RU2298416C2 - Pharmaceutical composition containing factor vii polypeptides and factor xi polypeptides - Google Patents

Abstract

FIELD: medicine, hematology, polypeptides, pharmacy.

SUBSTANCE: invention relates to composition containing factor VII or factor VII-related polypeptide and factor XI or factor XI-related-polypeptide, sets containing indicated compounds and to using composition used in treatment of bleeding. Pharmaceutical composition contains the effective dose of factor VII or factor VII-related polypeptide, and factor XI or factor XI-related polypeptide. Invention provides the following effects: (1) design of composition that can be used effectively in treatment or prophylaxis of bleeding and blood coagulation disorders being oral composition preferably; (2) design of compositions as the single dosed formulation that can be used effectively in treatment or prophylaxis of bleeding or as a procoagulant agent; (3) design of compositions possessing the synergetic effect, design of treatment methods or sets that promote to the development of synergetic effect; (4) design of compositions, methods for treatment and sets without adverse effects, such as the high level of systemic activation of the blood coagulation system.

EFFECT: valuable medicinal properties of pharmaceutical composition.

43 cl, 1 tbl, 4 dwg, 2 ex

Description

Field of Invention

The present invention relates to a pharmaceutical composition comprising a factor VII or factor VII related polypeptide and a factor XI or factor XI related polypeptide. The invention also relates to the use of a combination of factor VII or a factor VII related polypeptide and factor XI or a factor XI related polypeptide for the manufacture of a medicament for the treatment of subjects suffering from bleeding or the prevention of bleeding. The invention also relates to a method for treating bleeding in subjects and to a method for accelerating clot formation in a subject. The present invention also relates to kits containing these compounds.

BACKGROUND OF THE INVENTION

Hemostasis is initiated through the formation of a complex between tissue factor (TF), which is exposed in the bloodstream after damage to the vessel wall, and factor FVIIa, which is in the bloodstream in an amount corresponding to approximately 1% of the total weight of the FVII protein. This complex is firmly attached to the TF-bearing cell and activates FX (factor X) in FXa (factor Xa) and activates FIX (factor IX) in FIXa (factor IXa) on the cell surface. FXa activates prothrombin and turns it into thrombin, which in turn activates FVIII, FV, FXI FXIII. In addition, a limited amount of thrombin formed at this initial stage of hemostasis also activates platelets. After the action of thrombin on platelets, the latter change their shape and “charge” phospholipids to the surface. The activated platelet surface forms a matrix for further activation of FX and the complete formation of thrombin. Further activation of FX on the activated platelet surface occurs through the FIXa-FVIIIa complex formed on the surface of the activated platelet, and FXa then converts prothrombin to thrombin, while still remaining on the surface. Thrombin then converts fibrinogen to fibrin, which is insoluble and which stabilizes the original platelet plug. This process is isolated, i.e. localized at the site of expression or location of TF, thereby reducing the risk of systemic activation of the blood coagulation system. The insoluble “plug” fibrin is further stabilized by crosslinking fibrin fibers catalyzed by factor FXIII.

Factor FVII exists in plasma, mainly in the form of a single-chain zymogen, which is cleaved by factor FXa into a double-stranded, activated form, FVIIa. Recombinant activated factor VIIa (rFVIIa) has been developed as a progestostatic agent. Administration of rFVIIa results in a fast and highly effective prohemostatic response in hemophilia patients with bleeding who cannot be treated with coagulation factors due to antibody formation. In addition, subjects who suffer from bleeding, factor VII deficiency, or subjects in whom the blood coagulation system is normal, suffering from excessive bleeding, can be successfully treated with factor FVIIa. When studying this problem, the researchers did not encounter adverse side effects due to the administration of rFVIIa (in particular, the possibility of developing thromboembolism).

Additionally introduced factor FVIIa improves the formation of thrombin on the activated surface of the platelet. This phenomenon occurs in patients with hemophilia who have no FIX or FVIII factor and therefore the most effective way to complete the formation of thrombin is not carried out. Also, in the presence of a reduced platelet or platelet count with impaired function, the additional factor FVIIa increases the formation of thrombin.

Commercial recombinant human factor FVIIa preparations are marketed as NovoSeven ^® (Novo Nordisk A / S, Denmark). Novoseven ^{® is} indicated for the treatment of bleeding cases in patients with hemophilia A and B. Novoseven ^® is the only recombinant factor FVIIa available on the market for effective and reliable treatment of bleeding.

The FXI factor is one of the components of the blood coagulation pathway. FXI deficiency is associated with bleeding, from mild to moderate, especially from tissues with high local fibrinolytic activity. Researchers believe that compared with FXI deficiency, high FXI levels are a risk factor for venous thrombosis. Factor FXI is a trypsin-like serine protease zymogen that is activated by factor FXIIa, thrombin and factor FXIa. The activated factor FXI (FXIa) is involved in the activation of FIX, which in turn (in combination with FVIII) also activates FX and, thus, leads to the formation of thrombin.

It is well known that subjects who experience excessive bleeding due to surgery or extensive trauma and who need a blood transfusion develop more complications than subjects who do not suffer from bleeding. However, moderate bleeding in subjects requiring the introduction of human blood or blood elements (platelets, white blood cells, plasma-derived concentrates for the treatment of blood clotting disorders, etc.) can lead to complications associated with the risk of transmission of human viruses to the blood ( hepatitis virus, HIV, parvovirus and other, still unknown viruses). Massive bleeding in subjects requiring significant blood transfusions can lead to multiple organ failure, including impaired lung and kidney function. As soon as these serious complications begin to develop in the subject, a cascade of reactions is launched, in which a number of cytokines are involved, as well as inflammatory reactions, making any treatment extremely difficult and, unfortunately, often ineffective. Therefore, the main task in surgery, as well as in the treatment of extensive tissue damage, is to prevent bleeding or to minimize them. In order to avoid bleeding or minimize it, it is important to achieve the formation of stable and dense hemostatic “plugs” that are poorly soluble by fibrinolytic enzymes. In addition, it is important to achieve the rapid and effective formation of such “plugs” or clots.

To date, in cases of bleeding in subjects, including victims of various injuries and those who have bleeding associated with surgery, treatment is often carried out with several injections or infusions of factor FVIIa, because of the short half-life of FVIIa (2.5 hours) more than one administration may be required to maintain a certain level of hemostatic ability. Faster stopping of bleeding will bring more benefit to such subjects. Therefore, in such cases, the number of administrations should be reduced to stop bleeding and maintain hemostasis.

Japanese Patent Application No. 59-116213A relates to an aerosol composition for use as a tissue adhesive containing a blood coagulation reagent as an active component. The reagent that promotes blood coagulation can be selected from coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII and XIII, precallicrein, high polymer kininogen and thrombin. A combination of factor FXIII and thrombin is preferred.

European Patent No. 225160 (Novo Nordisk) relates to compositions FVIIa and methods for treating bleeding disorders that are not caused by defects in coagulation factors or coagulation factor inhibitors.

European Patent No. 82182 (Baxter Travenol Lab.) Relates to a composition of factor VIIa, which is used to compensate for the deficiency of coagulation factors or counteract coagulation factor inhibitors in a subject.

International Patent Publication No. WO 93/06855 (Novo Nordisk) relates to topical application of FVIIa.

U.S. Patent No. 5,252,217 relates to a method for producing a human factor XI concentrate for therapeutic purposes.

There is still a need in the art for improved treatment of subjects suffering from bleeding, including subjects in which cases of bleeding are caused by surgery, trauma or other types of tissue damage; coagulopathy, including coagulopathy in subjects undergoing multiple blood transfusions; caused by congenital or acquired disorders in the blood coagulation system or disorders accompanied by bleeding, including hypofunction of the liver (“liver disease”); due to impaired platelet function or a decrease in platelet count; the absence of significant “blood clotting compounds” or the presence of abnormal blood coagulation “compounds” (for example, platelets or von Willebrand protein factor); associated with increased fibrinolysis; due to anticoagulant therapy or thrombolytic therapy; or stem cell transplantation.

There is also a need in the art for an improved, reliable and widely used method that improves blood coagulation, improves or ensures the formation of stable hemostatic plugs, or increases the potential for beneficial treatment of a subject, or leads to complete hemostasis in subjects, in particular in subjects with reduced thrombin formation. There is also a need for methods in which the amount of FVIIa required to achieve complete hemostasis is reduced, and methods where the time for stopping bleeding is reduced.

SUMMARY OF THE INVENTION

One objective of the present invention is to provide compositions that can be effectively used to treat or prevent bleeding and blood clotting disorders.

The second objective of the present invention is the creation of compositions in the form of a single dosage form, which can be effectively used to treat or prevent bleeding or as a procoagulant. Another objective of the present invention is the creation of compositions having a synergistic effect, the creation of treatment methods or kits, the use of which contributes to the manifestation of a synergistic effect.

Another objective of the present invention is to provide compositions, methods of treatment and kits that do not have significant side effects, such as a high level of systemic activation of the blood coagulation system.

Other objectives of the present invention will be apparent upon reading the present description.

In a first aspect, the invention relates to a pharmaceutical composition comprising a factor VII or factor VII related polypeptide and a factor XI or factor XI related polypeptide.

In a second aspect, the invention relates to a kit of components for treating bleeding, comprising

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

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

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

In various aspects of the invention, the kit also contains an effective amount of a TFPI inhibitor and / or factor VIII; The TFPI inhibitor or factor VIII (or a combination thereof) may be in separate unit dosage forms or may be in one of the unit dosage forms containing either factor VII or a factor VII related polypeptide, factor XI or a factor XI related polypeptide.

In a third aspect, the invention relates to the use of a factor VII or factor VII-related polypeptide in combination with factor XI or a factor XI-related polypeptide for the manufacture of a medicament for the treatment of bleeding in a subject. In another aspect, the invention relates to the use of a composition as described in any one of claims 1-18 for the manufacture of a medicament for the treatment of bleeding in a subject.

In various aspects of the invention, drugs are intended to reduce blood clotting time, increase fibrinolysis time, and increase clot strength.

In another aspect, the medicament is prepared for intravenous administration, preferably as an injection or infusion, in particular as an injection.

In one aspect, the medicament is provided in a single dosage form; in another aspect, the drug is obtained in the form of a first single dosage form containing a preparation of factor VII or a factor VII related polypeptide, and in a second single dosage form containing a preparation of factor XI or a factor XI related polypeptide.

In various aspects, the medicaments are intended to treat subjects in cases of bleeding due to surgery, trauma, or other types of tissue damage; coagulopathy, including coagulopathy in subjects undergoing multiple blood transfusions; congenital or acquired disorders in the blood coagulation system or disorders accompanied by bleeding, including hypofunction of the liver (“liver disease”); impaired platelet function or decreased platelet count; the absence of significant “connections” of the blood coagulation system or the presence of abnormal “connections” of the blood coagulation system (for example, platelets or von Willebrand protein factor); increased fibrinolysis; therapy with anticoagulant agents and therapy with thrombolytic agents; stem cell transplantation. In one series of aspects, bleeding occurs in organs such as the brain, inner ear region, eyes, liver, lung, tumor tissue, gastrointestinal tract; in another series of aspects, bleeding is diffuse bleeding, such as hemorrhagic gastritis and profuse uterine bleeding. In another series of aspects, cases of bleeding are associated with surgery or trauma in subjects who have acute hemarthrosis (joint hemorrhage), chronic hemophilic arthropathy, hematomas (e.g., muscle, retroperitoneal, sublingual and pharyngeal), bleeding in another tissue, hematuria ( bleeding from the urinary tract), cerebral hemorrhage that underwent surgery (e.g. hepatectomy), tooth extraction and gastrointestinal hemorrhage (e.g. bleeding with UGI). In one aspect, the medicament is for treating bleeding due to trauma, surgery, or a reduced platelet count or decreased platelet activity in a subject.

In another aspect, the invention relates to a method for treating bleeding in a subject, comprising administering to the subject, if necessary, an amount of a first preparation of factor VII or a factor VII related polypeptide and an amount of a second preparation of factor XI or a factor XI related polypeptide, where the amounts of the first and second drugs taken together are effective in the treatment of bleeding.

In another aspect, the invention relates to a method for reducing blood clotting time in a subject, the method comprising administering to the subject, if necessary, an amount of a first preparation of factor VII or a factor VII related polypeptide and an amount of a second preparation of factor XI or a factor XI related polypeptide, where the amounts of the first and second drug, together taken are effective to reduce blood coagulation time.

In another aspect, the invention relates to a method for increasing hemostasis, comprising administering to the subject, if necessary, an amount of a first preparation of factor VII or a factor VII related polypeptide and an amount of a second preparation of factor XI or a factor XI related polypeptide, where the amounts of the first and second drugs taken together are effective for increasing hemostasis.

In another aspect, the invention relates to a method for increasing a fibrinolysis time period in a subject, comprising, if necessary, administering to the subject an amount of a first preparation of a factor VII or factor VII related polypeptide and an amount of a second preparation of a factor XI or factor XI related polypeptide, where the amounts of the first and second preparation taken together are effective for increasing fibrinolysis time.

