JP2005508985A - Pharmaceutical composition comprising a Factor VII polypeptide and an aprotinin polypeptide - Google Patents

Abstract

The present invention relates to pharmaceutical compositions comprising Factor VII or Factor VII-related polypeptides and aprotinin or aprotinin-related polypeptides, and their use to treat the development of bleeding.

Description

FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions comprising Factor VII or Factor VII related polypeptides and aprotinin or aprotinin related polypeptides. The present invention also relates to a factor VII or factor VII related polypeptide and aprotinin or aprotinin related polypeptide for the treatment of a subject suffering from the onset of bleeding or for the manufacture of a medicament for preventing it Related to the use of combinations. The invention also relates to a method for treating the onset of bleeding in a subject and to a method for enhancing clot formation in a subject. The present invention also relates to kits containing these compounds.

BACKGROUND OF THE INVENTION Hemostasis is the formation of a complex between tissue factor (TF) exposed to circulating blood after injury to the vessel wall and FVIIa present in the circulation in an amount corresponding to about 1% of the total FVII protein mass. Started by. This complex is anchored to cells with TF and activates FX to FXa and FIX to FIXa on the cell surface. FXa activates prothrombin to thrombin, which activates FVIII, FV, FXI, and FXIII. In addition, the limited amount of thrombin formed at this early stage in hemostasis activates platelets. Following the action of thrombin on platelets, they change shape and expose charged phospholipids on their surface. This activated platelet surface forms a template for further FX activation and complete thrombin generation. At the activated platelet surface, further FX activation occurs via the FIXa-FVIIIa complex formed on the surface of the activated platelet, and FXa then converts prothrombin to thrombin, but still on the surface It is in. Thrombin then converts fibrinogen into fibrin that is insoluble and stabilizes the initial platelet plug. This process compartmentalizes or localizes the site where TF is expressed or exposed, thereby minimizing the risk of systemic activation of the coagulation system. The insoluble fibrin that forms the plug is further stabilized by cross-linking of fibrin fibers catalyzed by FXIII.

  FVIIa exists mainly in plasma as a single-chain enzyme precursor, which is cleaved by FXa into its double-stranded active form FVIIa. Recombinant active factor VIIa (rFVIIa) has been developed as a pro-haemostatic drug. Administration of rFVIIa provides a rapid and highly effective pre-hemostatic response in hemophilia subjects with bleeding that cannot be treated with clotting factor products due to antibody formation. Also, bleeding subjects deficient in factor VII or subjects with a normal coagulation system but receiving excessive bleeding can be successfully treated with FVIIa. In these studies, undesirable rFVIIa side effects (particularly the occurrence of thromboembolism) were not encountered.

  Exogenously administered FVIIa increases the formation of thrombin on the activated platelet surface. This occurs in hemophilia subjects lacking FIX or FVIII and thus lacks the most powerful pathway for complete thrombin formation. Also, in the presence of platelets with reduced platelet count or defective function, excess FVIIa increases thrombin formation.

  A commercial formulation of recombinant human FVIIa is marketed as NovoSeven® (NovoNordiskA / S, Denmark). NovoSeven® is designated for the treatment of bleeding episodes in patients with hemophilia A and B and is the only set available on the market for effective and reliable treatment of bleeding episodes. It is a replacement FVIIa.

  Aprotinin (bovine pancreatic trypsin inhibitor) is a Kunitz-type inhibitor known as an inhibitor of various serine proteases, including trypsin, chymotrypsin plasmin, and kallikrein, and is associated with acute pancreatitis, shock syndrome, hyperfibrinolysis, and myocardium Used therapeutically in the treatment of infarctions (ycf., Eg JE Trapnell et al., Brit. J. Surg. 61, 1974, p.177; J. McMichan et al., Circular shock 9, 1982, p.107; LM Auer et al., Acta Neurochair. 49, 1979, 207; G, Sher Am. J. Obstet. Gynecol. 129, 1977, p. 164; and B. Schneider, Artzneim.-Forsch. 26, 1976, p. 1606) . Administration of high concentrations of aprotinin significantly reduces blood loss associated with cardiac surgery including cardiopulmonary manipulation (eg, BP Bidstrup et al., J. Thorac. Cardiovasc. Surg. 97, 1989, pp. 364-372 ; See W. van Oeversen et al., Ann. Thorac. Surg. 44, 1987, pp. 640-645). Certain aprotinin analogs, such as aprotinin (1-58, Val15), have previously been shown to have a relatively high selectivity for granulocyte elastase and an inhibitory effect on collagenase (HR (See Wenzel and H. Tschesche, Angew. Chem. Internat. Ed. 20, 1981, p.295). Aprotinin (1-58, Ala15) has a weak effect on elastase, but aprotinin (3-58, Arg15, Ala17, Ser42) shows an excellent plasma kallikrein inhibitory effect (see WO89 / 10374).

