being adequately generated. In addition, a recently described thrombin-activated fibrinolysis inhibitor, TAFI, requires considerably higher amounts of thrombin for its activation. Thus in the absence of sufficient thrombin formation, TAFI is not activated, resulting in the formation of a hemostatic plug that is more readily lysed than would normally be lysed by normal fibrinolytic activity. In cases of reduced platelet numbers, ie, thrombocytopenia, administration of exogenous extra Factor VIIa can initiate faster thrombin generation. However, even at high concentrations, Factor Vila fails to normalize overall thrombin production.

Sufficient thrombin activation did not occur in subjects with reduced plasma concentrations of fibrinogen (subjects with multiple blood transfusions due to multiple trauma or major surgery). More effective hemostasis was obtained by coadministration of factor VII and factor XI.

Subjects with thrombocytopenia have impaired thrombin generation as well as defects in fibrin thrombus stabilization, resulting in a predisposed premature dissolution of the hemostatic plug. Additionally, subjects who have suffered severe trauma or organ damage and thus frequently receive blood transfusions often have reduced platelet counts as well as reduced levels of fibrinogen, Factor VIII, and other clotting proteins. These subjects experience impaired (decreased) thrombin generation. In addition, its reduced fibrinogen levels negatively interfere with the activation of Factor XIII. Thus, these subjects had hemostatic deficiencies, or less efficient hemostasis, resulting in the formation of fibrin thrombi that were readily and prematurely lysed by proteolytic enzymes in the setting of extensive trauma and organ damage. The case was further widely released.

To assist in the formation of a sufficiently stable thrombus in a subject with full capacity to maintain hemostasis, a composition according to the invention may be administered. The composition is particularly beneficial in subjects with reduced platelet numbers and reduced plasma levels of fibrinogen and/or other blood clotting proteins.

In the presence of Factor XI, it is believed that lower concentrations of Factor Vila may be sufficient to ensure adequate hemostasis.

Factor VII Peptides:

In practicing the present invention, any Factor VII polypeptide that is effective in preventing or treating bleeding can be used. It includes Factor VII polypeptides derived from blood or plasma, or recombinantly produced.

The present invention includes Factor VII polypeptides, eg, polypeptides having the amino acid sequence disclosed in US Pat. No. 4,784,950 (wild-type human Factor VII). In some embodiments, the Factor VII polypeptide is human Factor VIIa disclosed, eg, in US Patent No. 4,784,950 (wild-type Factor VII). In one series of embodiments, the Factor VII polypeptide comprises a polypeptide exhibiting 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 VIIa . In one series of embodiments, the Factor VII polypeptide comprises 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% polypeptide. In one series of embodiments, the Factor VII polypeptide comprises at least about 70%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% identical peptides.

As used herein, "Factor VII polypeptide" includes, but is not limited to, Factor VII, and Factor VII-related polypeptides. The term "Factor VII" is intended to include, but is not limited to, polypeptides having the amino acid sequence 1-406 of wild-type human Factor VII (disclosed in U.S. Patent No. 4,784,950), as well as wild-type Factor VII from other species, such as bovine, Porcine, canine, murine, and salmon Factor VII derived from blood or plasma, or recombinantly produced. It also includes natural allelic variants of Factor VII that exist and occur from one individual to another. Additionally, the degree and location of glycosylation or other post-translational modifications may vary depending on the chosen host cell and the properties of the host cell environment. The term "Factor VII" is also intended to include Factor VII polypeptides in their uncleaved (zymogen) form, and those polypeptides that have been proteolytically processed to produce their respective biologically active forms, designated Factor VIIa. Generally, Factor VII is cleaved between residues 152 and 153 to generate Factor VIIa.

"Factor VII-related polypeptides" include, but are not limited to, chemically modified relative to human Factor VII and/or contain one or more amino acid sequence changes relative to human Factor VII (i.e., Factor VII variants), and/or Factor VII polypeptides comprising a truncated amino acid sequence (ie, a fragment of Factor VII) relative to human Factor VII. The Factor VII-related polypeptide may exhibit different properties relative to human Factor VII, including stability, phospholipid binding, changes in specific activity, and the like. The term "Factor VII-related polypeptide" is intended to include such polypeptides in their uncleaved (zymogen) form, and which have been proteolytically processed to produce their respective biologically active forms, and may be named "Factor VIIa-related polypeptides" or "Factor VIIa-related polypeptides" Activated factor VII-related polypeptides" are those polypeptides.

As used herein, a "factor VII-related polypeptide" includes, but is not limited to, a polypeptide exhibiting substantially the same or increased biological activity relative to wild-type human factor VII, and a biological activity of factor VIIa relative to wild-type human factor VII A polypeptide in which the activity of VIIa is substantially modified or reduced. These polypeptides include, but are not limited to, chemically modified Factor VII or Factor VIIa and Factor VII variants that introduce specific amino acid sequence changes that modify or destroy the biological activity of the polypeptide.

It also includes polypeptides with slightly modified amino acid sequences, eg, polypeptides with a modified N-terminus including deletions or additions of N-terminal amino acids, and/or polypeptides that are chemically modified relative to human Factor Vila.

Factor VII-related polypeptides comprising variants of Factor VII, whether exhibiting substantially the same or better biological activity than wild-type Factor VII, or selected for exhibiting substantially modified or reduced biological activity relative to wild-type Factor VII Include, but are not limited to, polypeptides having an amino acid sequence that differs from the wild-type Factor VII sequence by insertion, deletion, or substitution of one or more amino acids.

Factor VII-associated polypeptides (including variants) encompass those that exhibit a specific activity of wild-type Factor VIIa in the same cell type of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, or at least about 130% polypeptide.

Factor VII-related polypeptides (including variants) having substantially the same or improved biological activity relative to wild-type Factor VIIa, encompassing those detected in one or more of the above coagulation assays, proteolytic assays, or TF binding assays , 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 110% of the specific activity of wild-type Factor VIIa in the same cell type About 120%, and most preferably at least about 130% polypeptide.

Factor VII-related polypeptides (including variants) having substantially reduced biological activity relative to wild-type Factor VIIa are identical when tested in one or more of the coagulation assays, proteolysis assays, or TF binding assays described above The wild-type Factor Vila specific activity in the cell type is less than about 25%, preferably less than about 10%, more preferably less than about 5%, and most preferably less than about 1% of the polypeptide. Factor VII variants having substantially modified biological activity relative to wild-type Factor VII include, but are not limited to, Factor VII variants that exhibit TF-dependent Factor X proteolytic activity and those that bind TF but do not cleave Factor X variant.

In some embodiments, the Factor VII polypeptide is a Factor VII-related polypeptide, particularly a variant, wherein the activity of the Factor VII polypeptide and native human factor when tested in an "in vitro hydrolysis assay" (see "Assay" below) The ratio between the activities of VIIa (wild-type FVIIa) is at least about 1.25; in other embodiments, the ratio is at least about 2.0; in other embodiments, the ratio is at least about 4.0. In some embodiments of the invention, the Factor VII polypeptide is a Factor VII-related polypeptide, particularly a variant, wherein the activity of said Factor VII polypeptide when tested in an "in vitro proteolytic assay" (see "Assay" below) The ratio between the activity of natural human Factor VIIa (wild-type FVIIa) is at least about 1.25; in other embodiments, the ratio is at least about 2.0; in other embodiments, the ratio is at least about 4.0; in other embodiments In embodiments, the ratio is at least about 8.0.

In some embodiments, the Factor VII polypeptide is human Factor VII disclosed, eg, in US Patent No. 4,784,950 (wild-type Factor VII). In some embodiments, the Factor VII polypeptide is human Factor VIIa. In one series of embodiments, the Factor VII polypeptide is a factor 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 VIIa VII-related polypeptides. In some embodiments, the Factor VII polypeptide has an amino acid sequence that differs from the wild-type Factor VII sequence by the insertion, deletion, or substitution of one or more amino acids.

Non-limiting examples of Factor VII variants having substantially the same or better biological activity than wild-type Factor VIIa include, but are not limited to, those described in Danish Patent Application Nos. PA 2000 00734 and PA 2000 01360 (corresponding to WO 01/83725), and those variants described in PA 2000 01361 (corresponding to WO02/22776). Non-limiting examples of Factor VII variants having substantially the same or higher biological activity compared to wild-type Factor VII include S52A-FVII, S60A-FVII (lino et al., Arch. Biochem. Biophys. 352: 182- 192, 1998); L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q , V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298V/L305V/K337AII, 27FVII, K1 - FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII; FVIIa variants exhibiting increased proteolytic stability disclosed in US Pat. No. 5,580,560; at residues 290 and Between 291 or between residues 315 and 316 proteolytically cleaved Factor VIIa (Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); and the oxidized form of Factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54, 1999). Non-limiting examples of Factor VII variants having substantially reduced or modified biological activity relative to wild-type Factor VII include R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990), S344A-FVIIa (Kazama et al. , J.Biol.Chem.270:66-72, 1995), FFR-FVIIa (Holst et al., Eur.J.Vasc.Endovasc.Surg.15:515-520, 1998), and factor VIIa lacking the Gla domain (Nicolaisen et al., FEBS Letts. 317:245-249, 1993). Non-limiting examples of chemically modified Factor VII polypeptides and sequence variants are described, eg, in US Patent No. 5,997,864.