In another aspect, the invention relates to a method for increasing clot strength in a subject, the method comprising administering to the subject, if necessary, an amount of a first preparation of factor VII or a factor VII related polypeptide and an amount of a second preparation of factor XI or factor XI related polypeptide, where the amounts of the first and second drugs taken together are effective for increasing clot strength.

In one series of aspects of the methods, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are administered in a unit dosage form.

In another series of aspects, a factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide is administered in the form of a first single dosage form containing a factor VII or factor VII-related polypeptide preparation, and a second single dosage form containing a factor XI or related drug factor XI polypeptide. In a series of aspects of the invention, the first unit dosage form and the second unit dosage form are administered separately at a time interval of no more than 15 minutes.

In another aspect, the invention relates to a kit for treating bleeding, including

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

e) a container containing said single, unit dosage form.

In one series of aspects of the invention, the factor VII or factor VII-related polypeptide is a factor VII-related polypeptide. In one series of aspects of the invention, a factor VII-related polypeptide is a variant of the amino acid sequence of factor VII. In one aspect, the ratio of the activity of the factor VII-related polypeptide to the activity of native human factor VIIa (wild type FVIIa) is at least about 1.25, the data obtained from studies using an “in vitro hydrolysis assay” as described in the present description.

In one series of aspects of the invention, factor VII or a factor VII related polypeptide is factor VII. In one aspect, said factor VII is human factor VII. In one aspect, factor VII is factor VII of cattle, pig, dog, horse, mouse, or salmon. In another aspect, factor VII is a recombinant factor. In another aspect, factor VII is derived from plasma. In a preferred aspect, factor VII is a recombinant human factor VII. In one series of aspects of the invention, factor VII or a factor VII related polypeptide is in its activated form. In one preferred aspect of the invention, factor VII is a recombinant human factor VIIa.

In one series of aspects of the invention, the factor XI or factor XI-related polypeptide is a factor XI-related polypeptide. In one series of aspects of the invention, a factor XI-related polypeptide is a variant amino acid sequence of factor XI. In one aspect, the ratio of the activity of said factor XI-related polypeptide to the activity of native human plasma factor XI (wild-type FXI) is at least about 1.25, the data obtained from studies using the FXI chromogenic assay, as described in the present description. In one aspect, the factor XI or factor XI-related polypeptide is a factor XI-related polypeptide. In one aspect, factor XI is human factor XI. In one aspect, factor XI is factor XI of cattle, pig, dog, horse, mouse, or salmon. In a preferred aspect, factor XI is a recombinant factor. In another aspect, factor XI is derived from plasma. In another aspect, factor XI is platelet derived factor XI. In a preferred aspect, factor XI is a recombinant human plasma factor XI. In one series of aspects of the invention, factor XI or a factor XI-related polypeptide is in its activated form. In one aspect, a factor XI-related polypeptide is a fragment of factor XI. In one aspect, the factor XI-related polypeptide is a hybrid factor XI polypeptide, for example, a pig / human hybrid polypeptide. In one aspect, factor XI is an activated human plasma factor XI (FXIa).

In one aspect, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are present in a weight ratio of between about 100: 1 and 1: 100 (w / w factor VII: factor XI).

In one aspect, factor VII-related polypeptides are amino acid sequence variants in which no more than 20 amino acids are substituted, deleted, or inserted compared to wild-type factor VII (i.e., a polypeptide with the amino acid sequence described in US Pat. 4,784,950). In another aspect, factor VII variants have no more than 15 amino acids that are substituted, deleted, or inserted; in other aspects, factor VII variants have no more than 10 amino acids, such as 8, 6, 5, or 3 amino acids, which are substituted, deleted, or inserted compared to wild-type factor VII. In one aspect, factor VII variants are selected from the list of L305V-FVIIa, L305V / M306D / D309S-FVIIa, L305I-FVIIa, L305T-FVIIa, F374P-FVIIa, V158T / M298Q-FVIIa, V158D / E296-FVIIa, V158D / E296-FVIIV, FVIIa, M298Q-FVIIa, V158D / M298Q-FVIIa, L305V / K337A-FVIIa, V158D / E296V / M298Q / L305V-FVIIa, V158D / E296V / M298Q / K337A-FVIIa815VVA29V5AVVAaV15AVVAaV15AVVAaV15A5V8V5V8A / FVIIaV5a8 K157A-FVII, E296V-FVII, E296V / M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII and S336G-FVII.

In another aspect, factor VII-related polypeptides have increased activity independent of tissue factor compared with the activity of native human blood coagulation factor VIIa. In another aspect, increased activity is not accompanied by changes in substrate specificity. In another aspect of the invention, the binding of factor VII-related polypeptides to tissue factor is not impaired, and factor VII-related polypeptides exhibit at least wild type factor VIIa activity upon binding to tissue factor.

In a preferred aspect, 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 aspect, the clotting time of mammalian blood is reduced. In another aspect, hemostasis in the blood of mammals is enhanced. In another aspect, the time of fibrinolysis in the blood of mammals is increased. In another aspect, the strength of the clot in the blood of mammals is increased. In one aspect, the blood of mammals is human blood. In another aspect, mammalian blood is normal human blood; in one aspect, the blood is the blood of a subject in which the formation of thrombin is reduced. In one aspect, the blood is the blood of a subject with a deficiency of one or more coagulation factors; in another aspect, the blood is the blood of a subject containing inhibitors of one or more coagulation factors; in one aspect, the blood is the blood of a subject containing a reduced concentration of fibrinogen; in one aspect, the blood is human blood with a factor XI deficiency. In one series of aspects, blood is plasma.

In one aspect, factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide contribute to an increase in normal plasma fibrinolysis time. In another aspect, a factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide maximize clot strength and fibrinolysis time in normal human plasma in vitro. In another aspect, factor VII or a factor VII-related polypeptide and factor XI or a factor XI-related polypeptide contribute to a decrease in clotting time in normal human plasma in vitro.

In one aspect of the invention, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are the only hemostatic agents in the composition. In another aspect, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are the only active hemostatic agents in the composition. In another aspect, a factor VII or factor VII-related polypeptide and factor XI or a factor XI-related polypeptide are the only coagulation factors administered to a subject. In one aspect of the invention, a factor VII or factor VII-related polypeptide and factor XI or a factor XI-related polypeptide are the only active agents administered to a patient. In one aspect, the composition is substantially free of prothrombin; in another aspect, the composition is substantially free of FX; in another aspect, the composition is substantially free of FXa.

In another aspect, the pharmaceutical composition is prepared for intravenous administration, preferably in the form of an injection or infusion, in particular an injection. In one aspect, the composition comprises at least one pharmaceutically acceptable excipient or carrier.

In one aspect of the invention, the composition is in the form of a single dosage form, wherein a single, unit dosage form contains both coagulation factors. In one aspect of the invention, the composition is in the form of a kit containing a preparation of a factor VII or factor VII related polypeptide as a first unit dosage form and a preparation of a factor XI or factor XI related polypeptide as a second unit dosage form and containing a container containing said first and second standard dosage form. In one aspect, the composition or kit, if necessary, also contains instructions for administering the composition or individual components, respectively.

In one aspect of the invention, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are administered in a unit dosage form. In one aspect of the invention, a factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide is administered in a single unit dosage form containing a factor VII or factor VII-related polypeptide preparation, and a second unit dosage form containing a factor XI or related drug factor XI polypeptide.

In one aspect of the invention, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are administered simultaneously. In another aspect, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are administered sequentially. In one aspect, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are administered separately at a time interval of not more than 15 minutes, preferably 10, more preferably 5, most preferably 2 minutes. In one aspect, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are administered separately at intervals of up to 2 hours, preferably from 1 to 2 hours, more preferably up to 1 hour, more preferably from 30 minutes to 1 hours, more preferably up to 30 minutes, most preferably from 15 to 30 minutes.

In one aspect, an effective amount of a factor VII or factor VII-related polypeptide is from about 0.05 mg / day to about 500 mg / day (a subject weighing 70 kg). In one aspect, an effective amount of a factor XI drug or factor XI-related polypeptide is from about 0.01 mg / day to about 500 mg / day (a subject weighing 70 kg).

In one aspect, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are present in a weight ratio between about 100: 1 and 1: 100 (w / w factor VII: factor XI).

In one aspect of the present invention, the pharmaceutical composition is presented in a single dosage form and consists essentially of a preparation of factor VII or a factor VII related polypeptide and a preparation of factor XI or factor XI related polypeptide and one or more components selected from the list of pharmaceutically acceptable excipients or carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives and protease inhibitors. In a further aspect, the subject is a human being; in another aspect, the subject has a decrease in thrombin formation; in one aspect, the subject has a reduced plasma fibrinogen concentration (e.g., a subject undergoing multiple blood transfusions); in one aspect, the subject has a reduced plasma concentration of factor VIII.

In another aspect, the invention relates to a method designed to increase hemostasis in a subject suffering from a syndrome associated with factor VII, a method compared to a method of treating a subject with factor VII as the only coagulation protein, a method comprising administering to the subject an amount of a first drug as needed factor VII or factor VII-related polypeptide and the amount of the second drug of factor XI or factor XI-related polypeptide, where the amounts of the first and second drug are combined Contribute effectively improve hemostasis.

In another aspect, the invention relates to a method designed to increase the formation of thrombin in a subject, a method comprising administering to the subject, if necessary, the amount of the first preparation of factor VII or a factor VII-related drug and the amount of the second drug of factor XI or a factor XI-related polypeptide, where the amounts of the first and the second drug, taken together, contribute to an effective increase in the formation of thrombin.

In another aspect, the invention relates to a method designed to increase the formation of thrombin in a subject suffering from a syndrome associated with factor VII, a method compared to a method of treating a subject with factor VII as the only coagulation protein, a method comprising administering to the subject, if necessary, the amount of the first the preparation of factor VII or a factor VII related polypeptide and the amount of a second preparation of factor XI or a factor XI related polypeptide, where the amounts of the first and second preparation, Este combined, contribute to the effective increase in thrombin generation.

In another aspect, the invention relates to a method designed to reduce the number of administrations of the protein coagulation factor required to achieve hemostasis in a subject suffering from a syndrome associated with factor VII, compared with the number of administrations required when factor VII is administered to the subject in as the only protein coagulation factor, a method comprising administering to the subject, if necessary, a first amount of a factor VII preparation or a factor VII related polypeptide and a second amount an agent of factor XI or a factor XI-related polypeptide, where the first and second amounts together contribute to an effective reduction in the number of administrations of protein coagulation factor.

In another aspect, the invention relates to a method for treating bleeding in a subject suffering from a syndrome associated with factor VII, a method comprising administering to the subject, if necessary, an amount of a first preparation of factor VII or a factor VII related polypeptide and an amount of a second preparation of factor XI or a factor XI related polypeptide where the amounts of the first and second drug taken together are effective in treating bleeding.

In one aspect, factor VII is a recombinant human factor VIIa (rFVIIa). In another aspect, rFVIIa is ^{NovoSeven ® (Novo Nordisk A / S} , Bagsvaerd, Denmark).

In one aspect, a pharmaceutical composition is prepared for intravenous administration. In one aspect, the composition also comprises an inhibitor of the fibrinolytic system, including, without limitation, aprotinin, ε-aminocaproic acid, or tranexamic acid. In one aspect, the composition also comprises a TFPI inhibitor and / or FVIII.

In one aspect, the composition also comprises factor VIII. In one aspect, factor VIII is an activated factor VIII (factor VIIIa). In a further aspect, factor VIII is recombinant factor VIIIa. In a further aspect, factor VIII is a recombinant human factor VIIIa.

In another aspect, the invention relates to the use of factor VIIa in combination with factor XI for the manufacture of a medicament that enhances the formation of fibrin clot in mammalian plasma.

In another aspect, the invention relates to a method aimed at increasing the formation of a fibrin clot in a subject, a method that includes administering to the subject, if necessary, an amount of a first preparation of factor VII or a factor VII related polypeptide and an amount of a second preparation of factor XI or a factor XI related polypeptide, where the amounts of the first and second drug, taken together, are effective in treating bleeding.

In one aspect of the present invention, the pharmaceutical composition (in unitary form) consists essentially of factor VIIa and factor XI and, optionally, a pharmaceutically acceptable excipient or carrier and, optionally, a stabilizer and, possibly, a detergent and, possibly, a neutral salt and possibly an antioxidant and possibly a preservative and possibly a protease inhibitor.

In another aspect of the present invention, the pharmaceutical composition (in unitary form) consists essentially of factor VIIa and factor XI and, optionally, a pharmaceutically acceptable excipient or carrier and, optionally, a stabilizer and, possibly, a detergent and, possibly, a neutral salt and possibly an antioxidant and possibly a preservative and possibly a protease inhibitor and a TFPI inhibitor.

In another aspect of the present invention, the pharmaceutical composition (in unitary form) consists essentially of factor VIIa and factor XI and, optionally, a pharmaceutically acceptable excipient or carrier, and possibly a stabilizer and, possibly, a detergent and possibly neutral salts and possibly an antioxidant, and possibly a preservative, and, possibly, a protease inhibitor and factor VIII, and possibly a TFPI inhibitor.