  Kunitz inhibitors are generally basic, low molecular weight proteins that contain one or more inhibitory domains (“Kunitz domains”). The Kunitz domain is a folded domain of about 50-60 residues, forming a reverse transition beta sheet and a short C-terminal helix. This characteristic domain contains six cysteine residues that form three disulfide bonds, resulting in a double loop structure. There is an active inhibitory binding loop between the N-terminal region and the first beta strand. This binding loop is disulfide bonded to the hairpin loop formed between the latter two beta strands via the P2 cysteine residue. Isolated Kunitz domains derived from various proteinase inhibitors have been shown to have inhibitory activity (eg, Petersen et al., Eur. J. Biochem. 125: 310-316, 1996; Wagner et al., Biochem Biophys. Res. Comm. 186: 1138-1145, 1992; Dennis et al., J. Biol. Chem. 270: 25411-25417, 1995).

  It is well known that subjects who bleed excessively with surgery or major trauma and need blood transfusions develop more complications than people who have not had any bleeding. But also with moderate bleeding that requires the administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation defects, etc.), human viruses (hepatitis, HIV, May cause complications related to the risk of transmitting parvoviruses, and other currently unknown viruses. Massive bleeding that requires massive transfusions may lead to the development of multiple organ failure, including impaired lung and kidney function. Once a subject develops these serious complications, a cascade of events involving many cytokines and inflammatory responses is initiated, making any treatment extremely difficult and unfortunately often unsuccessful. Thus, the main goal in surgery as well as in the treatment of major tissue damage is to avoid or minimize bleeding. In order to avoid or minimize such bleeding, it is important to ensure the formation of a stable and firm stop thrombus that is not easily lysed by fibrinolytic enzymes. Furthermore, it is important to ensure the rapid and efficient formation of such plugs or clots.

  Today, subjects suffering from bleeding, including trauma victims and subjects bleeding with surgery, are often treated with some FVIIa injections or infusions, and the short half-life of FVIIa ( 2.5 hours), more than one dose is required to maintain a certain level of hemostasis. More rapid bleeding prevention would be an important benefit for such subjects. If so, the number of doses needed to stop bleeding and continue to stop bleeding will be reduced.

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

  EP 82.182 (Baxter Travenol Lab.) Relates to a composition of Factor VIIa for use in a subject in combating a defect in blood coagulation factor or the effect of an inhibitor on blood coagulation factor.

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

  US Pat. Nos. 5,206,161, 5,493,493, and 5,939,744 describe isolated monomeric human carboxypeptidase B (PCPB), expression vectors and host cells for making PCPB, and PCPB in the blood. The invention relates to a method for coagulating blood comprising adding.

  In the art, the occurrence of bleeding is surgery, trauma, or other forms of tissue damage; induction of coagulopathy including coagulopathy in subjects who have been transfused multiple times; decreased liver function (“liver disease”) Congenital or acquired coagulation or bleeding disorders, including: impaired platelet function or decreased platelet count; deficiency or abnormality of “compounds” (eg, platelets or von Willebrand factor protein) essential for coagulation; fibrin There is still a need to improve the treatment of subjects experiencing bleeding, including subjects who are due to increased lysis; anticoagulation or thrombolytic therapy; or stem cell transplantation.

  Improved, reliable and widely applicable in the art, enhances coagulation, ensures or enhances the formation of a stable antithrombotic, or convenience for a subject being treated There remains a need for methods to achieve complete hemostasis in subjects, particularly subjects with impaired thrombin production. There is also a need for a method with reduced bleeding prevention time.

Summary of the Invention One object of the present invention is to provide a composition that can be used effectively in the treatment or prevention of bleeding episodes and coagulation disorders.

  A second object of the present invention is to provide a composition in a single unit dosage form that can be used effectively in the treatment or prevention of bleeding episodes, or as a clot. Another object of the present invention is to provide a composition, method of treatment or kit that exhibits a synergistic effect.

  It is a further object of the present invention to provide a composition, method of treatment, or kit that does not exhibit substantial side effects, such as systemic high level activation of the coagulation system.

  Other objects of the present invention will become apparent by reading this specification.