The biological activity of factor VIIa in coagulation results from its (i) binding to tissue factor (TF) and (ii) catalyzing the proteolytic cleavage of factor IX or factor X to produce activated factor IX or X (factor IXa or Xa, respectively) Ability.

For the purposes of the present invention, the biological activity of Factor VII polypeptides can be quantified by measuring the ability of the preparation to promote coagulation using Factor VII-deficient plasma and thromboplastin as described, for example, in U.S. Patent No. 5,997,864 ("Factor VII Biological academic activity"). In this assay, biological activity is expressed as a reduction in clotting time relative to a control sample and is converted into "Factor VII units" by comparison with a pooled human blood standard containing 1 unit/ml of Factor VII activity. Additionally, the biological activity of Factor Vila can be quantified as follows:

(i) measuring the ability of Factor Vila or Factor Vila-related polypeptides to produce activated Factor X (Factor Xa) in a system containing TF and Factor X embedded in a lipid membrane. (Persson et al., J. Biol. Chem. 272:19919-19924, 1997);

(ii) measuring the hydrolysis of Factor X in an aqueous system ("in vitro proteolysis test", see below);

(iii) measuring physical binding of Factor Vila or Factor Vila-related polypeptides to TF using a surface plasmon resonance-based instrument (Persson, FEBS Letts. 413:359-363, 1997); and

(iv) measuring the hydrolysis of the synthetic substrate by Factor Vila and/or Factor Vila-related polypeptides ("in vitro hydrolysis assay", see below); and

(v) Measurement of thrombin generation in a TF-independent in vitro system.

The term "Factor VII biological activity" or "Factor VII activity" is intended to include the ability to generate thrombin; the term also includes the ability to generate thrombin on the surface of activated platelets in the absence of tissue factor.

A Factor VIIa preparation which can be used according to the invention is NovoSeven(R) (Novo Nordisk A/S, Bagsvaerd, Denmark), without being limited thereto.

Factor XI polypeptide:

In practicing the present invention, any Factor XI polypeptide that is effective in preventing or treating bleeding may be used. It includes Factor XI polypeptides derived from blood or plasma, or recombinantly produced. In addition, platelets may contain structurally distinct forms of FXI (possibly 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" as used herein includes, but is not limited to, Factor XI, and Factor XI-related polypeptides. The term "factor XI" is intended to include, but is not limited to, those described in Sun, Y. & Gailani, D. (1996), J.Biol.Chem. Amino acid sequence polypeptides, as well as wild-type factor XI from other species, eg, bovine, porcine, canine, murine, and salmon factor XI. In some embodiments, the Factor XI polypeptide is wild-type human Factor XI as disclosed in, eg, Sun, Y. & Gailani, D. (1996), J. Biol. Chem. 271:29023-29028.

It also includes natural allelic variants of Factor XI that exist and occur from one individual to another. Additionally, the degree and location of glycosylation or other post-translational modifications may vary depending on the chosen host cell and the properties of the host cell environment. The term "Factor XI" is also intended to include Factor XI polypeptides in their uncleaved (zymogen) form, and those polypeptides which have been proteolytically processed to produce their respective biologically active forms, which may be designated Factor XIa.

"Factor XI-related polypeptides" include, but are not limited to, chemically modified relative to human Factor XI and/or contain one or more amino acid sequence changes relative to human Factor XI (i.e., Factor XI variants), and/or A Factor XI polypeptide comprising a truncated amino acid sequence (ie, a Factor XI fragment) relative to human Factor XI. The Factor XI-related polypeptide may exhibit different properties relative to human Factor XI, including stability, phospholipid binding, changes in specific activity, and the like.

The term "Factor XI-related polypeptide" is intended to include such polypeptides in their uncleaved (zymogen) form, and which have been proteolytically processed to produce their respective biologically active forms, and may be named "Factor XIa-related polypeptides" or Those polypeptides that are "activated factor XI-related polypeptides".

As used herein, a "factor XI-related polypeptide" includes, but is not limited to, a polypeptide that exhibits substantially the same or increased biological activity relative to wild-type human factor XI, and the biological activity of factor XI is relative to that of wild-type human factor XI. A polypeptide in which the activity of XI is substantially modified or reduced. These polypeptides include, but are not limited to, chemically modified Factor XI or Factor XIa and Factor XI variants that introduce specific amino acid sequence changes that modify or destroy the biological activity of the polypeptide.

It also includes polypeptides with slightly modified amino acid sequences, eg, polypeptides with a modified N-terminus including deletions or additions of N-terminal amino acids, and/or polypeptides that are chemically modified relative to human Factor XI.

Factor XI-related polypeptides, including variants of Factor XI, whether exhibiting substantially the same or better biological activity than wild-type Factor XI, or selected for exhibiting substantially modified or reduced biological activity relative to wild-type Factor XI Include, but are not limited to, polypeptides having an amino acid sequence that differs from the wild-type Factor XI sequence by insertion, deletion, or substitution of one or more amino acids.

Factor XI-related polypeptides, including variants, exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, when detected in the factor XI activity assays described herein, 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 wild type produced in the same cell type Those polypeptides that have a specific activity of Factor XI.

Factor XI-related polypeptides, including variants, having substantially the same or improved biological activity relative to wild-type Factor XI comprise exhibiting at least about 25% when detected in said one or more specific factor XI activity assays, 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 wild-type cells produced in the same cell type Those polypeptides that have specific biological activity of human factor XI. For the purposes of the present invention, the biological activity of Factor XI can be quantified as described later in this specification ("assay section").

Factor XI-related polypeptides, including variants, that have substantially reduced biological activity relative to wild-type Factor XI exhibit no less than about 25%, preferably Those polypeptides having no less than about 10%, more preferably no less than about 5%, and most preferably no less than about 1% of the specific activity of wild-type Factor XI produced in the same cell type.

Non-limiting examples of Factor XI polypeptides include plasma as described in, for example, Gailani & Broze (1993), Blood Coagul. Fibrinolysis, 4: 15-20, or Kerbiriou & Griffin (1979), J. Biol. Chem., 254: 12020-12207 Source of Human Factor XI.

In some embodiments, Factor XI is a Factor XI-related polypeptide, wherein the activity of said Factor XI polypeptide is comparable to that of native human Factor XI (wild-type Factor XI) when tested in the "FXI Chromogenic Assay" (see below). The ratio between the activities is at least about 1.25; in other embodiments, the ratio is at least about 2.0; in other embodiments, the ratio is at least about 4.0.

Factor XI-related polypeptides also include factor XI or fragments of factor XI-related polypeptides that retain their characteristic hemostasis-related activity. For example, the hemostatic-related activity of a Factor XI polypeptide can be measured using the Factor XI activity assay described in this specification.

In a preferred embodiment, the Factor XI is human plasma Factor XI or activated human plasma Factor XIa. In one embodiment, FXI is platelet factor XI. In another embodiment, FXI is produced recombinantly.

definition

Herein, the three-letter or one-letter codes for amino acids are used according to their conventional meanings as shown in Table 1. Unless clearly indicated, amino acids mentioned herein are L-amino acids. It will be understood that, for example, the first letter in K337 represents the amino acid naturally occurring in wild-type Factor VII at that position, and that, for example, [K337A]-FVIIa represents a FVII-variant in which the naturally occurring amino acid at the position indicated is represented by The amino acid indicated by the one-letter code K is substituted with the amino acid indicated by the one-letter code A.

Table 1: Abbreviations for Amino Acids: amino acid three letter code single letter code Glycine Gly G proline Pro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine met m cysteine Cys C Phenylalanine Phe f Tyrosine Tyr Y Tryptophan Trp W Histidine His h Lysine Lys K arginine Arg R Glutamine Gln Q Asparagine Asn N glutamic acid Glu E. aspartic acid Asp D.