In another aspect, the pharmaceutical composition (in kit form) consists of a first unit dosage form containing, mainly, factor VIIa and, optionally, a pharmaceutically acceptable excipient or carrier and, optionally, a stabilizer and, possibly, a detergent and, possibly, a neutral salt and possibly an antioxidant and possibly a preservative and possibly a protease inhibitor; and a second unit dosage form containing, mainly, factor XI and possibly a pharmaceutically acceptable excipient or carrier and, possibly, a stabilizer and, possibly, a detergent and, possibly, a neutral salt and, possibly, an antioxidant and, possibly, a preservative and, possibly a protease inhibitor.

In another aspect, the pharmaceutical composition (in kit form) consists of a first unit dosage form containing, mainly, factor VIIa and, optionally, a pharmaceutically acceptable excipient or carrier and, optionally, a stabilizer and, possibly, a detergent and, possibly, a neutral salt and possibly an antioxidant and possibly a preservative and possibly a protease inhibitor; and a second unit dosage form containing, mainly, factor XI and possibly a pharmaceutically acceptable excipient or carrier and, possibly, a stabilizer and, possibly, a detergent and, possibly, a neutral salt and, possibly, an antioxidant and, possibly, a preservative and, possibly a protease inhibitor; where either the first unit dosage form or the second unit dosage form, or both dosage forms also contain a factor VIII and / or a TFPI inhibitor.

List of figures

Figure 1: The addition of FVIIa resulted in a dose-dependent increase in fibrinolysis time. The indicated effect was optimal at 10 nM FVIIa.

Figure 2: In the presence of 10 nM FVIIa, the addition of FXI led to a further increase in fibrinolysis time. The effect was dose dependent and optimal at 30 nM FXI.

Figure 3: Thromboelastography (roTEG) was used to analyze the effect of FVIIa and XI on maximum clot density (MCF), as well as analysis of clot resistance to dissolution mediated by t-PA.

Figure 4: The results obtained show that the addition of FVII and FXI to plasma in a synergistic manner reduces the clotting time in NHP (normal human plasma).

DETAILED DESCRIPTION OF THE INVENTION

Subjects who undergo profuse bleeding due to surgery or extensive trauma and who require blood transfusions due to blood loss, develop more complications than subjects who do not experience any bleeding. However, moderate bleeding can lead to complications if the administration of human blood or blood elements (platelets, leukocytes, concentrates obtained from blood plasma for the treatment of disorders in the blood coagulation system) is required in cases of such bleeding, since blood transfusions are associated with a risk of transmission with blood of human viruses (for example, hepatitis virus, HIV, parvovirus or other currently unknown viruses), as well as non-viral pathogens. Massive bleeding, requiring large blood transfusions, can lead to the development of multiple organ failure, including impaired lung and kidney function. As soon as the subject develops these serious complications, a cascade of reactions is launched, involving a number of cytokines and inflammatory reactions, which make any treatment extremely difficult and, unfortunately, often ineffective. A patient who has suffered a great loss of blood becomes clinically unstable. Such patients have a risk of developing atrial fibrillation, which can lead to fatal cardiac arrest; impaired renal function; or sweating fluid into the lungs (the so-called "pulmonary edema" or ARDS (shock lung)). Therefore, the main task in surgery, as well as in the treatment of significant tissue damage, is to prevent severe bleeding or to minimize it. In order to avoid unwanted bleeding or minimize them, it is necessary to achieve the formation of stable and solid hemostatic “plugs” that are poorly soluble by fibrinolytic enzymes. In addition, it is important to achieve the rapid and effective formation of such “plugs” or clots.

In subjects with thrombocytopenia (reduced platelet count or decreased platelet activity), the ability to form thrombin is reduced, as well as insufficient stabilization of fibrin “plugs”, which leads to the formation of hemostatic “plugs” subjected to premature dissolution. In addition, subjects who have suffered extensive trauma or organ damage, and who, as a result, undergo multiple blood transfusions, often have a reduced platelet count, as well as reduced levels of fibrinogen, factor VIII, and other coagulation system proteins. In such subjects, a violation (decrease) in the formation of thrombin is observed. Such subjects have imperfect, or less effective, hemostasis, leading to the formation of fibrin “plugs” that dissolve easily and prematurely under the action of proteolytic enzymes, which, moreover, are intensively released during extensive trauma and organ damage.

Bleeding in the tissues can also lead to the formation of hematomas. The size of (in particular, intracranial and cerebrospinal) hematomas closely correlates with the degree of loss of neurological function, with the difficulties encountered during rehabilitation and / or with the severity and degree of permanent deterioration of neurological function after rehabilitation. The most severe consequences of hematomas are observed in cases of their location in the brain, when they can even lead to the death of the patient.

Thus, the main tasks in the treatment of bleeding are to achieve hemostasis in a minimum time, which leads to minimal blood loss.

Thus, the present invention relates to effective compositions, the use of these compositions and methods of treatment for the treatment of bleeding in subjects in need of such treatment. Compositions, their use and methods can be associated with beneficial effects such as less blood loss before hemostasis, less blood demand during surgery, maintaining blood pressure at an acceptable level until hemostasis, faster stabilization of blood pressure, shorter recovery time the patient being treated, shorter rehabilitation time of the patient being treated, a decrease in the incidence of hematomas or the formation of smaller hematomas, including hematomas cataracts in the brain, faster stopping of bleeding, reducing the number of injections required to stop bleeding and maintain hemostasis.

Administration of a factor VII preparation or a factor VII related polypeptide, such as factor VIIa, in combination with a factor XI preparation or a factor XI related polypeptide helps to reduce blood clotting time, form a denser clot and increase fibrinolysis resistance compared to clotting time, clot density and resistance in cases where subjects are administered only factor VIIa or factor XI.

Administration of a factor VII preparation or a factor VII related polypeptide, for example, factor VIIa, in combination with a factor XI preparation or a factor XI related polypeptide also helps to reduce the time to stop bleeding and reduce the number of administrations to maintain hemostasis compared with the case when only either factor is administered VIIa, or factor XI. The present invention relates to the beneficial effect of simultaneous or sequential dosing of a factor XI preparation or a factor XI related polypeptide and a factor VII preparation or a factor VII related polypeptide. The pharmaceutical composition according to the invention may be in the form of a single composition or may be in the form of a multicomponent set (set of components). The composition of the present invention is used as a therapeutic and prophylactic procoagulant in mammals, including primates, such as humans. The present invention also relates to a method for treating (including prophylactic treatment or prophylaxis) bleeding in a subject, including humans.

Whenever the description refers to the first or second or third, etc. standard dose, this reference does not indicate the preferred order of administration, but is provided only for convenience.

The combination of the preparation of factor VII or factor VII related polypeptide and the preparation of factor XI or factor XI related polypeptide is a beneficial product that helps to reduce blood coagulation time, the rapid formation of hemostatic plugs and the formation of stable hemostatic plugs. The present inventor has found that the combination of a factor VII preparation or a factor VII related polypeptide and a factor XI preparation or a factor XI related polypeptide is a beneficial product that promotes the formation of solid, stable and rapidly forming “plugs”.

The authors of the present invention showed that the combination of factor VIIa and factor XI can reduce the clotting time of human plasma in normal with greater efficiency than either factor VIIa or factor XI alone. It has been shown that a combination of factor VIIa and factor XI can increase clot density with greater efficiency than either factor VIIa or factor XI alone. By combining factor VIIa at a concentration that does not lead to a further increase in clot density, with factor XI also at a concentration that does not lead to a further increase in clot density, it was unexpectedly revealed that a further increase in clot strength occurs. It was also shown that the combination of factor VIIa and factor XI can increase the in vitro time of fibrinolysis in human plasma, normally with greater efficiency than either factor VIIa or factor XI alone. It has also been shown that a combination of factor VIIa and factor XI can prolong the fibrinolysis half-life in normal human plasma with greater efficacy than either factor VIIa or factor XI alone. It has also been shown that the combination of factor VIIa and factor XI can protect the clot from fibrinolysis, in particular, from tPA-mediated fibrinolysis, in normal human plasma with greater efficacy than either factor VIIa or factor XI alone.

Thus, an increase in the level of coagulation contributes to a more effective treatment of bleeding in a subject.

Not limited to any theory, the inventors believe that the complete formation of thrombin is necessary for the formation of a solid, stable hemostatic “plug” and, thus, to maintain hemostasis. The fibrin structure of such a “plug” depends both on the amount of thrombin formed and on the rate of initial formation of thrombin. In the case of a decrease in the formation of thrombin, a loose fibrin “plug” is formed, which is highly permeable. Fibrinolytic enzymes, which are normally located on the surface of fibrin, easily dissolve such a fibrin “plug”. The formation of a stable fibrin “plug” also depends on the presence of factor XIIIa, which is formed as a result of the action of thrombin, and therefore also depends on the completed formation of thrombin. In addition, a thrombin-activated fibrinolysis inhibitor, TAFI, which requires high amounts of thrombin to be activated, has recently been described. In case of thrombin formation that has not completely occurred, TAFI is not activated, which leads to the formation of a hemostatic “plug”, which dissolves more easily than usual under normal fibrinolytic activity. In the case of a disorder characterized by a reduced platelet count, thrombocytopenia, the administration of exogenous adjuvant factor VIIa initiates a more rapid formation of thrombin. However, the complete formation of thrombin is not normalized by factor VIIa even at its high concentrations.

In subjects with low plasma fibrinogen concentrations (subjects with multiple blood transfusions due to complex trauma or extensive surgery), thrombin does not fully activate. More effective hemostasis is achieved by the introduction of a combination of factor VII and factor XI.

In subjects with thrombocytopenia, there is a weakening of the formation of thrombin, as well as insufficient stabilization of fibrin “plugs”, which leads to the formation of hemostatic “plugs” prone to premature dissolution. In addition, subjects who suffer injuries or organ damage and who undergo multiple blood transfusions as a result of this often have a reduced platelet count, as well as reduced levels of fibrinogen, factor VIII, and other blood coagulation system proteins. In such subjects, a weakening (or decrease) in the formation of thrombin is observed. In addition, a reduced level of fibrinogen in such subjects adversely affects the activation of factor XIII. Such subjects, therefore, have imperfect or less effective hemostasis, which leads to the formation of fibrin “plugs”, which easily and prematurely dissolve under the action of proteolytic enzymes, moreover, such enzymes are intensively released during extensive injury and organ damage.

In order to facilitate the formation of fully stabilized “plugs” with the full ability to maintain hemostasis in a subject, a composition according to the invention is administered to the subject. The specified composition is especially beneficial for subjects with a reduced platelet count and for subjects with reduced levels of fibrinogen in blood plasma and / or other proteins of the coagulation system.

It is believed that in the presence of factor XI, lower concentrations of factor VIIa may be required to achieve sufficient hemostasis.

Factor VII Polypeptides

For the implementation of the present invention, any factor VII polypeptide can be used, that is, a polypeptide that is an effective tool in the prevention or treatment of bleeding. Such polypeptides include factor VII polypeptides derived from blood or plasma or obtained by recombinant methods.

The present invention includes factor VII polypeptides, such as, for example, polypeptides whose amino acid sequence is described in US Pat. 4,784,950 (wild-type human factor VII). In some aspects, the factor VII polypeptide is human factor VIIa, as described, for example, in US Pat. 4,784,950 (wild-type factor VII). In one series of aspects, factor VII polypeptides include polypeptides that exhibit at least about 10%, preferably at least about 30%, more preferably at least about 50%, and most preferably at least at least about 70% of the specific biological activity of human factor VIIa. In one series of aspects, factor VII polypeptides include polypeptides that exhibit at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least , about 140% and most preferably at least about 160% of the specific activity of human factor VIIa. In one series of aspects, factor VII polypeptides include polypeptides whose amino acid sequence is at least about 70% identical, preferably at least about 80% identical, more preferably at least about 90%, and most preferably at least about 95% of the wild type factor VII sequence described in US Pat. 4,784,950.

Used in the text, the term "factor VII polypeptide" covers, without limitation, factor VII, as well as related to factor VII polypeptides. The term "factor VII" is intended to include, without limitation, polypeptides with a sequence of amino acids 1-406 of wild-type human factor VII (as described in US Pat. No. 4,784,950), as well as wild-type factor VII derived from other species, such as for example, cattle, pig, dog, mouse and salmon, said factor being obtained from blood or plasma or obtained by recombinant methods. This term also encompasses natural allelic variants of factor VII, which may exist and occur in different representatives. In addition, the degree and place of glycosylation or other post-translational modifications may vary depending on the host cells selected and the nature of the host cell environment. The term “factor VII” is also intended to encompass factor VII polypeptides in their unsplit form (zymogen), as well as polypeptides that have undergone proteolytic processing to form the corresponding bioactive forms designated as factor VIIa. Typically, a factor VII molecule is cleaved between 152 and 153 amino acid residues to give factor VIIa.