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

In a second aspect, the present invention is a kit of parts comprising a therapeutic agent for the onset of bleeding,
a) an effective amount of a preparation of Factor VII or a Factor VII-related polypeptide in a first unit dosage form, and a pharmaceutically acceptable carrier;
b) an effective amount of a preparation of aprotinin or aprotinin-related polypeptide in a second unit dosage form, and a pharmaceutically acceptable carrier; and
c) container means for containing said first and second dosage forms;
A kit of parts including

  In a third aspect, the invention provides the use of factor VII or a factor VII-related polypeptide in combination with aprotinin or an aprotinin-related polypeptide for the manufacture of a medicament for treating the development of bleeding in a subject To do. In a further aspect, the present invention provides the use of a composition according to any one of claims 1-18 for the manufacture of a medicament for treating the development of bleeding in a subject.

  In its different embodiments, the drug extends the clot lysis time to reduce the time needed to obtain complete hemostasis, thus reducing the time required to maintain hemostasis, and reducing the clotting time. To increase clot strength.

  In different embodiments, the drug may cause bleeding due to surgery, trauma, or other forms of tissue damage; induction of coagulopathy including coagulopathy in a subject who has been transfused multiple times; decreased liver function (“liver disease”) Congenital or acquired coagulation or bleeding disorders, including:) defective platelet function or decreased platelet count; deficiency or abnormality of essential coagulation “compounds” (eg, platelets or von Willebrand factor protein); fibrinolysis Anti-coagulation or thrombolytic therapy; or to treat a subject who has developed bleeding due to stem cell transplantation. In a series of embodiments, the bleeding occurs in organs such as the brain, inner ear region, eyes, liver, lungs, tumor tissue, gastrointestinal tract; in another series, it is in hemorrhagic gastritis and copious uterine bleeding Such as diffuse bleeding. In another set of embodiments, the onset of bleeding is acute haemarthroses (joint bleeding), chronic haemophilia arthropathy, hematoma (eg, muscle, retroperitoneum, sublingual, and posterior pharynx) ), Bleeding in other tissues, hematuria (bleeding from the renal tract), cerebral bleeding, surgery (eg, hepatectomy), tooth extraction, and surgery in subjects with gastrointestinal bleeding (eg, UGI bleeding) Or bleeding associated with trauma. In one embodiment, the drug is for treating the occurrence of trauma, surgery, or bleeding due to decreased platelet count or activity in a subject.

  In a further aspect, the present invention is a method for treating the development of bleeding in a subject, said method comprising, for a subject in need thereof, a label of factor VII or a factor VII-related polypeptide. Administering a first amount of the product and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts are both effective for treating bleeding Provide a method.

  In a further aspect, the present invention is a method for reducing clotting time in a subject, said method comprising, for a subject in need thereof, a preparation of factor VII or a factor VII-related polypeptide And a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts are both effective to reduce clotting time Provide a method.

  In a further aspect, the present invention is a method for enhancing hemostasis in a subject, said method comprising, for a subject in need thereof, a first of a preparation of factor VII or a factor VII-related polypeptide. Administering an amount and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts together provide a method that is effective to enhance hemostasis.

  In a further aspect, the present invention is a method for extending clot lysis time in a subject, said method comprising, for a subject in need thereof, a factor VII or a factor VII related polypeptide Administering a first amount of a preparation of and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, the first and second amounts both extending clot lysis time To provide a method that is effective for:

  In a further aspect, the present invention is a method for increasing clot strength in a subject, said method comprising subjecting a subject in need thereof to a factor VII or a factor VII-related polypeptide. Administering a first amount of the product and a second amount of a preparation of aprotinin or aprotinin-related polypeptide, wherein the first and second amounts are both effective to increase clot strength To provide a way to be.

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

  In another series of embodiments, the factor VII or factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide comprise a first unit dosage form comprising a preparation of a factor VII or factor VII-related polypeptide and an aprotinin or aprotinin related Administered in the form of a second unit dosage form containing a polypeptide preparation. In that series of embodiments, the first unit dosage form and the second unit dosage form are not administered more than 15 minutes apart.

In a further aspect, the present invention is a kit comprising a therapeutic agent for the development of bleeding,
d) an effective amount of Factor VII or a Factor VII-related polypeptide, and an effective amount of aprotinin or an aprotinin-related polypeptide, and a single dosage form of a pharmaceutically acceptable carrier; and
e) container means for containing said single unit dosage form;
A kit is provided.