The terms "Factor Vila" or "FVIIa" are used interchangeably. The term Factor Vila includes zymogen Factor VII (single-chain Factor VII). The terms "Factor XI" or "FXI" are used interchangeably. The terms "Factor VIII" or "FVIII" are used interchangeably. The term "Factor VIII" or "FVIII" includes activated Factor VIII (FVIIIa), variants and truncated forms that retain the characteristic FVIII-associated hemostatic activity; the term includes recombinantly produced FVIII and plasma-derived FVIII. Human FVIII and human recombinant FVIII are preferred.

As used herein, "subject with impaired thrombin generation" refers to a subject who is unable to generate a sufficient burst of thrombin on the surface of activated platelets and includes more than a fully functioning normal hemostatic system, including normal amounts and functions Subjects with clotting factors, platelets and fibrinogen (e.g., in collected normal human plasma) produced less thrombin, and include, but are not limited to, subjects deficient in Factor VIII Subjects with reduced platelet count or platelet function defects (e.g., subjects with thrombocytopenia or Grantsmann's platelet insufficiency or excessive bleeding); subjects with reduced levels of prothrombin, FX or FVII some subjects with reduced levels of clotting factors (eg, excessive bleeding due to trauma or extensive surgery); and subjects with decreased plasma concentrations of fibrinogen (eg, subjects with multiple transfusions).

"Thrombin generation level" or "normal thrombin generation" refers to the level of thrombin generation in a patient compared to the level in healthy subjects. This level is called the percent normal level. The terms are used interchangeably where appropriate.

The term "enhancement of the hemostatic system" refers to an increase in the ability to generate thrombin. The term "enhancing hemostasis" is intended to encompass the thrombin generation measured by a test sample containing a Factor VII or Factor VII-related polypeptide preparation and a Factor XI or Factor XI-related polypeptide preparation when measured in the same thrombin generation assay, Prolonged conditions relative to individual thrombin generation of a control sample containing only Factor VII or Factor VII-related polypeptide or Factor XI or Factor XI-related polypeptide, respectively. Thrombin generation can be determined as described in the Thrombin Generation Assay of this specification (see "Assay Section").

As used herein, "the only" agent or factor means that Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide together are the only hemostatic agent contained in the pharmaceutical composition or kit, or the active hemostatic agent , or a coagulation factor, or is the only hemostatic agent, or an active hemostatic agent, or a coagulation factor administered to a patient during a particular course of treatment, eg, during a particular bleeding episode. It is to be understood that such circumstances include those in which the amount or activity of other applicable hemostatic agents or coagulation factors is insufficient to appreciably affect one or more coagulation parameters.

Coagulation lysis time, coagulation strength, fibrin coagulation formation, and coagulation time are clinical parameters used to determine the status of a patient's hemostatic system. Blood samples are drawn from the patient at appropriate time intervals and one or more parameters are determined by methods such as thromboelastography, e.g., according to Meh et al., Blood Coagulation & Fibrinolysis 2001; 12:627-637; Vig et al., Hematology, Vol.6( 3), pages 205-213 (2001); Vig et al., Blood coagulation&fibrinolysis, Vol.12(7), pages 555-561 (2001) Oct; Glidden et al., Clinical and applied thrombosis/hemostasis, Vol.6(4 ), pp. 226-233 (2000) Oct; McKenzie et al., Cardiology, Vol.92(4), pp. 240-247 (1999) Apr; or Davis et al., Journal of the American Society of Nephrology, Vol.6(4 ), pp. 1250-1255 (1995).

The term "prolonged coagulation lysis time" is intended to encompass the coagulation lysis time measured for a test sample containing a Factor VII or Factor VII-related polypeptide preparation and a Factor XI or Factor XI-related polypeptide preparation when measured in the same coagulation lysis assay Individual coagulation lysis times are prolonged relative to control samples containing only Factor VII or Factor VII-related polypeptides or Factor XI or Factor XI-related polypeptides, respectively. Coagulation and lysis times can be determined as described above.

The term "enhanced coagulation intensity" is intended to include the coagulation intensity measured for a test sample containing a Factor VII or Factor VII-related polypeptide preparation and a Factor XI or Factor XI-related polypeptide preparation when measured in the same assay for coagulation intensity, e.g. mechanical The intensity is enhanced relative to the individual coagulation intensity of a control sample containing only Factor VII or Factor VII-related polypeptide or Factor XI or Factor XI-related polypeptide, respectively. Coagulation strength can be as described, for example, by Carr et al., 1991. (Carr ME, Zekert SL, Platelet-mediated strength formation during plasma coagulation formation, AM J MED SCI 1991; 302:13-8), or by coagulation elasticity as described above. Tracing assay.

The term "enhancing fibrin coagulation formation" is intended to include fibrin measured for test samples containing a Factor VII or Factor VII-related polypeptide preparation and a Factor XI or Factor XI-related polypeptide preparation when measured in the same coagulation assay An increase in the rate or extent of clot formation relative to the rate or extent of single fibrin clot formation in a control sample containing only Factor VII or Factor VII-related polypeptide or Factor XI or Factor XI-related polypeptide, respectively. Fibrin clot formation can be determined as described above.

The term "reduced clotting time" is intended to include the coagulation time (clotting time) measured for test samples containing Factor VII or Factor VII-related polypeptide preparations and Factor XI or Factor XI-related polypeptide preparations when measured in the same coagulation assay. ) relative to the individual coagulation time shortening (note: the original text is "increase", may be wrong) relative to the control samples containing only factor VII or factor VII-related polypeptides or factor XI or factor XI-related polypeptides respectively. Clotting times can be determined by means of standard PT or aPTT tests known to those of ordinary skill.

The term "reduced platelet count or activity" refers to the number of platelets (thrombocytes) present in the plasma of a subject and the biological, coagulation-related activity of such platelets. Decreased counts may be due to, for example, increased platelet destruction, decreased platelet production, and greater aggregation than the normal fraction of platelets in the spleen. For example, thrombocytopenia is defined as a platelet count of no less than 150,000 platelets per microliter; the upper limit of normal platelet counts is generally considered to be between 350,000 and 450,000 platelets per microliter. Platelet counts can be measured by an automated platelet counter; it is a method well known to those skilled in the art. Syndromes caused by low platelet counts include, but are not limited to thrombocytopenia, coagulopathy. "Activity" includes, but is not limited to, platelet aggregation, adhesion, and coagulation activity. Decreased activity may be due to, for example, abnormalities in glycoproteins, abnormalities in membrane-cytoskeleton interactions, abnormalities in platelet granules, abnormalities in platelet coagulation activity, and abnormalities in signal transduction and secretion. Platelet activity, including aggregation, adhesion and coagulation activity, is measured by standard methods known to the skilled person, see, for example, Platelets. A Practical Approach, Ed. S. P. Watson & K. S. Authi: Clinical Aspects of Platelet Disorders (K.J. Clemetson) 15: 299-318, 1996, Oxford University Press; Williams Hematology, Sixth Edition, Eds. Butler, Lichtman, Coller, Kipps & Seligsohn, 2001, McGraw-Hill. Syndromes caused by decreased platelet activity include, but are not limited to, Grantsmann's thrombocytopenia, Bert-Sueh syndrome, anticoagulant therapy, and thrombolytic therapy. "Reduced" refers to the count or activity of a test plasma sample compared to the count or activity in a normally collected plasma sample when measured in the same assay.

The term "bleeding disorder" as used herein refers to any defect of cellular or molecular origin, congenital, acquired or induced, occurring in bleeding episodes. Examples of bleeding disorders include, but are not limited to, deficiencies of coagulation factors (eg, deficiencies of coagulation factors VIII, IX, XI, or VII), inhibitors of coagulation factors, defective platelet function (eg, Glanzmann's thrombocytopenia and primary- Sue syndrome), thrombocytopenia, von Willebrand disease, and conditions such as dilution by coagulation proteins, increased fibrinolysis, and decreased platelet numbers due to bleeding and/or transfusion (eg, in multiple Coagulopathy caused by blood transfusion in subjects.