The term “factor VII-related polypeptides” includes, without limitation, factor VII polypeptides, which are chemical modifications of human factor VII, and / or include one or more changes in amino acid sequence compared to human factor VII (i.e., variants factor VII), and / or polypeptides with truncated amino acid sequences compared to human factor VII (i.e., fragments of factor VII). Such factor VII-related polypeptides may exhibit properties different from those of human factor VII, including stability, phospholipid binding, specific activity, and the like. The term “factor VII-related polypeptides” is intended to encompass polypeptides in their unsplit form (zymogen), as well as polypeptides that have undergone proteolytic processing to form the corresponding bioactive forms, which are designated as “factor VIIa-related polypeptides” or “activated, factor VII-related polypeptides” .

As used herein, the term “factor VII-related polypeptides” encompasses, without limitation, polypeptides that exhibit mainly equal or improved biological activity relative to wild-type human factor VII, as well as polypeptides in which the biological activity of factor VIIa has been substantially modified or decreased relative to wild type human factor VIIa activity. Such polypeptides include, but are not limited to, Factor VII or Factor VIIa chemically modified, and Factor VII variants that have introduced specific amino acid sequence changes that have resulted in a modification or impairment of the bioactivity of the polypeptide.

In addition, the term encompasses polypeptides with a slightly modified amino acid sequence, for example, polypeptides with a modified N-terminal portion, including deletions or additions of amino acids in the N-terminal portion of polypeptides, and / or polypeptides that have been chemically modified with respect to human factor VIIa.

Factor VII-related polypeptides that include factor VII variants that exhibit mainly either equal or improved biological activity compared to wild-type factor VII, or, alternatively, exhibit mainly modified or reduced bioactivity relative to wild-factor VII of type include, without limitation, polypeptides with an amino acid sequence that differs from the wild-type factor VII sequence by the inclusion, deletion or substitution of one or more amino acids.

Factor VII-related polypeptides, including variants, encompass those polypeptides that exhibit at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, or at least about 130% of the specific activity of wild-type factor VIIa that is produced in cells of the same type, the data being obtained from studies using one or more of these assays, as a blood coagulation assay, proteolysis assay, or TF binding assay as described above. Factor VII-related polypeptides comprising variants having substantially reduced biological activity compared to wild factor VIIa ipa are polypeptides that exhibit less than about 25%, preferably less than about 50%, more preferably less than about 75%, more preferably less than about 100%, more preferably less than about 110%, more preferably less than about 120%, more preferably less than about 130%, of the specific activity of wild-type factor VIIa produced in cells of the same type, the data being obtained from studies using one Do multiple assays, such as analysis of blood clotting, or analysis of a proteolytic aniliz TF binding as opisago above.

Factor VII-related polypeptides, including those exhibiting significantly reduced biological activity compared to wild-type factor VIIa, are polypeptides exhibiting less than 25%, preferably less than about 10%, more preferably less than about and most preferably less than about 1% the specific activity of wild-type factor VIIa, which is produced in cells of the same type, the data being obtained as a result of studies using one or more of lyses, such as a blood coagulation assay, a proteolysis assay, or a TF binding assay as described above. Variants of factor VII, which exhibit mainly modified biological activity against wild-type factor VII, include, without limitation, variants of factor VII that exhibit TF independent proteolytic activity with respect to factor X, and those variants that bind to TF but do not split factor X.

In some aspects, the factor VII polypeptides are related to factor VII polypeptides, in particular, variants in which the ratio between the activity of the specified factor VII polypeptide and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25, moreover, the data are obtained as a result of studies using the "analysis of in vitro hydrolysis" (see "analysis" below); in other aspects, the ratio is at least about 2.0; in the following aspects, the ratio is at least about 4.0. In some aspects of the invention, factor VII polypeptides are related to factor VII polypeptides, in particular, variants in which the ratio between the activity of the specified factor VII polypeptide and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25, moreover, the data are obtained as a result of studies using the "analysis by hydrolysis in vitro" (see "analyzes" below); in other aspects, the ratio is at least about 2.0; in the following aspects, the ratio is at least about 4.0; in the following aspects, the ratio is at least about 8.0. In some aspects, the factor VII polypeptide is human factor VII, as described, for example, in US Pat. 4,784,950 (wild-type factor VII). In some aspects, the factor VII polypeptide is human factor VIIa. In one series of aspects, factor VII polypeptides are factor VII-related polypeptides that exhibit at least about 10%, preferably at least about 30%, more preferably at least about 50%, and most preferably at least about 70% of the specific enzymatic activity of human factor VIIa. In some aspects, the factor VII polypeptides have an amino acid sequence that differs from the wild-type factor VII sequence by the introduction, deletion or substitution of one or more amino acids.

Non-limiting examples of factor VII variants exhibiting equal or greater biological activity compared to wild-type factor VIIa include, but are not limited to, the variants described in Danish patent applications Nos. PA 2000 00734 and RA 2000 01360 (corresponding to international publication WO 01/83725), and RA 2000 01361 (corresponding to international patent application WO 02/22776). Non-limiting examples of factor VII variants exhibiting substantially equal or improved 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-FVIIa, K337A-FV8-FVII, K337A-FV8-VV-FVIIa FVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVII, V158D / E296V / M298Q / K337A-FVII, V158D / E296V / M298Q / L305V / K337A-FVII, F15II, FVII, FVII, FVII, F15, M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII and S336G-FVII; FVIIa variants exhibiting increased resistance to proteolysis as described in US Pat. 5,580,560; factor VIIa, which was proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); and oxidized forms of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54, 1999). Non-limiting examples of factor VII variants exhibiting substantially reduced or modified biological activity compared to wild-type factor VII include R125E-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 with no 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, for example, in US Pat. 5997864.

The biological activity of factor VIIa in the blood coagulation system is manifested in its ability (i) to bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of factor IX or factor X to form activated factor IX or X (factor IXa or Xa, respectively).

For the purposes of the invention, the biological activity of factor VII polypeptides ("biological activity of factor VII") can be quantified by measuring the ability of the drug to stimulate blood coagulation using plasma with insufficient factor VII and thromboplastins, as described, for example, in US Pat. 5997864. In this analysis, biological activity is expressed as a reduction in clotting time compared to a control sample and converted to “factor VII units” when compared to a standard representing pooled human serum that exhibits factor VII activity of 1 unit / ml. Alternatively, the biological activity of factor VIIa can be quantified by

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

(ii) measuring the hydrolysis of factor X in the aqueous system (“in vitro proteolysis assay”, see below);

(iii) measuring the physical binding of factor VIIa or factor VIIa-related polypeptide to TF using an instrument based on plasmon surface resonance (Persson, FEBS Letts. 413: 359-363, 1997); and

(iv) measuring the hydrolysis of the synthetic substrate by factor VIIa and / or a factor VIIa-related polypeptide (“in vitro hydrolysis assay”, see below); and (v) measuring thrombin formation in a TF-independent in vitro system.

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

Factor VIIa preparation that may be used according to the invention is, without ^{limitation, NovoSeven ® (Novo Nordisk A /} S, Bagsvaerd, Denmark).

Factor XI Polypeptides

In the practice of the present invention, any factor XI polypeptide can be used, that is, a polypeptide that is effective in preventing or treating bleeding. Such polypeptides include factor XI polypeptides derived from blood or plasma, or obtained by recombinant methods. In addition, platelets may contain an excellent FXI structure in form (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.

Used in the text, the term "factor XI polypeptide" covers, without limitation, factor XI, as well as related factor XI polypeptides. The term “factor XI” is intended to encompass, without limitation, polypeptides with the amino acid sequence described in Sun, Y. & Gailani, D. (1996), J. Biol. Chem. 271: 29023-29028 (wild-type factor XI from human plasma), as well as wild-type factor XI produced from other species such as, for example, cattle, pig, dog, mouse and salmon. In some aspects, the factor XI polypeptide is a wild-type human factor XI, as described, for example, in Sun, Y. & Gailani, D. (1996), J. Biol. Chem. 271: 29023-29028.

The term also encompasses allelic variants of factor XI, which may exist and occur in different representatives. In addition, the degree and place of glycosylation or other post-translational modifications may vary depending on the host cells selected and the nature of the host cell environment. The term “factor XI” is also intended to encompass factor XI polypeptides in their unsplit form (zymogen), as well as polypeptides that have undergone proteolytic processing to form the corresponding bioactive forms designated as factor XIa.

The term “factor XI-related polypeptides” includes, but is not limited to, chemically modified factor XI polypeptides and / or polypeptides whose amino acid sequence contains one or more amino acid sequence changes compared to human factor XI (ie, variants of factor XI ), and / or polypeptides with truncated amino acid sequences relative to human factor XI (i.e., fragments of factor XI). Such factor XI-related polypeptides may exhibit properties different from those of human factor XI, including stability, phospholipid binding, specific activity, and the like. The term “factor XI-related polypeptides” is intended to encompass such polypeptides in their unsplit form (zymogen), as well as polypeptides that have undergone proteolytic processing to form the corresponding bioactive forms, which can be designated as “factor XIa-related polypeptides” or “activated, factor-related XI polypeptides. "

As used herein, the term “factor XI-related polypeptides” encompasses, without limitation, polypeptides that exhibit mainly equal or improved biological activity compared to wild-type human factor XI, as well as polypeptides whose biological activity has been substantially modified or decreased relative to activity wild-type human factor XI. Such polypeptides include, without limitation, factor XI or factor XIa, which has been chemically modified, and variants of factor XI, which have been made specific changes in the amino acid sequence, which led to the modification or violation of the bioactivity of the polypeptide.

In addition, the term encompasses polypeptides with a slightly modified amino acid sequence, for example, polypeptides with a modified N-terminal portion, including deletions or additions of amino acids in the N-terminal portion of the polypeptides, and / or polypeptides that are chemical modifications of human factor XI.

Factor XI-related polypeptides comprising factor XI variants that exhibit a biological activity substantially equal to that of wild-type factor XI, or better biological activity compared to wild-type factor XI, or which, as an alternative, have a substantially modified or reduced bioactivity compared to the activity of wild-type factor XI, include, without limitation, polypeptides with an amino acid sequence that differs from the wild-type factor XI sequence including expression, deletion or substitution of one or more amino acids.

Factor XI-related polypeptides, including variants, encompass those polypeptides that exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, and at least riblizitelno 130% specific activity of wild-type factor XI, which is produced in the same cell type, wherein the listed data obtained as a result of studies by analyzing activity of factor XI, given herein.

Factor XI-related polypeptides, including variants that have equal biological activity or improved activity compared to wild-type factor XI than wild-type factor XI, encompass polypeptides that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120% and most preferably at least about 130% of the biological activity of wild-type human factor XI, which is produced in cells of the same type, the data obtained from studies using one or more of the specific activity analyzes of factor XI, as described. For the purpose of the invention, the biological activity of factor XI can be quantified, as will be described later in this text (part "analyzes").

Factor XI-related polypeptides, including variants that have substantially reduced biological activity compared to wild-type factor XI, are polypeptides that exhibit approximately less than 25%, preferably less than 10%, more preferably less than 5% and most preferably less than 1% of the specific activity of wild-type factor XI, which is produced in cells of the same type, and the data are obtained as a result of studies using one or more of the specific ac ivnosti factor XI, as described above.

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

In some aspects, factor XI is a factor XI-related polypeptide in which the ratio between the activity of said factor XI polypeptide and the activity of native human factor XI (wild-type factor XI) is at least about 1.25 when studies are performed using “FXI chromogenic analysis” (see below); in other aspects, the ratio is at least about 2.0; in the following aspects, the ratio is at least about 4.0.

The term “factor XI-related polypeptides” also includes fragments of factor XI or factor XI-related polypeptides that retain characteristic hemostasis-related activity. Hemostasis-related activity of the factor XI polypeptide can be measured, for example, by analysis of the activity of factor XI described in this text.

In preferred aspects, factor XI is human plasma factor XI or activated human plasma factor XIa. In one aspect, FXI is a platelet factor XI. In another aspect, FXI is a recombinantly derived factor.

Definitions

In the present text, three-letter or one-letter designations of amino acids in their usual meaning were used, as indicated in Table 1. Unless otherwise indicated, the amino acids mentioned in the text are L-amino acids. It should be understood that the first letter, for example, in the designation K337 represents an amino acid that is naturally in the indicated position of the wild-type factor VII molecule and that, for example, [K337A] -FVIIa means the FVII variant, where the amino acid represented by the single-letter code K in the native molecule in the indicated position has been replaced by the amino acid represented by the one-letter code A.

Table 1
Amino acid reductions 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 term “factor VIIa” or “FVIIa” may be used interchangeably. The term factor VIIa includes the zymogen factor VII (single chain factor VII). The term “factor XI” or “FXI” may be used interchangeably. The term “factor VIII” or “FVIII” may be used interchangeably. The term “factor VIII” or “FVIII” includes activated factor VIII (FVIIIa), variants and truncated forms that retain the characteristic hemostatic activity associated with factor VIII; the term includes recombinantly derived factor FVIII and factor VIII from plasma. Human factor VIII and recombinant human factor VIII are preferred.