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

  In one series of embodiments of the invention, the factor VII or factor VII-related polypeptide is factor VII. In one embodiment, the factor VII is human factor VII. In one embodiment, the Factor VII is bovine, porcine, dog, mouse, horse, or salmon Factor VII. In another embodiment, factor VII is made recombinantly. In another embodiment, the factor VII is derived from plasma. In a preferred embodiment, the factor VII is recombinant human factor VII. In one series of embodiments of the invention, the Factor VII or Factor VII related polypeptide is its active form. In one preferred embodiment of the invention, the factor VII is recombinant human factor VIIa.

  In a series of embodiments, the aprotinin or aprotinin-related polypeptide is an aprotinin-related polypeptide. In one embodiment, the aprotinin-related polypeptide is a variant of the aprotinin amino acid sequence. In one embodiment, the ratio between the activity of an aprotinin-related polypeptide and the activity of native bovine plasma aprotinin (wild type aprotinin) when tested in an “aprotinin assay” as described in the present invention, At least about 1.25. In one embodiment, the aprotinin or aprotinin-related polypeptide is an aprotinin polypeptide. In one embodiment, the aprotinin is siacia protinin. In one embodiment, the aprotinin is pig, dog, horse, mouse, or salmon aprotinin. In a preferred embodiment, aprotinin is made recombinantly. In another embodiment, aprotinin is derived from plasma. In a preferred embodiment, the aprotinin is a recombinant siasia protinin. In one series of embodiments of the invention, the aprotinin or aprotinin-related polypeptide is its active form. In one embodiment, the aprotinin-related polypeptide is a fragment of aprotinin. In one embodiment, the aprotinin-related polypeptide is a hybrid aprotinin polypeptide, such as a pig / bovine hybrid. In one embodiment, the aprotinin polypeptide is a human aprotinin equivalent. In one embodiment, the aprotinin equivalent is an aprotinin amino acid sequence variant in which the total positive charge of the protein is reduced. In one embodiment, the aprotinin polypeptide is a Kunitz-type inhibitor domain with aprotinin activity.

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

  In one embodiment, the Factor VII-related polypeptide is an amino acid sequence variant in which no more than 2 amino acids have been substituted, deleted, or inserted compared to wild-type Factor VII (ie, US Pat. Polypeptide having the amino acid sequence disclosed in 4,784,950). In another embodiment, the Factor VII variant is an amino acid sequence variant in which 15 or fewer amino acids have been substituted, deleted, or inserted. In other embodiments, a Factor VII variant has 10 or fewer amino acids substituted, deleted, or inserted, such as 8, 6, 5, or 3 amino acids compared to wild type Factor VII. In one embodiment, the factor VII variant is selected from the following list: L305V-FVIIa, L305V / M306D / D309S-FVIIa, L3051-FVIIa, L305T-FVIIa, F374P-FVIIa, V158T / M298Q-FVIIa, V158D / E296V / M298Q-FVIIa, K337A-FVIIa, M298Q-FVIIa, V158D / M298Q-FVIIa, L305V / K337A-FVIIa, V158D / E296V / M298Q / L305V-FVIIa, V158D / E296V / M298Q / K337A-FVIIa, V158D / E296 / M298Q / L305V / K337A-FVIIa, K157A-FVII, E296V-FVII, E296V / M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII, and S336G-FVII.

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

  In a preferred embodiment, the Factor VII or Factor VII-related polypeptide and the Factor VII or Factor VII-related polypeptide are recombinant human Factor VIIa and recombinant siasia protinin.

  In one embodiment, clotting time is reduced in mammalian blood. In another embodiment, hemostasis is enhanced in mammalian blood. In another embodiment, clot lysis time is prolonged in mammalian blood. In another embodiment, clot strength is increased in mammalian blood. In one embodiment, the mammalian blood is human blood. In another embodiment, the mammalian blood is normal human blood; in one embodiment, the blood is from a subject with impaired thrombin generation; in one embodiment, the blood is Blood from a subject having a deficiency of one or more clotting factors; in another embodiment, the blood is blood from a subject having an inhibitor to one or more clotting factors; In one embodiment, the blood is from a subject with reduced levels of fibrinogen; in one embodiment, the blood is human blood deficient in an aprotinin polypeptide. In one series of embodiments, the blood is plasma.