Hemorrhage is the spilling of blood from any component of the circulatory system. The term "bleeding episode" is meant to include unwanted, uncontrolled, and often excessive bleeding associated with surgery, trauma, or other forms of tissue damage, and in subjects with a bleeding disorder that does not Bleeding needed. Bleeding episodes can occur in subjects with an essentially normal coagulation system but experiencing (transient) coagulopathy, as well as in subjects with congenital or acquired coagulation or bleeding disorders. In subjects with defective platelet function, bleeding resembles that caused by hemophilia because, as in hemophilia, the hemostatic system lacks or has abnormal essential coagulation "compounds" (eg, platelets or von . Willebrand factor protein). In subjects experiencing extensive tissue damage, such as associated with surgery or severe trauma, the requirement for immediate hemostasis may exceed the normal hemostatic mechanisms and they are independent regardless of the presence or absence of substantially (pre-trauma or pre-operative) normal hemostatic mechanisms. Excessive bleeding may form. The subject typically also received multiple blood transfusions and developed a (transient) coagulopathy due to bleeding and/or transfusion (ie, increased fibrinolysis and decreased platelet number due to bleeding and/or transfusion dilution of coagulation proteins). Bleeding may also occur in organs such as the brain, inner ear region and eyes, which are areas where the possibility of surgical hemostasis is limited and therefore achieving satisfactory hemostasis is problematic. The same problem arises during biopsy from various organs (liver, lung, tumor tissue, gastrointestinal tract) and during laparoscopic surgery and free retropubic prostatectomy. Common to all of these conditions is difficulty in hemostasis with surgical techniques (sutures, hemostats, etc.), which is also the case when the bleeding is diffuse (eg, hemorrhagic gastritis and excessive uterine bleeding). Hemorrhages may also occur in anticoagulant-treated subjects; where hemostasis defects are induced by the provided therapy; these hemorrhages are usually acute and excessive. Anticoagulant therapy is commonly used to prevent thromboembolic disease. The therapy may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists as well as aspirin and other inhibitors of platelet aggregation, eg, antibodies or other inhibitors of GP IIb/IIIa activity. Bleeding can also be due to so-called lytic agents involving combination therapy with antiplatelet agents (e.g., acetylsalicylic acid), anticoagulants (e.g., heparin), and fibrinolytic agents (e.g., tissue plasminogen activator, tPA). Caused by thrombotherapy. Bleeding episodes are also meant to include, but are not limited to, in subjects with acute hemorrhagic arthropathy (joint bleeding), chronic hemophilic arthropathy, hematoma, (e.g., muscle, retroperitoneal, sublingual, and retropharyngeal) with Uncontrolled and excessive bleeding associated with surgery or trauma, bleeding in other tissues, hematuria (renal tract bleeding), cerebral hemorrhage, surgery (eg, liver resection), tooth extraction, and gastrointestinal bleeding (eg, , UGI bleeding). Bleeding episodes can be associated with inhibitors against factor VIII; hemophilia A; hemophilia A with inhibitors; hemophilia B; factor VII deficiency; factor XI deficiency; thrombocytopenia; von Willebrand factor Deficiency (von Willebrand disease); severe tissue damage; severe trauma; surgery; laparoscopic surgery; hemorrhagic gastritis; biopsy performed; anticoagulant therapy; associated with stem cell transplantation. Bleeding episodes can be excessive uterine bleeding; occur in organs where mechanical hemostasis is limited; occur in the brain; occur in the inner ear region; or occur in the eye. The terms "bleeding episode" and "bleeding" are used interchangeably as appropriate.

As used herein, the term "treating" is meant to include arresting anticipated bleeding (eg, bleeding in surgery) and regulating bleeding that has occurred (eg, bleeding in trauma) in order to inhibit or minimize bleeding. The "anticipated bleeding" mentioned above may be bleeding expected to occur in a specific tissue or organ, or may be non-specific bleeding. The prophylactic administration of Factor VII or Factor VII-related polypeptide preparations and Factor XI or Factor XI-related polypeptide preparations is thus included in the term "treatment".

The term "subject" as used herein refers to any animal, especially a mammal, such as a human, and is used interchangeably with the term "patient" if appropriate.

The factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide defined in this specification can be administered simultaneously or sequentially. The factor may be presented as a single dosage form, wherein a single dosage form contains both coagulation factors, or as a preparation containing Factor VII or a Factor VII-related polypeptide as the first unit dosage form and a preparation of Factor XI or a Factor XI-related polypeptide as a The second unit dosage form is provided as an assembled kit. Whenever a first or second or third, etc., unit dose is mentioned in this specification, it does not imply a preferred order of administration, but is merely for descriptive convenience.

Administration of Factor VII or a preparation of Factor VII-related polypeptide and a preparation of Factor XI or Factor XI-related polypeptide "simultaneously" means administration of a coagulation factor protein in a single dose, or administration of a first coagulation factor protein followed by a dose not exceeding 15 Minutes, preferably 10, more preferably 5, more preferably 2 minutes intervals of administering the second coagulation factor protein. Either factor may be administered first.

"Sequential" administration refers to administering the first coagulation factor protein first, followed by up to 2 hours, preferably 1 to 2 hours, more preferably up to 1 hour, more preferably 30 minutes to 1 hour, more preferably up to 30 minutes, more preferably Preferably the second coagulation factor protein is administered at intervals of 15 to 30 minutes. Either of the two unit dosage forms or the clotting factor protein may be administered first. Preferably, both products are injected by the same intravenous route.

"Level of Factor XI" or "Factor XI level" refers to the level of coagulation Factor XI activity in a patient compared to the level of a healthy subject. This level is called the percent of normal. The terms are used interchangeably as appropriate.

"Reduced level of factor XI" or "reduced factor XI level" means the factor present in the bloodstream as compared to the mean level of factor XI in a population of subjects without coagulation factor XI deficiency or inhibitors against coagulation factor XI XI or a reduction in its activity. Levels of circulating factor XI can be measured by either coagulation or immunological assays. The procoagulant activity of Factor XI is measured by the ability of patient plasma to correct the clotting time of Factor XI-deficient plasma (eg, APTT test, see below; see also "Assays Section" of this specification).

One unit of Factor XI is defined as the amount of Factor XI present in 1 ml of normal (collected) human plasma (corresponding to 100% of the Factor XI level).

One unit of Factor VII is defined as the amount of Factor VII present in 1 ml of normal plasma, corresponding to approximately 0.5 μg of protein. After activation, 50 units correspond to approximately 1 μg protein.

"Deficiency" refers to a decreased presence or activity of, for example, Factor XI in plasma compared to normal healthy individuals. The term is used interchangeably with "decreased factor XI level" as appropriate.

"APTT" or "aPTT" refers to activated partial thromboplastin time (e.g., Proctor RR, Rapaport SI: Partial thromboplastin time with kaolin; simple screen for first-stage plasma coagulation factor deficiencies Trial. Am J Clin Pathol 36:212, 1961).

"Factor XI responsive syndrome" refers to a syndrome in which administration of exogenous Factor XI to a subject in need thereof prevents, cures or ameliorates any symptoms, conditions or diseases expected or present that result from the syndrome. Including, but not limited to, syndromes caused by reduced levels of Factor XI, eg, bleeding disorders caused by inhibitors of Factor XI. Factor XI response syndrome can also be treated with compositions according to the invention.

"Factor VII response syndrome" means that administration of exogenous Factor VII, preferably Factor VIIa, to a subject in need thereof prevents, cures or ameliorates any symptoms, conditions or diseases expected or present that result from the syndrome syndrome. Including, but not limited to, syndromes caused by decreased levels of coagulation factors VIII, IX, XI, or VII, coagulation factor inhibitors, defective platelet function (eg, Grantsmann's thrombocytopenia and Bert-Sue syndrome), Thrombocytopenia, von Willebrand disease, and symptoms such as dilution by coagulation proteins, increased fibrinolysis, and decreased platelet count due to bleeding and/or transfusion (e.g., in subjects undergoing multiple transfusions from surgery or trauma coagulopathy caused by).

"Half-life" refers to the time required for the plasma concentration of Factor VII or Factor VII-related polypeptide or Factor XI or Factor XI-related polypeptide to decrease from a specified value to half of that value.

"Primary hemostasis" refers to the initial generation of thrombin by FXa and TF:Factor VIIa, followed by activation of platelets and activated, attached platelets to form a preliminary loose thrombus, which has not yet been stabilized by fibrin and eventually cross-linked fibrils Protein is firm. The thrombus is readily dissolved by the fibrinolytic system if not stabilized (maintains hemostasis) by the fibrin formed during the second step of the hemostatic process.

"Secondary hemostasis" or "maintained hemostasis" refers to the secondary, complete, and primary burst or generation of thrombin that occurs on the surface of activated platelets and is catalyzed by Factor VIIa and Factor VIIIa, followed by formation of fibrin and stabilization Preliminary platelet thrombus. Fibrin stabilizes the thrombus leading to complete hemostasis.

"Complete hemostasis" refers to the formation of a stable and firm fibrin clot or thrombus at the site of injury that effectively stops bleeding and is not easily dissolved by the fibrinolytic system. In this specification, the term hemostasis may be used to mean complete hemostasis as described above.