In this text, the term "subjects with a violation of the formation of thrombin" means subjects who do not fully form thrombin on the activated surface of platelets, and also includes subjects in which thrombin is formed to a lesser extent than in subjects with a fully functioning normal hemostasis system , including the normal number of coagulation factors, platelets and fibrinogen and their function is normal (for example, as in the combined, human plasma is normal), and includes, without limitation, subjects who are absent factor VIII (e.g., thrombocytopenia or Glanzmann thrombasthenia or subjects with extensive bleeding); subjects with reduced levels of prothrombin, FX, or FVII; subjects with a reduced level of certain coagulation factors (for example, due to excessive bleeding due to trauma or extensive surgery), and subjects with reduced plasma fibrinogen concentrations (for example, in subjects who underwent multiple blood transfusions).

By “thrombin formation level” or “normal thrombin formation” is meant the level of thrombin formation in a patient compared with the level of thrombin formation in healthy subjects. The level is designated as a percentage of the normal level. The terms may, where appropriate, be used interchangeably.

The term "improvement of the hemostatic system" means an increase in the ability to form thrombin. The term “improved hemostasis” is intended to encompass cases where a specific thrombin formation for a test sample containing a factor VII preparation or a factor VII related polypeptide and a factor XI or factor XI related polypeptide preparation is extended over time compared to the formation of a thrombin control sample containing only factor VII or factor VII-related polypeptide or factor XI or factor XI-related polypeptide, respectively, when studies are carried out using the same analysis thrombin Ia. The formation of thrombin can be analyzed as described in the analysis of the formation of thrombin of the present invention (see the part "analyzes").

The "sole" agents or factors used in this invention are those cases where factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide, taken together, are the only hemostatic agents or coagulation factors contained in the pharmaceutical compositions or kit, or are the only hemostatic agents or active hemostatic agents or coagulation factors that are administered to the patient during an individual treatment an ailment, such as, for example, individual treatment of bleeding. It should be understood that such cases cover situations where other hemostatic agents or coagulation factors that are commonly used are not present in sufficient quantity or lack sufficient activity to significantly affect one or more parameters of blood coagulation.

Fibrinolysis time, clot strength, fibrin clot formation and blood coagulation time are clinical parameters used to characterize the patient's hemostatic system. Blood samples are taken from a patient at suitable time intervals and one or more parameters are examined by methods, for example, thromboelastography, as described in Meh et al., Blood Coagulation & Fibrinolysis 2001, 12: 627-637; Vig et al., Hematology, Vol. 6 (3) pp. 205-213 (2001); Vig et al., Blood coagulation & fibrinolysis, Vol. 12 (7) pp. 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 “increased fibrinolysis time” is intended to encompass cases where the measured clot dissolution time for a test sample containing a factor VII preparation or factor VII-related polypeptide and factor XI drug or factor XI-related polypeptide increases compared to the characteristic dissolution time of the clot of the control sample only factor VII or a factor VII related polypeptide or factor XI or factor XI related polypeptide, respectively, when studies are carried out using t The same analysis of clot dissolution. The clot dissolution time can be determined as described above.

The term “increasing clot strength” is intended to encompass cases where the measured clot strength, for example, mechanical strength, for a test sample containing a factor VII preparation or factor VII-related polypeptide and a factor XI preparation or factor XI-related polypeptide increases relative to the characteristic clot strength of the control sample containing only factor VII or a factor VII related polypeptide or factor XI or factor XI related polypeptide, respectively, when studies are conducted with using the same analysis of the strength of the bunch. Clot strength can be determined as described, for example, in Carr et al., 1991. (Carr ME, Zekert SL. Measurement of platelet-mediated force development during plasma clot formation. AM. J. MED. SCI. 1991, 302 : 13-8), or as described above, that is, by thromboelastography.

The term “enhancing fibrin clot formation” is intended to encompass cases where the measured rate or degree of clot formation for a test sample containing a factor VII preparation or factor VII-related polypeptide and a factor XI drug or factor XI-related polypeptide increases compared to a characteristic rate or degree of formation a fibrin clot in a control sample containing only factor VII or a factor VII related polypeptide or factor XI or factor XI related polypeptide, respectively governmental, when research is carried out using the same analysis of blood clotting. The formation of a fibrin clot can be measured as described above.

The term "reduced blood coagulation time" is intended to cover cases where the measured clot formation time (coagulation time) for a test sample containing a factor VII preparation or factor VII-related polypeptide and a factor XI preparation or factor XI-related polypeptide is reduced relative to the characteristic clotting time of the control sample containing only factor VII or a factor VII related polypeptide or factor XI or factor XI related polypeptide, respectively, when conducting studies using the same clotting assay. Blood coagulation time can be measured using standard PT or aPTT assays, which are well known to those skilled in the art.

The term "reduced platelet count or decreased platelet activity" refers to the number of platelets (platelets) present in the plasma of a subject and the biological associated with coagulation of the activity of such platelets. A decrease in platelet count may be due, for example, to an increase in platelet disruption, a decrease in platelet formation and a combination of the platelet fraction more than the normal platelet fraction in the spleen. Thrombocytopenia, for example, is defined as a platelet count of less than 150,000 platelets per microliter; the upper limit of the normal platelet count is usually between 350,000 and 450,000 platelets per microliter. Platelet counts can be measured using automatic platelet counters, that is, a method well known to those skilled in the art. Syndromes, as a consequence of a decrease in platelet count, include, without limitation, thrombocytopenia, coagulopathy. “Activity” includes, but is not limited to, platelet aggregation, adhesive and coagulation activity. A decrease in activity may be due, for example, to changes in glycoproteins, an abnormal interaction of the membrane and the cytoskeleton, changes in platelet granules, changes in clotting activity of platelets, changes in the signal of transduction and secretion. Platelet activity, including aggregation, adhesive and coagulation activity, is measured by standard methods known to the skilled person, see, for example, Platelets publication. 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. Beutler, Lichtman, Coller, Kipps & Seligsohn, 2001, McGraw-Hill. Syndromes due to decreased platelet activity include, but are not limited to, Glanzmann thrombasthenia, Bernard-Soulier syndrome, anticoagulant treatment, and thrombolytic treatment. “Reduced” refers to the platelet count or platelet activity of a sample of the test plasma compared to the platelet count and platelet activity of the combined plasma sample when the tests are performed using the same assay.

As used in the text, the term “bleeding disorder” refers to any defect, congenital, acquired, or induced, of cellular or molecular origin, which is manifested by bleeding. Examples of bleeding disorders include, but are not limited to, insufficiency of coagulation factors (e.g., deficiency of coagulation factors VIII, IX, XI or VII), inhibition of coagulation factors, impaired platelet function (e.g., Glanzmann thrombasthenia and Bernard-Soulier syndrome) , thrombocytopenia, von Willebrand disease, and coagulopathy, such as that caused by dilution of coagulation proteins, increased fibrinolysis, and decreased platelet count due to bleeding and / or blood transfusion (e.g. for example, in subjects undergoing multiple blood transfusions due to surgery or injury).

Bleeding refers to extravasation of blood from any component of the blood circulation system. The term "bleeding" is intended to include unwanted, uncontrolled, and often excessive bleeding due to surgery, trauma, or other types of tissue damage, as well as unwanted bleeding in subjects with bleeding disorders. Cases of bleeding can occur in subjects whose coagulation system is mostly normal, but who experience (temporary) coagulopathy, as well as in subjects with congenital or acquired coagulation disorders or bleeding disorders. In subjects with impaired platelet function, hemorrhages can be compared to hemorrhages caused by hemophilia, since the hemostatic system, as in hemophilia, is absent or has abnormal significant “substances” of blood coagulation (for example, platelets or von Willebrand protein factor). In subjects with significant tissue damage, for example, due to surgery or significant trauma, if the bleeding stops immediately, the normal mechanism of hemostasis may be weakened, and such subjects may experience excessive bleeding, despite the fact that mainly (before surgery and before injury), hemostasis was carried out according to the normal mechanism. Such subjects, who also often undergo multiple blood transfusions, experience (temporary) coagulopathy as a result of bleeding and / or blood transfusions (i.e., dilution of coagulation system proteins, increased fibrinolysis and decreased platelet count due to bleeding and / or blood transfusion) . Bleeding can also occur in organs such as the brain, inner ear and eye; these organs are areas of surgical disability for hemostasis, and therefore satisfactory hemostasis is problematic. Similar problems can arise during the biopsy of various organs (liver, lungs, tumor tissue, gastrointestinal tract), as well as during laparoscopic surgery and radical postoperative prostatectomy. A common factor for all these cases is the difficulty of achieving hemostasis using surgical equipment (surgical sutures, clamps, etc.), and in the case of diffuse bleeding (for example, hemorrhagic gastritis and massive uterine bleeding), they face the same difficulty. Bleeding can also occur in subjects with anticoagulant therapy in which hemostasis is caused by this treatment; these bleeding is often sharp and profuse. Anticoagulant therapy is often prescribed to prevent thromboembolic disease. Such therapy may include administration of heparin, other types of proteoglycans, warfarin or other types of vitamin K antagonists, as well as aspirin and other platelet aggregation inhibitors, such as, for example, antibodies or other inhibitors of GP IIb / IIIa activity. Bleeding can also be caused by so-called thrombolytic therapy, which includes combined treatment with an antiplatelet agent (e.g. acetylsalicylic acid), an anticoagulant (e.g. heparin), and a fibrinolytic agent (e.g. tissue plasminogen activator, tPA). Cases of bleeding also include, without limitation, uncontrolled and heavy bleeding due to surgery or trauma in subjects who suffer from acute hemarthrosis (joint hemorrhage), chronic hemophilic arthropathy, hematomas (e.g., muscle, retroperitoneal, sublingual and pharyngeal) bleeding in other tissues, hematuria (bleeding from the urinary tract), cerebral hemorrhage that undergo surgery (e.g., hepatectomy), tooth extraction and that suffer gastrointestinally intestinal bleeding (e.g., blood loss with UGI). Cases of bleeding may be associated with factor VIII inhibitors; 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; surgical operation; laparoscopic surgery; hemorrhagic gastritis; biopsy; anticoagulant therapy; bleeding in the upper gastrointestinal tract (UGI); or stem cell transplantation. Cases of bleeding can be profuse uterine bleeding; bleeding that occurs in organs with a limited ability to achieve hemostasis mechanically; arising in the brain; arising in the region of the inner ear or arising in the eyes. The terms “cases of bleeding” and “bleeding” may, when appropriate, be used interchangeably.

In this context, it is understood that the term “treatment” includes both the prevention of possible bleeding, such as, for example, during surgery, and the regulation of existing bleeding, such as, for example, trauma, in order to stop or minimize bleeding. The above term “probable bleeding” may mean bleeding that is likely to occur in a single tissue or organ, or it may be undefined bleeding. Thus, the term “treatment” includes prophylactic administration of a factor VII or factor VII related polypeptide preparation and a factor XI or factor XI related polypeptide preparation.

Used in the text, the term "subject" means any animal, in particular mammals, such as humans, and may, when appropriate, be used interchangeably with the term "patient".

Factor VII or a factor VII related polypeptide and factor XI or a factor XI related polypeptide, as defined herein, may be administered simultaneously or sequentially. Factors can be used in the form of a single dosage form, where the single dosage form contains both coagulation factors, or as a kit of components containing a preparation of factor VII or a factor VII related polypeptide as the first single dosage form, and a preparation of factor XI or a factor XI related polypeptide as a second single dosage form. Whenever the description refers to the first or second or third, etc. standard dose, while the preferred order of administration is not indicated, these designations are made mainly for convenience.

By "simultaneous" dosing of a factor VII drug or factor VII related polypeptide and a factor XI or factor XI polypeptide preparation is meant the administration of protein coagulation factors in a single dosage form, or the administration of the first protein coagulation factor, followed by the introduction of a second protein coagulation factor with an interval of time of administration more than 15 minutes, preferably 10, more preferably 5, more preferably 2 minutes. Any factor can be entered first.

By "sequential" dosing is meant the administration of a first protein coagulation factor followed by the administration of a second protein coagulation factor with an administration time interval of up to 2 hours, preferably from 1 to 2 hours, more preferably up to 1 hour, more preferably from 30 minutes to 1 hour, more preferably up to 30 minutes, more preferably 15 to 30 minutes. Any of two standard dosage forms, or protein coagulation factors, may be administered first. Preferably, both products are administered by the same intravenous route.

By "factor XI content" is meant the level of activity of factor XI of the coagulation system compared with the level of activity in healthy subjects. The level is designated as a percentage of the normal level. The terms may, when appropriate, be used interchangeably.

By "lowering the level of factor XI" or "reduced content of factor XI" is meant a decrease in the amount or activity of factor XI in the bloodstream compared with the average content of factor XI in a population of subjects not having factor XI deficiency of the coagulation system or factor XI inhibitors of the coagulation system. The level of circulating factor XI can be measured using either a blood coagulation assay or an immunological assay. The procoagulant activity of factor XI is determined by the patient’s plasma ability to adjust the coagulation time of plasma with factor XI deficiency (for example, ARTT analysis, see below; see also the “Analyzes” section of this description).