  In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide are the only hemostatic agents included in the composition. In another embodiment, Factor VII or Factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide are the only active hemostatic agents included in the composition. In another embodiment, Factor VII or Factor VII-related polypeptide, and aprotinin or aprotinin-related polypeptide are the only clotting factors that are administered to the subject. In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide are the only active agents administered to the patient. In one embodiment, the composition is substantially free of thrombin or prothrombin; in another embodiment, the composition is substantially free of FX; in another embodiment, the composition is substantially free of FX. Does not include FXa.

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

  In one embodiment of the invention, the composition is a single unit dosage form, wherein the single unit dosage form comprises both clotting factors. In one embodiment of the invention, the composition comprises a preparation of factor VII or a factor VII-related polypeptide as a first unit dosage form and a preparation of aprotinin or an aprotinin-related polypeptide as a second unit dosage form. And is in the form of a kit of parts including container means for containing the first and second dosage forms. In one embodiment, the composition or kit further includes instructions for administering each of the compositions or separated components as applicable.

  In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide are administered in a single dosage form. In one embodiment of the invention, the factor VII or factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide comprise a first unit dosage form comprising a preparation of factor VII or factor VII-related polypeptide and aprotinin or aprotinin related Administered in the form of a second unit dosage form containing a polypeptide preparation.

  In one embodiment of the invention, Factor VII or Factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide are administered simultaneously. In another embodiment, the Factor VII or Factor VII related polypeptide and the aprotinin or aprotinin related polypeptide are administered sequentially. In one embodiment, the Factor VII or Factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide are administered 15 minutes apart, preferably 10 minutes, more preferably 5 minutes, more preferably more than 2 minutes apart. Not. In one embodiment, the factor VII or factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide are up to 2 hours, preferably 1-2 hours, more preferably up to 1 hour, more preferably 30 minutes to 1 hour, More preferably up to 30 minutes, more preferably 15-30 minutes apart.

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

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

  In one embodiment of the invention, the pharmaceutical composition is a single unit dosage form and consists essentially of a preparation of Factor VII or a Factor VII-related polypeptide, as well as a preparation of protein C inhibition, and a pharmaceutical Consisting of one or more ingredients selected from the list of acceptable carriers, stabilizers, surfactants, neutral salts, antioxidants, preservatives, and protease inhibitors.

  In another embodiment of the invention, the pharmaceutical composition comprises essentially a preparation of Factor VII or a Factor VII-related polypeptide as well as a pharmaceutically acceptable carrier, stabilizer, surfactant, neutral salt, With a first unit dosage form consisting of one or more components selected from the list of oxidizing agents, preservatives, and protease inhibitors, as well as preparations of aprotinin or aprotinin-related polypeptides and pharmaceutically acceptable Of a part with a second unit dosage form consisting of one or more ingredients selected from the list of carriers, stabilizers, surfactants, neutral salts, antioxidants, preservatives, and protease inhibitors It is in the form of a kit.

  In a further embodiment, the subject is a human; in another embodiment, the subject has impaired thrombin generation; in one embodiment, the subject has a reduced plasma concentration of fibrinogen. (Eg, a subject who has been transfused multiple times); In one embodiment, the subject has a reduced factor VIII or FIX plasma concentration of the factor.

  In another aspect, the present invention is a method of enhancing hemostasis in a subject suffering from a factor VII responsive syndrome as compared to treating the subject with factor VII as the only coagulation protein. The method comprises, for a subject in need thereof, a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, Wherein the first and second amounts are both effective to enhance hemostasis.

  In another aspect, the present invention is a method for enhancing thrombin formation in a subject, said method comprising, for a subject in need thereof, a factor VII or a factor VII-related polypeptide. Administering a first amount of a preparation and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts are both effective to enhance thrombin formation Is related to the method.

  In another aspect, the present invention provides a method for enhancing thrombin formation in a subject suffering from a factor VII responsive syndrome as compared to treating the subject with factor VII as the only coagulation protein. The method includes, for a subject in need thereof, a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide; Wherein the first and second amounts are both effective to enhance thrombin formation.

  In another aspect, the present invention provides hemostasis in a subject suffering from a factor VII responsive syndrome as compared to the number of doses required when the subject is administered factor VII as the only coagulation factor protein. A method for reducing the number of coagulation factor protein doses required to achieve the above, wherein the method comprises, for a subject in need thereof, a preparation of a factor VII or factor VII-related polypeptide preparation. Administering a first amount and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, said first and second amounts together to reduce the number of coagulation factor protein doses It relates to a method that is effective.