The total amount of protein in a preparation can be measured by known methods, for example, by measuring optical density. The amount of coagulation Factor XI or Factor VII protein ("antigen") can be measured by well known methods such as standard ELISA immunoassays. Generally, the assay is carried out by contacting, for example, a solution containing a Factor XI protein preparation with an anti-FXI antibody immobilized on an ELISA plate, followed by contacting the immobilized antibody-Factor XI complex with a second labeled anti-FXI antibody, This is done in a third step by measuring the amount of this label. The amount of each coagulation factor can be measured in a similar manner using appropriate antibodies. The total amount of clotting factor proteins present in the preparation can be determined by adding the amount of each clotting factor protein. In one embodiment, the preparation comprises isolated coagulation factors. In another embodiment, the preparation is free of Factor II and Factor Ila (prothrombin and thrombin) and/or Factor X or Xa.

As used herein, the term "isolated" refers to coagulation factors such as Factor XI or Factor XI-related polypeptides that are isolated from the cells in which they are synthesized or from the medium in which they are found naturally (eg, plasma or blood). Separation of cells from their source is accomplished by any method known in the art, including, but not limited to, removal of cell culture medium containing the desired product from adherent cell culture; centrifugation or filtration to remove non-adherent cells; etc. peptide. By any method known in the art, including, but not limited to, such as affinity chromatography on anti-Factor VII or anti-Factor XI antibody columns, respectively; hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography ; Electrophoretic methods (eg, preparative isoelectric focusing (IEF)), differential dissolution (eg, ammonium sulfate precipitation), or extraction can be accomplished to separate polypeptides from their naturally occurring medium.

The term "TFPI inhibitor" refers to a compound that inhibits the anticoagulant 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) are used interchangeably.

The "effective amount" of the factor VII polypeptide and the factor XI polypeptide in the present invention is defined as the factor VII polypeptide, such as FVIIa, and the factor XI polypeptide together are sufficient to prevent or reduce bleeding or blood loss, and are sufficient to cure, alleviate or partially control the disease and its complications amount.

The term "Factor Vila activity" 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.

The compositions according to the invention also comprise TFPI-inhibitors. The composition according to the invention also comprises Factor VIII. The composition is preferably administered to a subject who does not have an inhibitor against Factor VIII.

abbreviation

TF tissue factor

FVII in its single-chain, unactivated form of Factor VII

FVIIa in its activated form Factor VII

rFVIIa Recombinant Factor VII in its activated form

FXI Factor XI in its zymogen, inactive form

FXIa Factor XI in its activated form

rFXI Recombinant FXI

rFXIa Recombinant FXIa

FVIII as its zymogen, inactive form of factor VIII

rFVIII Recombinant FVIII

FVIIIa Factor VIII in its activated form

rFVIIIa Recombinant FVIIIa

TFPI tissue factor pathway inhibitor

Compound preparation:

The human purified Factor VIIa suitable for use in the present invention is preferably obtained by recombinant DNA technology, such as described in Hagen et al., Proc . ) described in the preparation.

Factor VII can also be produced as described by Broze and Majerus, J. Biol. Chem. 255 (4):1242-1247, 1980 and Hedner and Kisiel, J. Clin. Invest . These methods yield Factor VII without detectable amounts of other coagulation factors. Even more purified Factor VII preparations can be obtained by including an additional gel filtration as a final purification step. Factor VII can then be converted to activated factor FVIIa by known methods, for example by several different plasma proteins, eg Factor XIIa, IX or Xa. Alternatively, Factor VII can be activated by passing it through an ion exchange column such as Mono Q(R) (Pharmacia fine Chemicals) as described by Bjoern et al. (Research Disclosure, 269 , September 1986, pp. 564-565).

Factor VII-related polypeptides can be produced by modifying wild-type Factor VII or by recombinant techniques. Factor VII-related polypeptides having an altered amino acid sequence compared to wild-type Factor VII can be modified by known methods, such as by site-directed mutagenesis to change the amino acid codons in the nucleic acid encoding native Factor VII or to remove some amino acid codons to modify the coding sequence. The nucleic acid sequence of wild-type factor VII was used for production.

It will be apparent to those skilled in the art that substitutions can be made outside of functionally critical regions of the Factor Vila or Factor XI molecule and still result in an active polypeptide. Substitutions essential for the activity of Factor VII or Factor VII-related polypeptides or Factor XI or Factor XI-related polypeptides, and thus are preferably not made, can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue of the molecule, and the resulting mutant molecules are tested for coagulation, cross-linking activity, respectively, to identify amino acid residues critical to the activity of the molecule. Substrate-enzyme interaction sites can also be determined by analysis of three-dimensional structures determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, Science 255:306-312 ; Smith et al., 1992, Journal of Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

Introduction of mutations into a nucleic acid sequence such that one nucleotide is replaced by another can be accomplished by site-directed mutagenesis using any method known in the art. Particularly useful is the method utilizing a supercoiled double-stranded DNA vector containing the insert of interest and two synthetic primers containing the desired mutation. Oligonucleotide primers, respectively complementary to opposite strands of the vector, are extended during temperature cycling with the aid of Pfu DNA polymerase. When incorporated, this primer produces a mutant plasmid containing staggered gaps. After temperature cycling, the product is treated with DpnI specific for methylated and hemimethylated DNA to digest the parental DNA template and select for synthetic DNA containing mutations. Other methods known in the art for generating, identifying and isolating variants, eg, gene shuffling or phage display techniques, can also be used.

Isolation from its cellular source can be accomplished by any method known in the art, including, but not limited to, removal of cell culture medium containing the desired product from adherent cell culture; centrifugation or filtration to remove non-adherent cells; etc. peptide.

Optionally, Factor VII or Factor VII-related polypeptides can be further purified. Using any method known in the art, including, but not limited to, such as affinity chromatography on an anti-Factor VII antibody column (see, e.g., Wakabayashi et al., J. Biol. Chem. 261: 11097, 1986; and Thim et al., Biochem.27:7785,1988); hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography; electrophoretic methods (for example, preparative isoelectric focusing (IEF)), differential dissolution (for example, sulfuric acid Ammonium precipitation), or extraction, etc. can complete the purification. In general, see Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, J.C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. After purification, the preparation preferably contains less than about 10%, more preferably less than about 5%, and most preferably less than about 1% by weight of non-Factor VII or Factor VII-related polypeptides from the host cell.

Factor VII or Factor VII-related polypeptides can be activated by proteolytic cleavage using Factor XIIa or other proteases with trypsin-like specificity, eg, Factor IXa, kallikrein, Factor Xa, and thrombin. See, eg, Osterud et al., Biochem. 11:2853 (1972); Thomas, US Patent No. 4,456,591; and Hedner et al., J. Clin. Invest. 71:1836 (1983). Alternatively, Factor VII or Factor VII-related polypeptides can be activated by passing them over ion-exchange chromatography columns such as Mono Q(R) (Pharmacia). The resulting activated Factor VII or Factor VII-related polypeptides can then be formulated and administered as described below.

Factor XI used in the present invention can be used according to known methods, such as Koide et al. (Biochemistry 16:2279-2286, 1977) and Bouma et al. (J.Biol.Chem.252:6432-6437, 1977) cited herein as references. Those methods disclosed are prepared from blood plasma. However, the use of recombinant factor XI is preferred to avoid the use of blood or tissue derived products which carry the risk of disease transmission. Methods of making recombinant Factor XI are known in the art. See, e.g., 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) , cited herein in its entirety.

Factor XI-related polypeptides can be produced by modifying wild-type Factor XI or by recombinant techniques. Factor XI-related polypeptides having an altered amino acid sequence compared to wild-type Factor XI may be altered or removed by known methods, such as by site-directed mutagenesis, in nucleic acids encoding native Factor XI, as described in more detail above. Some amino acid codons were modified to produce the nucleic acid sequence encoding wild-type Factor XI. Isolation from its cellular source can be accomplished by any method known in the art, including, but not limited to, removal of cell culture medium containing the desired product from adherent cell culture; centrifugation or filtration to remove non-adherent cells; etc. peptide. Optionally, Factor XI or Factor XI-related polypeptides can be further purified. Any method known in the art may be used as described in more detail above, including, but not limited to, such as affinity chromatography on an anti-Factor XI antibody column; hydrophobic interaction chromatography; ion exchange chromatography; size sorting Purification is accomplished by resistance chromatography; electrophoretic methods (eg, preparative isoelectric focusing (IEF)), differential dissolution (eg, ammonium sulfate precipitation), or extraction. After purification, the preparation preferably contains less than about 10%, more preferably less than about 5%, and most preferably less than about 1% by weight of non-Factor XI or Factor XI-related polypeptides from the host cell. The resulting activated Factor XI or Factor XI-related polypeptides can then be formulated and administered as described below.