For one unit of factor XI, take the amount of factor XI located in one milliliter of normal (combined) human plasma (corresponding to a 100% level of factor XI).

For one factor VII unit, take the amount of factor VII in 1 ml of normal plasma, corresponding to a protein content of approximately 0.5 μg of protein. After activation, 50 units correspond to approximately 1 μg of protein.

By "deficiency" is meant a decrease in the amount or activity of, for example, factor XI in plasma compared with the amount or activity of this factor in the plasma of normal healthy individuals. The term may, when appropriate, be replaced by the term “reduced level of factor XI”.

By “ARTT” or “ARTT” is meant partially activated thromboplastin time (described, for example, in Proctor RR, Rapaport SI: The partial thromboplastin time with kaolin; a simple screening test for first-stage plasma clotting factor deficiencies. Am. J Clin. Pathol. 36: 212, 1961).

By “factor XI-associated syndrome” is meant a syndrome where, if necessary, exogenous factor XI, administered to a subject, can prevent, heal, or improve any symptoms, conditions, or diseases that are possible or already present that are caused by the syndrome. Syndromes include, without limitation, syndromes caused by a decrease in factor XI levels, for example, bleeding disorders caused by factor XI inhibitors. The syndrome associated with factor XI can also be treated with the composition of the present invention.

By “factor VII related syndrome” is meant a syndrome where, if necessary, exogenous factor VII, preferably VIIa, administered to a subject can prevent, heal or improve any symptoms, conditions or diseases that are possible or already present caused by the syndrome. Syndromes include, without limitation, syndromes caused by a decrease in the level of coagulation factors VIII, IX, XI or VII, coagulation factor inhibitors, impaired platelet function (e.g., Glanzmann thrombasthenia and Bernard-Soulier syndrome), thrombocytopenia, von Willebrand disease and coagulopathy, which caused by dilution of coagulation system proteins, increased fibrinolysis and a decrease in platelet count due to bleeding and / or blood transfusions (for example, in subjects who underwent multiple blood transfusions after surgery and or injury).

The term "half-life" refers to the time it takes for the concentration of factor VII or factor VII-related polypeptide or factor XI or factor XI-related polypeptide to decrease from a given value to half of this value.

By “primary hemostasis” is meant the initial formation of thrombin under the action of FXa and the TF complex: factor VIIa, the subsequent activation of platelets and the formation of the initial loose “plug” from activated, tightly connected platelets, which has not yet been stabilized by fibrin and, finally, stabilization cross-linked fibrin. If the “plug” is not stabilized by fibrin formed in the second stage of the hemostatic process (supported hemostasis), it is easily dissolved by the fibrinolytic system.

By “secondary hemostasis” or “sustained hemostasis” is meant secondary, complete and significant, “flash” thrombin formation occurring on the surface of activated platelets and catalyzed by factor VIIa and factor VIIIa, subsequent fibrin formation and stabilization of the original platelet “plug” from platelets . Stabilization of the cork with fibrin leads to complete hemostasis.

By “complete hemostasis” is meant the formation of a stable and solid fibrin clot or “cork” in the lesion that effectively stops bleeding and which does not readily dissolve under the action of the fibrinolytic system. In this context, the term hemostasis will be used to represent complete hemostasis, as described above.

The total amount of protein in the preparation can be measured by well-known methods, for example, by measuring the optical density. The amounts of coagulation factor XI or protein factor VII (“antigen”) can be measured by well-known methods such as standard Elisa immunoassays. In general terms, such an analysis is carried out by the interaction of, for example, a solution of a preparation containing protein factor XI with an anti-FXI antibody immobilized on an Elisa plate, subsequent interaction of the complex of immobilized antibody-factor XI with a second anti-FXI antibody carrying a marker of which in the third stage, measure. The amounts of each coagulation factor can be measured in a similar manner using appropriate antibodies. The total amount of the protein factor of the coagulation system present in the preparation is determined by summing the amounts of the individual protein factors of the coagulation system. In one aspect, the preparation comprises an isolated coagulation factor. In another aspect, the preparation does not contain factor II and factor IIa coagulation (prothrombin and thrombin) and / or factor X or XA.

As used herein, the term “isolated” refers to coagulation factors of the blood, for example, factor XI or factor XI-related polypeptides that are isolated from the cell in which they are synthesized, or from the environment in which they are found in nature (eg, plasma or blood ) Separation of the polypeptides from the cell in which they are synthesized can be achieved by any method known in the art, including, without limitation, separating the cell culture medium containing the desired product from the attached cell culture; centrifugation or filtration to remove loose cells and the like. Separation of the polypeptides from the environment in which they are naturally located is achieved by any method known in the art, including, without limitation, affinity chromatography, such as, for example, on a column with an antibody against factor VII or factor XI, respectively; hydrophobic chromatography; ion exchange chromatography; gel chromatography; electrophoretic procedures (for example, preparative isoelectric focusing (IEF)), differential solubility (for example, precipitation with ammonium sulfate) or extraction and the like. The term "TFPI inhibitor" means compounds that inhibit the anticoagulant activity of TFPI (tissue factor exchange pathway inhibitor). The term includes compounds such as those described in European Patent No. 558529, international publication WO 96/28153 and US patent 5622988. "TFPI" and "EPI" (external pathway inhibitor) can be used interchangeably.

In the present invention, an “effective amount” of a Factor VII polypeptide and a Factor XI polypeptide is defined as an amount of a Factor VII polypeptide, for example, FVIIa, and an amount of a Factor XI polypeptide, which together are sufficient to prevent or reduce bleeding or blood loss for treatment, relief or partial inhibition of the disease and its complications.

The term “factor VIIa activity” or “factor VIIa activity” includes the ability to form thrombin; the term also includes the ability to form thrombin on the surface of activated platelets in the absence of tissue factor.

The composition according to the invention may also contain a TFPI inhibitor. The composition according to the invention may also contain factor VIII. Such a composition is preferably administered to subjects who lack factor VIII inhibitors.

Abbreviations

TF tissue factor

FVII factor VII in the form of a single chain, activated form

FVIIa factor VII in its activated form

rFVIIa recombinant factor VII in its activated form; FXI factor XI in its zymogenic, inactive form

FXIa factor XI in its activated form

rFXI recombinant FXI

rFXIa recombinant FXIa

FVIII factor VIII in its zymogenic, non-activated form

rFVIII recombinant FVIII

FVIIIa factor VIII in its activated form

rFVIIIa recombinant FVIIIa

TFPI tissue factor exchange pathway inhibitor

Preparation of compounds:

Purified human factor VIIa suitable for use in the present invention is preferably obtained by recombinant DNA method, for example, described in the publication Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986, or described in European Patent No. 200421 (ZymoGenetics, Inc.).

Factor VII can also be obtained by the methods described in Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. Invest. 71: 1836-1841, 1983. These methods make it possible to obtain a factor VII preparation without detectable amounts of other blood coagulation factors. In addition, a purified factor VII preparation can be obtained by incorporating additional gel filtration as the final purification step. Factor VII is then converted to activated factor VIIa by known methods, for example, using several different plasma proteins, such as factor XIIa, IX or Xa. Alternatively, as described in Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565), factor VII can be activated by passing it through an ion exchange chromatographic column, such as Mono ^Q® (Pharmacia fine Chemicals) or the like.

Factor VII-related polypeptides can be obtained by modification of wild-type factor VII or by recombinant technology. Factor VII-related polypeptides with a modified amino acid sequence relative to the wild-type factor VII sequence can be obtained by modifying the nucleic acid sequence encoding the wild-type factor VII, or by changing the codons determining the amino acids, or by removing some of the codons for amino acids in the nucleic acid molecule encoding the natural factor VII, by known methods, for example, site-specific mutagenesis.

It will be apparent to those skilled in the art that substitutions can be made outside areas that are important for the function of factor VIIa or factor XI molecule, and these substitutions will lead to the formation of an active polypeptide. Amino acid residues essential for the activity of factor VII or factor VII related polypeptide or factor XI or factor XI related polypeptide and therefore, preferably not subject to substitution, can be identified according to procedures 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 method, mutations are introduced at each positively charged residue in the molecule, and the obtained mutant molecules are examined for coagulation, respectively, cross-linking activity to identify amino acid residues essential for the activity of the molecule. Plots of enzyme-substrate interaction can also be determined by analysis of the three-dimensional structure by methods such as nuclear magnetic resonance, crystallography or photoaffinity labeling (see, for example, publications 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).

The introduction of a mutation into a nucleic acid sequence in order to exchange one nucleotide for another nucleotide can be carried out by site-directed mutagenesis using any of the methods known in the art. The most used is a method in which a supercoiled, double-stranded DNA vector is used with inserted primers of interest and two synthetic primers including the desired mutation. Oligonucleotide primers, each of which is complementary to the opposite chains of the vector, are extended during the passage of the temperature cycle using Pfu DNA polymerase. When primers are introduced, a mutated plasmid is formed, which includes unstable single-stranded breaks. After going through the temperature cycle, the product is treated with the enzyme DpnI, which is specific for methylated and semi-methylated DNA, in order to digest the maternal form of DNA and select the synthesized DNA, including mutations. Other methods known in the art for creating, identifying and isolating variants can be used, such as, for example, gene permutation or phage reproduction methods.

Separation of the polypeptides from the cell in which they are synthesized can be achieved by any method known in the art, including, without limitation, separating the cell culture medium containing the desired product from the attached cell culture; centrifugation or filtration to remove loose cells and the like.

It is possible that factor VII or factor VII-related polypeptides can be further purified. The purification is carried out by any method known in the art, including, but not limited to, affinity chromatography, such as, for example, column chromatography with an anti-factor VII antibody (see, for example, Wakabayashi et al., J. Biol. Chem. 261: 11097 , 1986 and Thim et al., Biochem. 27: 7785, 1988); hydrophobic chromatography; ion exchange chromatography; gel chromatography; electrophoretic procedures (e.g., preparative electro-focusing (IEF)), differential solubility (e.g., precipitation with ammonium sulfate) or extraction, and the like. See, in general, Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, JC Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. After purification, the preparation preferably contains less than 10 wt.%, more preferably less than 5% and most preferably less than 1% of products other than factor VII or related factor VII polypeptides derived from a host cell.

Factor VII or related factor VII polypeptides can be activated by proteolytic cleavage using factor XIIa or other proteases having trypsin-like specificity, such as, for example, factor IXa, kallikrein, factor Xa and thrombin. See, for example, Osterud et al., Biochem. 11: 2853 (1972); Thomas, U.S. Pat. 4,456,591 and Hender et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, factor VII or factor VII-related polypeptides can be activated by passing them through an ion exchange chromatographic column, such as Mono ^Q® (Pharmacia) or the like. Thus, activated factor VII or factor VII-related polypeptides can be prepared and administered as described below.

For use in the present invention, factor XI can be obtained from plasma according to known methods, such as those described by Koide et al. (Biochemistry 16: 2279-2286, 1977) and Bouma et al. (J. Biol. Chem. 252: 6432-6437, 1977), incorporated herein by reference. However, it is preferable to use recombinant factor XI in order to avoid the use of products made from blood and tissue that carry a risk of transmission of the disease. Methods for producing recombinant factor XI are known in the art. See, for example, Kemball-Cook et al. (Gene 139 (2): 275-279, 1994), Fujikawa et al. (Biochemistry 25: 2417-2424, 1986), Meijers et al. (Blood 79 (6): 1435-1440, 1992), which are incorporated herein by reference in their entirety.

Factor XI-related polypeptides can be obtained by modification of wild-type factor XI or by recombinant technology. Factor XI-related polypeptides whose amino acid sequence is changed relative to the amino acid sequence of wild-type factor XI can be obtained by modifying the nucleic acid sequence encoding wild-type factor XI, either by changing the codons that determine the amino acids, or by removing some of the amino acid codons in the molecule nucleic acid encoding the natural factor XI, by known methods, for example, site-specific mutagenesis, as described in more detail but higher. Separation of the polypeptides from the cell in which they are synthesized can be achieved by any method known in the art, including, without limitation, separating the cell culture medium containing the desired product from the attached cell culture; centrifugation or filtration to remove loose cells and the like. Perhaps factor XI or polypeptides related to factor XI may be further purified. The purification is carried out by any method known in the art, including, without limitation, affinity chromatography, such as, for example, column chromatography with an antibody against factor XI; hydrophobic chromatography; ion exchange chromatography; gel chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., precipitation with ammonium sulfate) or extraction and the like, as described in more detail above. After purification, the preparation preferably contains less than 10 wt.%, More preferably less than 5% and most preferably less than 1% of products other than factor XI or factor XI-related polypeptides derived from the host cell. Activated factor XI or a factor XI-related polypeptide can then be prepared and administered as described below.

As will be appreciated by those skilled in the art, it is preferable to use the protein factor XI and factor VIIa syngeneic to the subject to reduce the risk of an immune response. The preparation and characterization of factor XI other than human is described, for example, in Gailani publication (Blood 90 (3): 1055-1064, 1997). The present invention also encompasses the use of proteins such as factor XI and factor VIIa in veterinary medicine.