  In another aspect, the present invention is a method of treating bleeding in a subject having a factor VII responsive syndrome, wherein the method comprises: Administering a first amount of a preparation of Factor VII-related polypeptide and a second amount of aprotinin or a preparation of aprotinin-related polypeptide, wherein the first and second amounts together cause bleeding. It relates to a method that is effective for treating.

  In one embodiment, the factor VII is human recombinant factor VIIa (rFVIIa). In another embodiment, the rFVIIa is Novo Seven® (Novo Nordisk A / S, Bagsvaerd, Denmark).

  In another aspect, the invention relates to the use of factor VII or a factor VII-related polypeptide in combination with aprotinin or an aprotinin-related polypeptide for the manufacture of a medicament for enhancing fibrin clot formation in mammalian plasma. .

  In another aspect, the present invention is a method of enhancing fibrin clot formation in a subject, said method comprising, for a subject in need thereof, a factor VII or a factor VII-related polypeptide. Administering a first amount of a preparation and a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts are both effective for treating bleeding. Regarding a certain method.

Detailed Description of the Invention Subjects who bleed excessively in connection with surgery or serious trauma and need blood transfusions develop more complications than those who have not experienced any bleeding. However, if they require administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates, etc. for the treatment of coagulation defects), this may cause human viruses (hepatitis, HIV , Parvovirus, and other currently unknown viruses) and non-viral pathogens, so even moderate bleeding may cause complications. Once a subject develops these serious complications, a cascade of events involving many cytokines and inflammatory responses is initiated, making any treatment extremely difficult and unfortunately often unsuccessful. Patients who experience most blood loss become clinically unstable. Such patients are at risk of undergoing atrial fibrillation, which can result in fatal cessation of cardiac activity; renal dysfunction; or fluid extravasation in the lungs (so-called “wet lungs” or ARDS) There is also. Thus, the main goal in surgery as well as in the treatment of major tissue damage is to avoid or minimize bleeding. In order to avoid or minimize such undesired bleeding, it is important to ensure the formation of a stable and solid hemostasis that is not easily dissolved by fibrinolytic enzymes. Furthermore, it is important to ensure the rapid and efficient formation of such plugs or clots.

  In addition, subjects with thrombocytopenia (with reduced platelet count or activity) are more likely to be lysed in the middle due to impaired thrombin generation and incomplete fibrin plug stabilization. Causes blood clots. In addition, subjects with major trauma or organ damage often result in frequent transfusions, decreased platelet counts, and decreased levels of fibrinogen, factor VIII, and other clotting proteins . These subjects experience an impairment (or reduction) in thrombin generation. Thus, in these subjects, hemostasis is defective or has little effect and will form a fibrin plug that is easily and partially dissolved by proteolytic enzymes, and such enzymes Released extensively in aspects characterized by extensive trauma and organ damage.

  There is also the possibility that hematoma may form due to tissue bleeding. The size of the hematoma (especially in the skull and spinal cord) indicates the degree of neurological loss, difficulty of rehabilitation, and / or the severity and extent of permanent loss of neurological function after loss and rehabilitation Closely related to The most severe hematoma results are seen when they are located in the brain, which will cause even the death of the patient.

  Thus, the primary goal in the treatment of bleeding is to provide hemostasis in the shortest possible time, thus minimizing blood loss.

  Thus, the present invention provides beneficial compositions, uses, and methods of treatment for the treatment of bleeding episodes in subjects in need of such treatment. Useful compositions, uses, and methods reduce blood loss before hemostasis is performed, reduce blood required during surgery, and maintain blood pressure at an acceptable level until hemostasis occurs More rapid stabilization of blood pressure, shortening the recovery time of treated patients, reducing rehabilitation time of treated patients, reducing the formation of hematomas including brain hematomas or reducing the formation of hematomas Will be involved in beneficial effects such as rapid deterrence of bleeding, cessation of bleeding and reduction in the number of doses required to maintain hemostasis.

  Administration of either factor VIIa or aprotinin or aprotinin-related polypeptide alone by administering factor VII or a factor VII-related polypeptide, such as a sample of factor VIIa, in combination with a protinin or aprotinin-related polypeptide preparation When compared to clotting time, clot firmness, and tolerance, it results in reduced clotting time, firm clots, and increased resistance to fibrinolysis.