Those skilled in the art will appreciate that it is preferable to use Factor XI and Factor Vila proteins that are homologous to the subject to reduce the risk of inducing an immune response. The preparation and characterization of non-human factor XI has been disclosed, for example, by Gailani (Blood 90(3):1055-1064, 1997). The invention also includes the use of the Factor XI and Factor Vila proteins in veterinary methods.

Pharmaceutical compositions and methods of use

The articles of manufacture of the present invention may be used to treat any factor VII responsive syndrome, such as a bleeding disorder, including, but not limited to, syndromes caused by reduced levels of coagulation factors VIII, IX, XI or VII, coagulation factor inhibitors, defective platelet function ( eg, Grantsmann's thrombocytopenia and Bert-Sau syndrome), thrombocytopenia, von Willebrand disease, and disorders such as dilution by coagulation proteins, increased fibrinolysis, and platelets due to bleeding and/or transfusion Coagulopathy due to reduced numbers (eg, in subjects undergoing multiple transfusions from surgery or trauma). The pharmaceutical composition containing Factor VII or Factor VII-related polypeptide preparation and Factor XI or Factor XI-related polypeptide preparation according to the present invention is mainly used for parenteral administration for preventive and/or therapeutic treatment. Preferably, the pharmaceutical composition is administered parenterally, ie intravenously, subcutaneously, or intramuscularly; most preferably intravenously. They can also be administered by continuous or pulsatile infusion.

The pharmaceutical composition or formulation according to the invention comprises Factor VII or Factor VII, preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent, formulated in single unit dosage form or in the form of a kit of parts. VII-related polypeptides, and factor XI or factor XI-related polypeptides. Briefly, a pharmaceutical composition suitable for use according to the invention may be obtained by combining Factor Vila, or Factor XI, or a combination of Factor Vila and Factor XI, preferably in purified form, with a suitable adjuvant and a suitable carrier or Diluents are mixed to prepare. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, etc. Preparations of the invention may also be formulated using non-aqueous vehicles, for example, in the form of gels or as liposomal preparations for administration or targeting to the site of injury. Liposome preparations are generally as described, for example, in US Patent Nos. 4,837,028, 4,501,728, and 4,975,282. The composition can be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solution is packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized product is mixed with a sterile aqueous solution before administration.

The composition may contain pharmaceutically acceptable auxiliary substances or adjuvants, including, but not limited to, pH adjusting and buffering agents and/or tonicity adjusting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

Formulations may also include one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in forms such as liquids, powders, emulsions, sustained release formulations, and the like. Compositions of the present invention can be formulated by those skilled in the art in a suitable manner and according to accepted practice such as disclosed in Remington's Pharmaceutical Sciences . Gennaro, ed., Mack Publishing Co., Easton, PA, 1990 . Thus, a typical pharmaceutical composition for intravenous infusion may be formulated to contain 250 ml of sterile Ringer's solution and 10 mg of the preparation.

Compositions containing the preparations of the invention may be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, a subject already suffering from a disease as described above is administered an amount of the composition sufficient to cure, alleviate or partially arrest the clinical manifestations of the disease and its complications. An amount sufficient to accomplish this is defined as a "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 appreciated that determining an appropriate dosage can be accomplished using routine experimentation by constructing a matrix of values and testing different points in the matrix.

Topical administration of the articles of the invention, for example, topical application can be by, for example, by means of spraying, infusion, double balloon catheters, stents, insertion of vascular grafts or stents, hydrogels for covering balloon catheters, or other well-defined method. In either case, the pharmaceutical composition should provide an amount of preparation sufficient to effectively treat the condition.

The concentration of Factor VII or Factor VII-related polypeptides, Factor XI or Factor XI-related polypeptides, or combinations of Factor VII or Factor VII-related polypeptides and Factor XI or Factor XI-related polypeptides in these formulations can be within a wide range Variations, ie from less than about 0.5% by weight, usually or at least about 1% by weight, up to as much as 15 or 20% by weight are selected primarily by fluid volume, viscosity, etc., depending on the particular mode of administration selected. Administration is preferably by injection or infusion, especially injection. Accordingly, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are prepared in a form suitable for intravenous administration, for example, the preparation may contain both Factor VII or Factor VII-related polypeptide in one dosage form A dissolved lyophilized powder or liquid formulation of the polypeptide and Factor XI or Factor XI-related polypeptide, or a dissolved lyophilized powder or liquid formulation containing Factor VII or Factor VII-related polypeptide in one dosage form and in another Dosage forms contain dissolved lyophilized powder or liquid formulations of Factor XI or Factor XI-related polypeptides.

It will be understood that the amount of Factor VII or Factor VII-related polypeptide and the amount of Factor XI or Factor XI-related polypeptide together comprise a combined effective amount for treating a bleeding episode.

It must be borne in mind that the substances of the invention are generally useful in serious disease or injury states, ie in life-threatening or potentially life-threatening situations. In this case, given the minimization of exogenous material and the general lack of immunogenicity of human Factor Vila and Factor XI, the attending physician may and believes it necessary to administer a substantial excess of this composition.

In prophylactic applications, a composition comprising a Factor VII or Factor VII-related polypeptide preparation and a Factor XI or Factor XI-related polypeptide preparation can be administered to a subject susceptible to or at risk of a disease state or injury to enhance the immune response. The subject's own blood coagulation ability. This amount is defined as a "prophylactically effective amount". It will be understood that the amount of Factor VII or Factor VII-related polypeptide and the amount of Factor XI or Factor XI-related polypeptide together comprise a combined effective amount to prevent a bleeding episode.

Single or multiple administrations of the composition can be carried out at dosage levels and in a manner selected by the attending physician. The composition can be administered one or more times daily or weekly. An effective amount of the pharmaceutical composition is an amount that provides a clinically significant effect on bleeding episodes. The amount will depend in part on the particular condition being treated, the age, weight, and general health of the subject, as well as other factors apparent to those skilled in the art.

Compositions of the invention are generally administered in a single dose just before bleeding is expected or at the onset of bleeding. However, repeated doses (as multiple doses) may also be given, preferably at intervals of 2-4-6-12 hours, depending on the dose provided and the condition of the subject.

In therapy associated with the preparation of intervention, Factor VII or Factor VII-related polypeptide and Factor XI or Factor XI-related polypeptide are generally administered within about 24 hours prior to the intervention and for up to 7 days or longer thereafter. Administration as an anticoagulant can be by various routes as described herein.

The composition may be in the form of a single preparation (single dosage form) containing both a preparation of Factor VII or a Factor VII-related polypeptide and a preparation of Factor XI or a Factor XI-related polypeptide at appropriate concentrations. The composition may also be in the form of an assembly kit consisting of a first unit dosage form comprising a Factor VII or Factor VII-related polypeptide preparation and a second unit dosage form comprising a Factor XI or Factor XI-related polypeptide preparation. In this case, the Factor VII or Factor VII-related polypeptide and the Factor XI or Factor XI-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 Within 5 minutes, or more preferably within 2 minutes of each other. Either of the two unit dosage forms may be administered first.

The kit comprises at least two separate pharmaceutical compositions. The kit includes container means, such as a divided bottle or a divided foil pack, containing the divided compositions. Generally, the kit will include instructions for the administration of the separate components. This kit form is particularly advantageous when the separate components are preferably administered in different dosage forms, at different dosing intervals, or when titrations of the individual components of the combination are desired by the attending physician.

The amount of Factor VII or Factor VII-related polypeptide and the amount of Factor XI or Factor XI-related polypeptide administered according to the invention may vary in a ratio of between about 1:100 to about 100:1 (w/w). Thus the ratio of Factor VII to Factor XI may be, for example, 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 and about 1:1 , or between about 1:80 and about 1:2, or between about 1:70 and about 1:5, or between about 1:60 and about 1:10, or between about 1:50 and between about 1:25, or between about 1:40 and about 1:30, or between about 90:1 and about 1:1, or between about 80:1 and about 2:1, or Between about 70:1 and about 5:1, or between about 60:1 and about 10:1, or between about 50:1 and about 25:1, or between about 40:1 and about 30 : between 1.

Doses of Factor VII or Factor VII-related polypeptides may range from about 0.05 mg to about 500 mg/day for a 70-kg subject as loading and maintenance doses, depending on the subject's weight, condition and severity of symptoms The dosage of wild-type factor VII of the present invention varies, for example, from about 1 mg to about 200 mg/day, or, for example, from about 5 mg to about 175 mg/day.

Doses of Factor XI or Factor XI-related polypeptides may range from about 0.05 mg to about 500 mg/day for a 70-kg subject as loading and maintenance doses, depending on the subject's weight, condition and severity of symptoms The dose of wild-type factor XI of the present invention varies, for example, from about 1 mg to about 200 mg/day, or, for example, from about 1 mg to about 175 mg/day.