Pharmaceutical compositions and methods for their use

The preparations according to the present invention can be used to treat any syndrome associated with factor VII, such as, for example, disorders accompanied by bleeding, including, without limitation, syndromes caused by a decrease in the level of coagulation factors VII, IX, XI or VII, coagulation factor inhibitors, platelet dysfunction (e.g., Glanzmann thrombasthenia and Bernard-Soulier syndrome), thrombocytopenia, von Willebrand disease and coagulopathy, such as is caused by dilution of coagulation system proteins, p Vyshen fibrinolysis and lowered number of platelets due to bleedings and / or transfusions (e.g., in subjects which have undergone multiple transfusions of blood following surgery or trauma). Pharmaceutical compositions comprising a preparation of a factor VII or factor VII-related polypeptide and a preparation of factor XI or a factor XI-related polypeptide of the present invention are intended primarily for parenteral administration for prophylactic and / or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously or intramuscularly; intravenous administration is most preferred. Compositions can also be administered by continuous or intermittent infusion.

The pharmaceutical compositions or preparations according to the invention comprise a factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide, either prepared as a unit dosage form or as a set of components, preferably dissolved in a pharmaceutically acceptable carrier, preferably in an aqueous carrier or diluent. Briefly, pharmaceutical compositions suitable for use in accordance with the present invention are prepared by mixing factor VIIa, or factor XI, or factor VIIa in combination with factor XI, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent. A number of aqueous vehicles can be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like. The preparations according to the invention can also be formulated using non-aqueous vehicles, such as, for example, in the form of a gel or as liposome preparations for delivery to the lesion sites or targeting said sites. Liposome preparations are described mainly in US Pat. Nos. 4837028, 4501728 and 4975282. The compositions can be sterilized by suitable, well known sterilization methods. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation is combined with a sterile aqueous solution prior to administration.

The compositions may contain pharmaceutically acceptable excipients or adjuvants, including, but not limited to, pH adjusting agents and buffering agents and / or tone adjusting agents, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and etc.

Compositions may also include one or more diluents, emulsifiers, preservatives, buffers, fillers, etc. and can be obtained in the form of liquids, powders, controlled-release emulsions, etc. One of skill in the art can formulate the compositions of the invention appropriately and in accordance with accepted practice, as described in Remington ^'s Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990. Thus, a conventional pharmaceutical composition for intravenous infusion should be formulated so that it contains 250 ml of sterile ringer's solution and 10 mg of the drug.

Compositions containing the preparations of the present invention may be administered for prophylactic and / or therapeutic treatment. For therapeutic purposes, the compositions are administered to a subject who is already suffering from a disease, as described above, in an amount sufficient to treat, alleviate or partially stop the clinical manifestations of the disease and its complications. An amount sufficient to achieve such results is defined as a “therapeutically effective amount”. Effective amounts to achieve each goal will depend on the severity of the disease or damage, as well as the weight and general condition of the subject. It should be understood that the determination of the appropriate dosage can be carried out using standard experimentation, creating a matrix of quantities and checking different points in the matrix.

Local delivery of drugs according to the present invention, such as, for example, topical application, can be accomplished, for example, by spraying, perfusion, double balloon catheters or other well-established methods. In any case, the pharmaceutical composition should contain an amount of the drug sufficient to effectively treat the condition.

The concentration of factor VII or factor VII related polypeptide, factor XI or factor XI related polypeptide, or factor VII or factor VII related polypeptide in combination with factor XI or factor XI related polypeptide in these compositions can vary significantly, i.e., from concentration, component of approximately less than 0.5 wt.%, or at least about 1 wt.%, to a concentration of 15 or 20 wt.%, and will be selected mainly on the basis of fluid volume, viscosity, etc. . in accordance with a separate, selected route of administration. Administration by injection or infusion, in particular injection, is preferred. Thus, factor VII or a factor VII related polypeptide and factor XI or factor XI related polypeptide are prepared in a form suitable for intravenous administration, such as a preparation that is either a dissolved lyophilized powder or a liquid composition containing as factor VII or related to factor VII a polypeptide or factor XI or a factor XI-related polypeptide in a single dosage form, either a dissolved lyophilized powder or a liquid composition containing factor VII or a factor VII-related field peptide in one dosage form and dissolved lyophilized powder or a liquid formulation containing the factor XI or factor XI-related polypeptide in another dosage form.

It is understood that the amount of factor VII or factor VII-related polypeptide and the amount of factor XI or factor XI-related polypeptide together represent the total effective amount for treating bleeding.

It should be borne in mind that the materials of the present invention can mainly be used in cases of serious illness or damage, that is, life-threatening or possibly life-threatening cases. In such cases, due to the reduction of foreign substances to a minimum and the general lack of immunogenicity of factor VIIa and factor XI in humans, it is desirable to administer to the subject a substantial excess of these compositions by the attending physician.

For prophylaxis purposes, compositions containing a preparation of a factor VII or factor VII-related polypeptide and a preparation of factor XI or a factor XI-related polypeptide are administered to a subject susceptible to the disease or damage or in other cases at the risk of the disease or damage, in order to increase the subject's own coagulation ability blood. This amount is defined as a "prophylactically effective dose." It is understood that the amount of factor VII or factor VII-related polypeptide and the amount of factor XI or factor XI-related polypeptide together represents the total effective amount to prevent bleeding.

Single or multiple injections of the compositions can be performed at such dose levels and regimens as selected by the attending physician. The compositions may be administered one or more times a day or week. An effective amount of such a pharmaceutical composition is an amount that promotes a clinically significant effect against bleeding. Such amounts will depend, in part, on the individual condition to be treated, the age, weight and general health of the subject and other factors obvious to those skilled in the art.

The composition according to the invention is usually administered in a single dose before possible bleeding or at the beginning of bleeding. However, the composition can be re-administered (in the form of multiple doses), preferably at intervals of 2-4-6-12 hours, depending on the dose and the condition of the subject.

For the purpose of treatment associated with a preventive intervention, a factor VII or factor VII-related polypeptide and a factor XI or factor XI-related polypeptide are usually administered within about 24 hours before the intervention and up to 7 days or more after it. The introduction of the drug as a coagulant can be performed using a number of methods described in this text.

The composition may be presented in the form of a single preparation (single dosage form) containing both preparations, namely, a preparation of factor VII or a factor VII related polypeptide and a preparation of factor XI or factor XI related polypeptide in suitable concentrations. The composition may also be presented in the form of a kit of components consisting of a first single dosage form containing a preparation of factor VII or a factor VII related polypeptide, and a second single dosage form containing a preparation of factor XI or a factor XI related polypeptide. In this case, the factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide should be administered one after another, preferably within about 15 minutes from each other, for example, within 10 minutes from each other or, preferably, within 5 minutes or, more preferably, within 2 minutes of each other. Any of the two unit dosage forms may be administered first.

The kit includes at least two separate pharmaceutical compositions. The kit includes containers for containing individual compositions, such as a split bottle or split foil bag. Typically, the kit includes instructions for administering the individual components. The use of the kit is particularly advantageous in cases where the individual components are preferably administered in different dosage forms, administered at different intervals between doses of the drug, or when titration of the individual components of the combination is prescribed by a physician.

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

The dose of factor VII or a factor VII related polypeptide ranges from a dose that corresponds to approximately 0.05 mg to a dose corresponding to 500 mg / day of wild-type factor VII, for example, from about 1 mg to 200 mg / day, or, for example, approximately from 5 mg to 175 mg / day for a subject weighing 70 kg, and the dose may be a loading and maintenance dose and depend on the weight of the subject, the condition and severity of the disease.

The dose of factor XI or a factor XI-related polypeptide ranges from a dose that corresponds to approximately 0.05 mg to a dose corresponding to 500 mg / day of wild-type factor XI, for example, from about 1 mg to 200 mg / day, or, for example, approximately from 5 mg to 175 mg / day for a subject weighing 70 kg, and the dose may be a loading and maintenance dose and depend on the weight of the subject, the condition and severity of the disease.

The combination of factor VIIa and factor XI exhibits a synergistic effect when studies are carried out in vitro by analysis of clot density and fibrinolysis time. In addition, the combination of factor VIIa and factor XI exhibits a synergistic effect in the formation of stable fibrin clots, an increase in the half-life of fibrinolysis, an increase in the strength of the clot, and an increase in resistance to fibrinolysis.

The composition may be presented in the form of a single preparation containing both factor VIIa and factor XI in suitable concentrations. The composition may be presented in the form of a kit consisting of a first single dosage form containing factor VIIa, and a second single dosage form containing factor XI, and optionally one or more unit dosage forms containing factor VIII and / or a TFPI inhibitor. In this case, factor VIIa and factor XI should be administered sequentially, preferably within 1 to 2 hours from each other, for example, within 30 minutes from each other, or, preferably, within 10 minutes, or, more preferably, within 5 minutes of each other. Any of the two unit dosage forms may be administered first.

Since the present invention relates to the prevention or treatment of bleeding or to the treatment of disorders of the blood coagulation system by treating with a combination of active ingredients that can be administered separately, the invention also relates to combining the individual pharmaceutical compositions as a kit. The kit includes at least two separate pharmaceutical compositions. The kit includes containers for containing individual compositions, such as a split bottle or split foil bag. Typically, the kit includes instructions for administering the individual components. The use of the kit is particularly advantageous in cases where the individual components are preferably administered in different dosage forms, administered at different intervals between doses of the drug, or when titration of the individual components of the combination is prescribed by a physician.

Analyzes

The study of the activity of factor VIIa

A suitable assay to verify the activity of factor VIIa and, therefore, the selection of suitable variants of factor VIIa can be carried out in the form of a simple preliminary in vitro test:

In vitro hydrolysis assay

The native (wild type) factor VIIa and a variant of factor VIIa (both referred to in the text as “factor VIIa”) can be investigated with respect to their specific activity. They can be investigated in parallel for the convenience of comparing their specific activities. The analysis is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). Chromogenic substrate D-Ile-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mM, added to factor VIIa (final concentration 100 nM) in 50 mM Hepes buffer, pH 7.4, containing 0, 1 M NaCl, 5 mM CaCl ₂ and 1 mg / ml bovine serum albumin. Absorption is measured at 405 nm continuously in a SpectraMax ^™ 340 plate reader (Molecular Devices, USA). The amount of absorption that develops during a 20-minute incubation, after subtracting the absorption in a control well containing no enzyme, is used to calculate the relationship between the activities of the variant factor VIIa and wild-type factor VIIa variants:

Ratio = (A _{405 nm} variant of factor VIIa) / (A _{405 nm} of wild type factor VIIa)

Based on the results of this calculation, variants of factor VIIa can be identified with activity comparable to the activity of native factor VIIa or higher of the indicated activity, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is approximately higher 1,0.

The activity of factor VIIa or factor VIIa variants can be measured using a physiological substrate, such as factor X, appropriately at a concentration of 100-1000 nM, where the formed factor Xa is measured after adding the appropriate chromogenic substrate (for example, S-2765). In addition, activity analysis can be performed at physiological temperature.

In vitro proteolysis assay

The native (wild type) factor VIIa and the variant of factor VIIa (both named in the text as "factor VIIa") are examined in parallel for the convenience of comparing their specific activities. The analysis is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) and factor X (0.8 μM) in 100 microliters of 50 mm Hepes buffer, pH 7.4, containing 0.1 M NaCl, 5 mm CaCl ₂ and 1 mg / ml bovine serum albumin, incubated for 15 minutes. The factor X cleavage reaction is then stopped by adding 50 microliters of 50 mM Hepes buffer, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg / ml bovine serum albumin. The amount of factor Xa formed is measured by adding the chromogenic substrate ZD-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Absorption is measured at 405 nm continuously in a SpectraMax ^™ 340 plate reader (Molecular Devices, USA). The amount of absorption that develops within 10 minutes, after subtracting the absorption in the control well containing no FVIIa, is used to calculate the relationship between the proteolytic activities of the variant factor VIIa and wild-type factor VIIa variants:

Ratio = (A _{405 nm} variant of factor VIIa) / (A _{405 nm} of wild type factor VIIa)

Based on the results of this calculation, variants of factor VIIa can be identified with activity comparable to the activity of native factor VIIa or higher of the indicated activity, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is approximately higher 1,0.

Thrombin Formation Analysis

The ability of factor VII or factor VII-related polypeptides or factor XI or factor XI-related polypeptides (e.g. variants) to produce thrombin can be measured using an assay that is performed using all relevant blood coagulation factors and inhibitors at physiological concentrations and activated platelets (as described in p. 543 in Monroe et al. (1997) Brit. J. Haemotol. 99, 542-547, which is incorporated herein by reference).

Check factor XI activity

A suitable assay to verify the amidolytic activity of factor XI and thereby select the appropriate variants of factor XI can be performed as a simple in vitro test using a chromogenic substrate, as described, for example, in Gailani et al. (Blood 97 (10): 3117-3122, 2001) ("FXI chromogenic analysis").

The biological activity of factor XI can be determined using a simple in vitro test by measuring the activation of factor IX to factor IXa, as described, for example, in the publication of Gailani et al. (Blood 97 (10): 3117-3122, 2001).