  Alternatively, either factor VIIa or aprotinin or aprotinin-related polypeptide alone is administered by administering a factor VII or factor VII-related polypeptide, such as a factor VIIa preparation, in combination with a preparation of aprotinin or aprotinin-related polypeptide. Compared to the situation when administered at a time, there is a reduction in time to control bleeding and a reduction in the number of doses to maintain hemostasis. The present invention provides a beneficial effect in the simultaneous or sequential dosing of an aprotinin or aprotinin related polypeptide preparation and a factor VII or factor VII related polypeptide preparation. The pharmaceutical composition according to the present invention may be in the form of a single composition or may be in the form of a multi-component kit (part kit). The compositions according to the present invention are useful as therapeutic and prophylactic clots in mammals including primates such as humans. The present invention further provides a method for treating (including prophylactically treating or preventing) the development of bleeding in a subject, including a human.

  First, second or third other unit doses are referred to throughout this specification, but do not indicate the preferred order of administration, but merely for convenience.

  The combination of a Factor VII or Factor VII-related polypeptide preparation and an aprotinin or aprotinin-related polypeptide preparation is an advantageous product that ensures a short clotting time, rapid formation of a stop thrombus, and the formation of a stable stop thrombus It is. The fact that the combination of factor VII or a factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide is an advantageous product that ensures the formation of a robust, stable and rapidly formed hemostatic thrombus Found by the inventor.

  The inventors of the present invention have shown that the combination of factor VIIa and aprotinin can increase clot firmness more efficiently than either factor VIIa or aprotinin alone. It was also expected that a further increase in clot firmness was observed by combining factor VII or a factor VII-related polypeptide at a concentration at which no further increase in clot firmness was observed with aprotinin It was outside. Also, factor VII or a combination of factor VII-related polypeptides and aprotinin or aprotinin-related polypeptides can prolong the in vitro clot lysis time in normal human plasma more efficiently than either factor VIIa or aprotinin alone. It was shown that it can be done. Also, factor VII or a factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide combination can prolong half clot lysis time in normal human plasma more efficiently than either factor VIIa or aprotinin alone It has been shown. Also, factor VII or a combination of factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide is more efficient than either factor VIIa or aprotinin alone in fibrinolysis in normal human plasma, especially tPA-mediated fibrin It has been shown that clots can be protected from lysis.

  Therefore, by enhancing coagulation, a more effective treatment of the subject's bleeding can be obtained.

  While not wishing to be obligated by theory, it is believed that complete thrombin generation is necessary to form a robust and stable hemostatic clot and, consequently, to maintain hemostasis. The fibrin structure of such plugs is dependent on the amount of thrombin formed and the initial rate of thrombin generation. In the presence of obstructive thrombin generation, a highly permeable porous fibrin plug is formed. The fibrinolytic enzyme normally present on the surface of fibrin readily dissolves such fibrin plugs. The formation of a stable fibrin plug is also dependent on the presence of factor XIIIa activated by thrombin and thus also on complete thrombin generation. Furthermore, the recently described fibrinolytic inhibitor aprotinin, which can activate thrombin, requires rather large amounts of thrombin for its activation. In the presence of thrombin formation, which is not fully sufficient, aprotinin is not activated, thus resulting in the formation of a stopped thrombus that is easier than normal degradation due to normal fibrinolytic activity. In thrombocytopenia, a situation where the platelet count has decreased, administration of extra exogenous factor VIIa initiates more rapid thrombin generation. However, total thrombin production is not normalized even by high concentrations of factor VIIa.

  Complete thrombin activation does not occur in subjects with reduced plasma concentrations of fibrinogen (subjects who have been transfused multiple times as a result of many traumas or extensive surgery). A more effective hemostasis is then performed by administering a combination of Factor VII and aprotinin.

  Subjects with thrombocytopenia result in impaired thrombin generation as well as fibrin plug stabilization defects, resulting in a thrombus that is prone to dissolution in the middle. In addition, subjects who have undergone major trauma or tissue damage have resulted in frequent transfusions, reduced platelet counts, and decreased levels of fibrinogen, factor VIII, and other clotting proteins Will be. These subjects experience an impairment (or reduction) in thrombin generation. In addition, these reduced fibrinogen levels negatively prevent factor XIII activation. Thus, these subjects are either deficient in hemostasis or are not very efficient, and proteolytic enzymes, in addition to such enzymes, are widely used in situations characterized by extensive trauma and organ damage. By being released, it causes the formation of a fibrin plug that is easily and prematurely dissolved.

  In order to facilitate the formation of a fully stabilized plug with full ability to maintain the subject's hemostasis, the composition according to the invention is administered. This composition is particularly beneficial in subjects with reduced platelet counts and in subjects with reduced plasma levels of fibrinogen and / or other clotting proteins.