The combination of factor VIIa and factor XI showed a synergistic effect in in vitro coagulation hardness and fibrinolysis time assays. Furthermore, the combination of factor VIIa and factor XI exhibited synergistic effects in forming stable fibrin coagulation, prolonging hemicoagulation lysis time, increasing coagulation strength and enhancing resistance to fibrinolysis.

The composition may be in the form of a single preparation containing both Factor Vila and Factor XI at appropriate concentrations. The composition may also be a reagent consisting of a first unit dosage form comprising Factor Vila and a second unit dosage form comprising Factor XI and optionally one or more other unit dosage forms comprising Factor VIII and/or a TFPI inhibitor box form. In this case, Factor Vila and 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 preferably within 5 minutes of each other . Either of the two unit dosage forms may be administered first.

Since the present invention relates to the prophylaxis or treatment of bleeding episodes or for the treatment of blood coagulation by combined treatment with active ingredients which can be administered separately, the present invention also relates to combining separate pharmaceutical compositions in the form of a kit. The kit comprises at least two separate pharmaceutical compositions. The kit includes container means, such as a divided bottle or a divided foil pack, containing the divided compositions. Generally, the kit will include instructions for the administration of the separate components. This kit form is particularly advantageous when the separate components are preferably administered in different dosage forms, at different dosing intervals, or when titrations of the individual components of the combination are desired by the attending physician.

test:

Detection of Factor VIIa activity:

Suitable assays to detect Factor VIIa activity in in vitro assays with simple pilot tests to select appropriate Factor VIIa variants:

In vitro hydrolysis test

The specific activity of native (wild-type) Factor Vila and Factor Vila variants (both hereinafter referred to as "Factor Vila") can be determined. They can also be assayed in parallel to directly compare their specific activities. The assay was performed on microtiter plates (MaxiSorp, Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden) was added at a final concentration of 1 mM in 50 mM Hepes containing 0.1 M NaCl, 5 _mM CaCl and 1 mg/ml bovine serum albumin. , in Factor Vila (100 nM final concentration) at pH 7.4. Absorbance at 405 nm was measured continuously on a SpectraMax ^™ 340 plate reader (Molecular Devices, USA). Absorbance in blank wells without enzyme was subtracted from the absorbance developed during the 20 min incubation to calculate the ratio between variant and wild-type Factor VIIa activity:

Ratio = (A _405nm Factor Vila variant)/(A _405nm Factor Vila wild type).

On this basis, factor VIIa variants are identified that have an activity equivalent to or higher than that of native factor VIIa, for example, the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is about, or higher than 1.0 variant.

The activity of Factor Vila or Factor Vila variants can also be measured using a physiological substrate such as Factor X at a suitable concentration of 100-1000 nM, wherein the factor produced is measured after addition of a suitable chromogenic substrate (e.g., S-2765) Xa. Alternatively, activity assays can be performed at physiological temperatures.

In vitro proteolysis test

Native (wild-type) Factor Vila and Factor Vila variants (both hereinafter referred to as "Factor Vila") were assayed in parallel to directly compare their specific activities. The assay was performed on microtiter plates (MaxiSorp, Nunc, Denmark). Factor Vila (10 nM) and Factor X (0.8 micromolar) were incubated for 15 minutes in 100 microliters of 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl ₂ and 1 mg/ml bovine serum albumin. Cleavage of Factor X was then terminated by adding 50 microliters of 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 produced was measured by adding the chromogenic substrate ZD-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden) at a final concentration of 0.5 mM. Absorbance at 405 nm was measured continuously on a SpectraMax ^™ 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes minus the absorbance in blank wells without FVIIa was used to calculate the ratio between the proteolytic activity of the variant and wild-type Factor VIIa:

Ratio = (A405nm Factor Vila variant)/(A405nm Factor Vila wild type).

On this basis, factor VIIa variants are identified that have an activity equivalent to or higher than that of native factor VIIa, for example, the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is about, or higher than 1.0 variant.

Thrombin generation test:

The ability of Factor VII or Factor VII-related polypeptides or Factor XI or Factor XI-related polypeptides (e.g., variants) to generate thrombin can be measured in assays involving all relevant coagulation factors and inhibitors at physiological concentrations and activated platelets ( As described on page 543 of Monroe et al. (1997) Brit. J. Haematol. 99, 542-547, incorporated herein by reference).

Detection of Factor XI activity:

A suitable assay to detect the amide of Factor XI can be simply performed in an in vitro assay using, for example, a chromogenic substrate as described by Gailani et al. Disaggregate activity to select appropriate Factor XI variants.

Factor XI biological activity assays are also readily performed in in vitro assays measuring the activation of Factor IX to IXa as described, for example, by Gailani et al. (Blood 97(10):3117-3122, 2001).

Detection of Factor VIII activity:

By eg, Kirkwood TBL, Rizza CR, Snape TJ, Rhymes IL, Austen DEG. Identifying sources of interlaboratory variability in factor VIII assays. B J Haematol 1981; 37; 559-68; or Kessels et al., British Journal of Haematology, Vol. 76 (Suppl. 1) pp. 16 (1990) A suitable assay can simply be performed to detect the factor in an in vitro assay. VIII activity, thereby providing a means of selecting suitable Factor VIII variants for use in the present invention. Factor VIII activity can also be measured by a two-step chromogenic assay based on the amidolytic activity of FXa produced (Wagenvoord et al., 1989, Haemostasis, 19(4):196-204).

By measuring the product correction factor as described e.g. in Nilsson et al., 1959. (Nilsson IM, Blombaeck M, Thilen A, von Francken I., Carriers of Hemophilia A - Laboratory Studies, Acta Med Scan 1959; 165:357) The ability to clotting time of VIII-deficient plasma can also quantify the biological activity of Factor VIII. In this assay, biological activity is expressed as units/ml of plasma (1 unit corresponds to the amount of FVIII present in normally collected plasma).

Aspects of the invention:

Aspect 1: A pharmaceutical composition comprising Factor VII or a Factor VII-related polypeptide, and Factor XI or a Factor XI-related polypeptide.

Embodiment 2: The composition of aspect 1, wherein said Factor VII or Factor VII-related polypeptide is a Factor VII-related polypeptide.

Embodiment 3: The composition of Aspect 1, wherein Factor Vila is human Factor Vila.

Embodiment 4: The composition of Aspect 1 or Aspect 3, wherein Factor Vila and Factor XI are recombinant human Factor Vila and recombinant human Factor XI.

Embodiment 5: The composition of any of aspects 1-4, wherein Factor XI is platelet Factor XI.

Embodiment 6: The composition of any of Aspects 1-5, wherein Factor XI is activated Factor XI.

Embodiment 7: The composition of any of Aspects 1-6, wherein the composition further comprises a TFPI inhibitor.

Embodiment 8: The composition of any of Aspects 1-7, wherein the composition further comprises Factor VIII.

Aspect 2: A kit comprising the treatment of bleeding episodes comprising

a) an effective amount of Factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form;

b) an effective amount of Factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; and

c) container means containing said first and second dosage forms.

Embodiment 10: The composition of Aspect 2, comprising

a) an effective amount of Factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form;

b) an effective amount of Factor XI and a pharmaceutically acceptable carrier in a second unit dosage form;

c) an effective amount of a TFPI inhibitor in a third unit dosage form and a pharmaceutically acceptable carrier; and

d) container means containing said first, second and third dosage forms.

Aspect 3: A kit comprising a treatment for a bleeding episode comprising

a) an effective amount of a factor VIIa and TFPI inhibitor in a first unit dosage form and a pharmaceutically acceptable carrier;

b) an effective amount of Factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; and

c) container means containing said first and second dosage forms.

Aspect 4: A kit comprising a treatment for bleeding episodes comprising

a) an effective amount of Factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form;

b) an effective amount of a factor XI and TFPI inhibitor in a second unit dosage form and a pharmaceutically acceptable carrier; and

c) container means containing said first and second dosage forms.

Embodiment 9: The kit according to any one of Embodiments 2-4, further comprising one or more agents selected from the group consisting of Factor VIIa, Factor XI or TFPI inhibitors formulated in separate unit dosage form. Factor VIII in a unit dosage form of a compound.

Aspect 6: Use of the combination of Factor Vila and Factor XI for the manufacture of a medicament for treating bleeding episodes in a subject.

Aspect 7: Use of a combination of Factor Vila and Factor XI for the manufacture of a medicament for reducing clotting time in a subject.

Aspect 8: Use of the combination of Factor Vila and Factor XI for the manufacture of a medicament for prolonging the coagulation and lysis time in normal mammalian plasma.

Aspect 9: Use of the combination of Factor Vila and Factor XI for the manufacture of a medicament for increasing the strength of coagulation in normal mammalian plasma.