Testing the activity of factor VIII

Suitable assays to verify the activity of factor VIII and thereby enable selection of appropriate factor VIII variants for use in the present invention can be carried out in the form of simple in vitro tests, as described, for example, in Kirkwood TBL, Rizza CR, Snape TJ, Rhymes IL, Austen DEG. Identification of sources of interlaboratory variation in factor VIII assay. B. J. Haemotol. 1981; 37; 559-68 or Kessels eta l., British Journal of Haemotology, Vol. 76 (Suppl. 1) pp. 16 (1990)). Factor VIII activity can also be measured using a two-step chromogenic assay based on the amidolytic activity of the formed FXa (Wagenvoord et al., 1989, Haemostasis, 19 (4): 196-204).

The biological activity of factor VIII can also be quantified by measuring the ability of the drug to adjust the clotting time of plasma with factor VIII deficiency, for example, as described in Nilsson et al., 1959. (Nilsson IM, Blombaeck M, Thilen A, von Francken I., Carriers of haemophilia A - A laboratory study, Acta Med. Scan. 1959; 165: 357). In this analysis, biological activity was expressed as units / ml of plasma (1 unit corresponds to the amount of FVIII present in normal pooled plasma).

Aspects of the invention:

Aspect 1: A pharmaceutical composition comprising a factor VII or factor VII related polypeptide and a factor XI or factor XI related polypeptide.

Option 2: a composition as in aspect 1, wherein said factor VII or factor VII-related polypeptide is a factor VII-related polypeptide.

Option 3: The composition as in aspect 1, where factor VIIa is human factor VIIa.

Option 4: The composition as in aspect 1 or aspect 3, where factor VIIa and factor XI is a recombinant human factor VIIa and recombinant human factor XI, respectively.

Option 5: The composition, as in any one of aspects 1-4, where factor XI is platelet factor XI.

Option 6: The composition, as in any one of aspects 1-5, where factor XI is activated factor XI.

Option 7: The composition as in any one of aspects 1-6, where the composition also contains a TFPI inhibitor.

Option 8: The composition, as in any one of aspects 1-7, where the composition also contains factor VIII.

Aspect 2: A kit for treating bleeding, containing

a) an effective amount of factor VIIa 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) a container containing said first and second dosage forms.

Option 10: The composition as in aspect 2, containing

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

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

Aspect 3: A kit designed to treat bleeding, containing

a) an effective amount of a factor VIIa and TFPI inhibitor 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) a container containing said first and second dosage forms.

Aspect 4: A kit for treating bleeding, containing

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

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

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

Option 9: A kit, as in any one of options 2-4, also containing factor VIII, either prepared in a separate single dosage form, or in a single dosage form, also containing one or more compounds selected from the list of factor VIIa, factor XI or a TFPI inhibitor.

Aspect 6: Use of factor VIIa in combination with factor XI for the manufacture of a medicament for the treatment of bleeding in a subject.

Aspect 7: Use of factor VIIa in combination with factor XI for the manufacture of a medicament to reduce clotting time in a subject.

Aspect 8: The use of factor VIIa in combination with factor XI for the manufacture of a medicament in order to increase the time of fibrinolysis in mammalian plasma is normal.

Aspect 9: The use of factor VIIa in combination with factor XI for the manufacture of a medicament in order to increase the stability of a clot in a plasma of a mammal is normal.

Aspect 10: The use of factor VIIa in combination with factor XI for the manufacture of a medicament in order to increase the formation of fibrin clot in the plasma of a mammal is normal.

Aspect 11: A method for increasing the formation of a fibrin clot in a subject, the method comprising administering to the subject an effective amount of factor VIIa in combination with an effective amount of factor XI.

Aspect 12: A method for treating bleeding in subjects comprising administering to the subject an effective amount of factor VIIa in combination with an effective amount of factor XI.

Option 10: The method as in aspect 19 or 20, where factor VIIa and factor XI are administered in the same dosage form.

Option 11: A method as in aspect 19 or 20, where factor VIIa and factor XI are administered sequentially.

The present invention is also illustrated by the following examples, which, however, are not considered to limit the scope of protection. The characteristic features presented in the above description and in the following examples may, either individually or in any combination thereof, be involved in carrying out the invention in its various forms.

EXAMPLES

Example 1

Improving the stability of a hemostatic clot by combining coagulation factors VIIa and XI

METHODS:

Clot Dissolution Assay: Normal human plasma diluted 10-fold with buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ , pH 7.4) containing innovin (Dade Behring, 2000-fold dilution), rFVIIa (Novo Nordisk A / S, Bagsvaerd, Denmark; various concentrations) and t-PA (American Diagnostics, 8 nM) were added to 96-well ELISA plates and turbidity was measured at 650 nm over time at room temperature. Where indicated, purified human FXI factor (Haemotological Tachnologies, various concentrations) was added.

Rotational thromboelastography (roTEG): Measurements were performed using citrated, normal human plasma with 5 nM t-PA added, and the effect of adding 1 nM FVIIa factor alone or in combination with 30 nM FXI factor (Haemotological Tachnologies) was analyzed. Coagulation was initiated by adding innovin (final concentration 2000-fold dilution, Dade Behring # 526945) and calcium (final concentration 15 mM) in 20 mM HEPES buffer, 150 mM NaCl, pH 7.4.

RESULTS:

Analysis of clot dissolution: Addition of FVIIa factor resulted in a dose-dependent increase in clot dissolution time (Figure 1). This effect was optimal at 10 nM FVIIa. In the presence of 10 nM FVIIa, the addition of FXI factor led to a further increase in clot dissolution time (Figure 2). The effect was dose dependent and optimal at 30 nM FXI.

Thromboelastography: roTEG measurements were used to study the effect of FVIIa and FXI on maximum clot density (MCF), as well as on the resistance of clots to dissolution mediated by t-PA. Prior to the addition of FVIIa / FXI, the maximum clot density (MCF) was 25 mm, and the clot dissolution half-life was 12.3 minutes (FIG. 3). The addition of FXI (30 nM) did not alter the MCF, but increased the clot dissolution half-life to 16.1 minutes (Figure 3). Similarly, the addition of FVIIa (1 nM) led to the protection of the clot from fibrinolysis mediated by t-PA (half period of clot dissolution 16.7 min), without any effect on MCF (Figure 3). However, the addition of FVIIa (1 nM) together with FXI (30 nM) increases MCF (29 mm), as well as the half-period of clot dissolution (24, 2 min., FIG. 3).

CONCLUSION

The results show that the addition of FVIIa and FXI to plasma improves synergistically the mechanical strength and resistance to t-PA mediated fibrinolysis.

Example 2

Reducing coagulation time in human plasma is normal by combining coagulation factors VIIa and XI

METHODS

Clot analysis: Aliquots (55 μl) of rFVIIa (1 μg / ml) only, FXI (25 nM) only, or rFVIIa and FXI in 50 mM Pipes buffer, 100 mM NaCl, 2 mM EDTA, 1% BSA, pH 7.2 incubated for 5 min with 55 μl aliquots containing 100 μM PC / PS bubbles and 50 mM CaCl ₂ in the same buffer. Then 55 μl aliquots of normal human plasma (NHP) were added and clotting was monitored for 400 seconds in an ACL coagulation apparatus using an ARTT program.

RESULTS

Clot analysis: Prior to the addition of rFVIIa or FXI, NHP was not coagulated during a control time of 400 seconds. After adding FXI (25 nM), the clotting time was still longer than 400 s. The addition of FVIIa (1 μg / ml) reduced the clotting time to 159.4 ± 1.4 seconds (Figure 4). The addition of both FVIIa (1 μg / ml) and FXI (25 nM) reduced the clotting time to 95.0 ± 1.4 seconds (Figure 4).

CONCLUSION

The results show that the addition of FVIIa and FXI to plasma in a synergistic manner reduces coagulation time in NHP.

Claims (43)

1. A pharmaceutical composition comprising an effective amount of a factor VII preparation or a factor VII related polypeptide and a factor XI preparation or a factor XI related polypeptide. 2. The composition according to claim 1, where the specified factor VII or factor VII-related polypeptide is a factor VII-related polypeptide. 3. The composition according to claim 2, where the specified related factor VII polypeptide is a variant of the amino acid sequence of factor VII. 4. The composition according to claim 2 or 3, wherein the ratio between the activity of said factor VII-related polypeptide and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25 when the studies are carried out by analysis by hydrolysis in vitro as described herein. 5. The composition according to claim 1, where the specified factor VII or a related factor VII polypeptide is factor VII. 6. The composition according to claim 5, where the specified factor VII is human factor VII. 7. The composition according to claim 6, where the specified factor VII is a recombinant human factor VII. 8. The composition according to claim 1, where the specified factor VII or related to factor VII polypeptide is in activated form. 9. The composition of claim 8, where the specified factor VII is recombinant human factor VIIa. 10. The composition according to claim 1, where the specified factor XI or related factor XI polypeptide is a polypeptide related to factor XI. 11. The composition of claim 10, where the specified related factor XI polypeptide is a variant of the amino acid sequence of factor XI. 12. The composition of claim 10 or 11, wherein the ratio between the activity of said factor XI-related polypeptide and the activity of native human plasma factor XI (wild-type FXI) is at least about 1.25 when studies are performed using FXI- chromogenic analysis as described in the present description. 13. The composition according to claim 1 or 9, where the specified factor XI or a related factor XI polypeptide is a factor XI polypeptide. 14. The composition of claim 13, wherein said factor XI is human factor XI. 15. The composition according to 14, where the specified factor XI is a recombinant factor XI of human plasma. 16. The composition of claim 14, wherein said factor XI is a recombinant human platelet factor XI. 17. The composition according to claim 1, where the specified factor VII or related to factor VII polypeptide and the specified factor XI or related to factor XI polypeptide are present in a ratio by weight between approximately 100: 1 and 1: 100 (wt./wt. Factor VII: factor Xi). 18. The composition according to claim 1, where the composition also contains pharmaceutically acceptable excipients suitable for injection or infusion, especially for injection. 19. A set of components for the treatment of bleeding, containing a) an effective amount of a factor VII preparation or a factor VII related polypeptide and a pharmaceutically acceptable carrier in a first unit dosage form; c) an effective amount of a preparation of factor XI or a factor XI-related polypeptide and a pharmaceutically acceptable carrier in a second unit dosage form; and c) a container containing said first and second dosage forms. 20. The kit according to claim 19, where the specified factor VII or factor VII-related polypeptide is a factor VII-related polypeptide. 21. The kit of claim 20, wherein said factor VII-related polypeptides are variants of the amino acid sequence of factor VII. 22. The kit of claim 20 or 21, wherein the ratio between the activity of said factor VII-related polypeptide and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25 when the assays are performed by analysis by hydrolysis in vitro as described herein. 23. The kit according to claim 19, where the specified factor VII or a related factor VII polypeptide is factor VII. 24. The kit of claim 23, wherein said factor VII is human factor VII. 25. The kit according to paragraph 24, where the specified factor VII polypeptide is a recombinant human factor VII. 26. The kit according to claim 19, where the specified factor VII or related to factor VII polypeptide is in activated form. 27. The kit of claim 26, wherein said factor VII is a recombinant human factor VIIa. 28. The kit according to claim 19, where the specified factor XI or related factor XI polypeptide is a polypeptide related to factor XI. 29. The kit of claim 28, wherein said related factor XI polypeptide is a variant of the amino acid sequence of factor XI. 30. The kit of claim 28 or 29, wherein the ratio between the activity of said factor XI-related polypeptide and the activity of native human plasma factor XI (wild-type FXI) is at least about 1.25 when studies are performed using FXI-chromogenic analysis as described in the present description. 31. The kit according to claim 19 or 27, where the specified factor XI or a related factor XI polypeptide is factor XI. 32. The kit of claim 31, wherein said factor XI is human factor XI. 33. The kit according to p, where the specified factor XI is a recombinant factor XI of human plasma. 34. The kit of claim 32, wherein said factor XI is a recombinant human platelet factor XI. 35. The kit according to claim 19, wherein said factor VII or factor VII-related polypeptide and said factor XI or factor XI-related polypeptide are present in a weight ratio between about 100: 1 and 1: 100 (w / w factor VII: factor Xi). 36. The use of factor VII or a factor VII related polypeptide in combination with factor XI or a factor XI related polypeptide for the manufacture of a medicament for treating bleeding in a subject. 37. The application of clause 36, where the drug is intended to reduce coagulation time. 38. The application of clause 36, where the drug is intended to increase the time of fibrinolysis. 39. The application of clause 36, where the drug is intended to increase the strength of the clot. 40. The application of clause 36, where the drug is prepared for injection or infusion, especially for injection. 41. The application of clause 36, where the bleeding is the result of an injury or surgery or a decrease in the number or activity of platelets. 42. The application of clause 36, where the drug is presented in the form of a single dosage form. 43. The application of clause 36, where the drug is obtained in the form of a first single dosage form containing a preparation of factor VII or a factor VII related polypeptide, and a second single dosage form containing a preparation of factor XI or a factor XI related polypeptide.

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