Aspect 10: Use of the combination of Factor Vila and Factor XI for the manufacture of a medicament for enhancing fibrin coagulation formation in normal human plasma.

Aspect 11: A method of enhancing fibrin clot formation in a subject, the method comprising administering to the subject an effective amount of Factor Vila in combination with an effective amount of Factor XI.

Aspect 12: A method of treating a bleeding episode in a subject comprising administering to the subject an effective amount of Factor Vila in combination with an effective amount of Factor XI.

Embodiment 10: The method of Aspect 19 or 20, wherein Factor Vila and Factor XI are administered in a single dosage form.

Embodiment 11: The method of Aspect 19 or 20, wherein Factor Vila and Factor XI are administered sequentially.

The invention is further illustrated by the following examples, however, they are not to be construed as limiting the scope of protection. The features disclosed in the above description and in the following examples can be used separately and in any combination as a basis for realizing the invention in its different forms.

Example

Example 1

Improved stability of hemostatic coagulation by combining coagulation factors VIIa and XI

method:

Coagulation and lysis test: the buffer solution (20mM HEPES, 20mM HEPES, 10-fold diluted normal human plasma (150mM NaCl, 5mM CaCl, pH7.4) was added to a 96-well ELISA plate and the turbidity at 650nm was measured at room temperature for a period of time. Where indicated, purified human FXI (Haematologic Technologies, various concentrations) was included.

Rotational (Rotatonal) Thromboelastography (roTEG): Measured in citrated normal human plasma supplemented with 5nM t-PA, the effect of 1nM FVIIa alone or in combination with 30nM FXI (Haematologic Technologies) was analyzed. Coagulation was initiated by the addition of Innovin (2000-fold diluted final concentration, Dade Behring #526945) and calcium (15 mM final concentration) in 20 mM HEPES, 150 mM NaCl, pH 7.4 buffer.

result:

Coagulation and lysis assay: Addition of FVIIa resulted in a dose-dependent prolongation of the coagulation and lysis time (Figure 1). This effect is optimal at 10 nM FVIIa. In the presence of 10 nM FVIIa, the addition of FXI resulted in a further prolongation of the coagulation and lysis time (Figure 2). This effect is dose dependent and optimal at 30 nM FXI.

Thromboelastography: roTEG measurements were used to analyze the effect of FVIIa and FXI on maximum coagulation firmness (MCF), and resistance of coagulation to t-PA-mediated lysis. Before the addition of FVIIa/FXI, the MCF was 25 mm and the time required for half of the coagulation to lyse was 12.3 minutes (Figure 3). Addition of FXI (30 nM) did not change the MCF but extended the hemicoagulation lysis time to 16.1 minutes (Figure 3). Likewise, addition of FVIIa (1 nM) resulted in protection of coagulation from t-PA-mediated fibrinolysis (half coagulation lysis time: 16.7 min), without any effect on MCF (Fig. 3). However, addition of FVIIa (1 nM) together with FXI (30 nM) increased MCF (29 mm), as well as hemicoagulation and lysis time (24.2 minutes, Figure 3).

in conclusion:

These results demonstrate that addition of FVIIa and FXI to plasma synergistically increases the mechanical strength of coagulation and resistance to t-PA-mediated fibrinolysis.

Example 2

Reduction of coagulation time in normal human plasma by combining coagulation factors VIIa and XI

method:

Coagulation assay: rFVIIa alone (1 μg/ml), FXI alone (25 nM), or aliquots (55 μl) of rFVIIa and FXI in 50 mM Pipes, 100 mM NaCl, 2 mM EDTA, 1% BSA, pH 7.2 and in A 55 μl aliquot containing 100 μM PC/PS vesicles and 50 mM CaCl ₂ in the same buffer was incubated for 5 minutes. A 55 [mu]l aliquot of normal human plasma (NHP) was then added and coagulated for 400 seconds in an ACL coagulator using standard APTT procedures.

result:

Coagulation test: Before adding rFVIIa or FXI, NHP did not coagulate within a monitoring time of 400 seconds. After adding FXI (25nM), the coagulation time was still longer than 400 seconds. Addition of FVIIa (1 μg/ml) shortened the coagulation time to 159.4±1.4 seconds ( FIG. 4 ). Simultaneous addition of FVIIa (1 μg/ml) and FXI (25 nM) shortened the coagulation time to 95.0 ± 1.4 seconds (Fig. 4).

in conclusion:

These results demonstrate that addition of FVIIa and FXI to plasma shortens clotting time in NHP in a synergistic manner.

Claims (35)

CLAIMS 1. A pharmaceutical composition comprising coagulation factor VII and coagulation factor XI. 2. The composition according to claim 1, wherein said coagulation factor VII is human coagulation factor VII. 3. The composition according to claim 2, wherein said coagulation factor VII is recombinant human coagulation factor VII. 4. Composition according to any one of claims 1-3, wherein said coagulation factor VII is in its activated form. 5. The composition according to claim 4, wherein said coagulation factor VII is recombinant human coagulation factor VIIa. 6. The composition according to claim 1, wherein said coagulation factor XI is human coagulation factor XI. 7. The composition according to claim 6, wherein said coagulation factor XI is recombinant human plasma coagulation factor XI. 8. The composition according to claim 6, wherein said coagulation factor XI is recombinant human platelet coagulation factor XI. 9. The composition according to any one of claims 1 to 8, wherein said coagulation factor VII and said coagulation factor XI are present in a mass ratio between 100:1 and 1:100. 10. The composition according to claim 9, wherein the composition further comprises a pharmaceutically acceptable excipient suitable for injection or infusion. 11. Use of a composition according to any one of claims 1 to 10 for the manufacture of a medicament for treating bleeding episodes in a subject. 12. The use according to claim 11, wherein the medicament is used to shorten the coagulation time. 13. The use according to claim 11, wherein the medicament is used to prolong the coagulation and lysis time. 14. The use according to claim 11, wherein the medicament is used to increase the strength of coagulation. 15. The use according to any one of claims 11-14, wherein the medicament is formulated for injection or infusion. 16. The use according to any one of claims 11-14, wherein the bleeding episode is due to trauma, or surgery, or a decrease in platelet count or activity. 17. Use according to any one of claims 11-14, wherein the medicament is in single dose form. 18. Kits for use in the treatment of bleeding episodes, comprising f) a preparation of an effective amount of Factor VII in a first unit dosage form and a pharmaceutically acceptable carrier; g) a preparation of an effective amount of Factor XI in a second unit dosage form and a pharmaceutically acceptable carrier; and h) container means containing said first and second dosage forms. 19. The kit according to claim 18, wherein said coagulation factor VII is human coagulation factor VII. 20. The kit according to claim 19, wherein said coagulation factor VII polypeptide is recombinant human coagulation factor VII. 21. A kit according to any one of claims 18 to 20, wherein said factor VII is in its activated form. 22. The kit according to claim 21, wherein said coagulation factor VII is recombinant human coagulation factor VIIa. 23. The kit according to claim 18, wherein said coagulation factor XI is human coagulation factor XI. 24. The kit according to claim 23, wherein said coagulation factor XI is recombinant human plasma coagulation factor XI. 25. The kit according to claim 23, wherein said coagulation factor XI is recombinant human platelet coagulation factor XI. 26. The kit according to claim 18, wherein said coagulation factor VII and coagulation factor XI are present in a mass ratio between 100:1 and 1:100. 27. Use of a combination of coagulation factor VII and coagulation factor XI for the manufacture of a medicament for treating bleeding episodes in a subject. 28. The use according to claim 27, wherein the medicament is used to shorten blood clotting time. 29. The use according to claim 27, wherein the medicament is used to prolong the coagulation and lysis time. 30. The use according to claim 27, wherein the medicament is used to increase the strength of coagulation. 31. The use according to any one of claims 27 to 30, wherein the medicament is formulated for injection or infusion. 32. Use according to any one of claims 27 to 30, wherein the bleeding episode is due to trauma, or surgery, or a decrease in platelet count or activity. 33. Use according to any one of claims 27 to 30, wherein the medicament is prepared in the form of a first unit dosage form comprising a preparation of factor VII and a second unit dosage form comprising a preparation of factor XI. 34. Use of a first amount of a preparation of Factor VII and a second amount of a preparation of Factor XI in the manufacture of a medicament for enhancing hemostasis in a subject, wherein the first and second amounts together are effective to enhance hemostasis. 35. A kit for treating bleeding episodes, comprising a) an effective amount of Factor VII and an effective amount of Factor XI and a pharmaceutically acceptable carrier in a unit dosage form; and b) container means containing said one unit dosage form.

Images