Factor VII polypeptide:
In practicing the present invention, any Factor VII polypeptide effective to prevent or treat bleeding may be used. This includes factor VII polypeptides derived from blood or plasma, or produced by recombinant means.

  The present invention includes Factor VII polypeptides such as those having the amino acid sequence disclosed, for example, in US Pat. No. 4,784,950 (wild type human Factor VII). In some embodiments, the factor VII is a human factor VIIa polypeptide as disclosed, for example, in US Pat. No. 4,784,950 (wild type factor VII). In a series of embodiments, the Factor VII polypeptide has a specific biological activity of human Factor VIIa of at least about 10%, preferably at least about 30%, more preferably at least about 50%, and most preferably at least about 70%. The polypeptide shown is included. In a series of embodiments, the Factor VII polypeptide is 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% human Factor VIIa Including a polypeptide exhibiting a specific biological activity. In a series of embodiments, the Factor VII polypeptide has at least about 70%, preferably at least about 80%, more preferably at least about 90%, with the sequence of wild type Factor VII as disclosed in U.S. Pat.No. 4,784,950, and Most preferably, it comprises a polypeptide that exhibits at least about 95% identity.

  As used herein, a “Factor VII polypeptide” includes, but is not limited to, Factor VII as well as Factor VII related polypeptides. The term “Factor VII” refers to wild type human Factor VII (disclosed in US Pat. No. 4,784,950) and other species such as bovine, pig, dog, mouse, and salmon factor VII. It is contemplated to include, but is not limited to, a polypeptide having the amino acid sequence 1-406 of Factor VII, said Factor VII being derived from blood or plasma or produced by recombinant means. This further includes natural allelic variations of Factor VII that may exist and occur from one individual to another. Also, the extent and site of glycosylation or other post-translational modifications will vary depending on the host cell chosen and the nature of the host cell's environment. The term “Factor VII” is also referred to as the uncut (enzyme precursor) form of the Factor VII polypeptide as well as Factor VIIa, which is proteolytically processed to produce each of these bioactive forms. It is intended to include Typically, factor VII is cleaved between residues 152 and 153 to produce factor VIIa.

  A “Factor VII-related polypeptide” is chemically modified compared to human Factor VII and / or includes one or more amino acid sequence changes compared to human Factor VII (ie, a Factor VII variant) And / or any factor VII polypeptide, including, but not limited to, a truncated amino acid sequence compared to human factor VII (ie, a factor VII fragment). Such factor VII-related polypeptides may exhibit different properties, including stability, phospholipid binding, altered specific activity, etc., compared to human factor VII. The term “factor VII-related polypeptide” refers to the uncut (enzyme precursor) form of such a polypeptide as well as the “factor VIIa” that has been proteolytically processed and produced into their respective bioactive forms. It is contemplated to include what are referred to as “related polypeptides” or “activated factor VII-related polypeptides”.

  As used herein, a “Factor VII-related polypeptide” is a polypeptide that exhibits substantially the same or improved biological activity as compared to wild-type human Factor VII, as well as wild-type human VIIa. Including, but not limited to, a polypeptide in which factor VIIa biological activity is substantially modified or reduced compared to factor activity. These polypeptides include chemically modified Factor VII or VIIa and Factor VII variants in which the bioactivity of the polypeptide has been modified or disrupted by introduction of specific amino acid sequence changes. It is not limited.

  Polypeptides having a slightly modified amino acid sequence, such as polypeptides having a modified N-terminus including deletion or addition of the N-terminal amino acid, and / or chemically modified poly compared to human factor VIIa Contains peptides.

  Factor VII-related polypeptides, including variants of factor VII, have substantially the same or better bioactivity as wild-type factor VII, or alternatively have been modified or reduced compared to wild-type factor VII Including a polypeptide having a different amino acid sequence from that of wild-type factor VII by insertion, deletion, or substitution of one or more amino acids, regardless of whether it exhibits biological activity However, it is not limited to these.

  A Factor VII-related polypeptide comprising a variant is a wild-type Factor VIIa produced in the same cell type when tested in one or more of a clotting assay, proteolytic assay, or TF binding experiment as described above. 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 70%, at least about 75%, at least about 80%, at least Including those exhibiting about 90%, at least about 100%, at least about 110%, at least about 120%, or at least about 130%.

  Factor VII-related polypeptides, including variants, have substantially the same or improved biological activity as compared to wild-type factor VIIa, and as described above, clotting assay, proteolytic assay, or TF binding assay At least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 25% of the specific activity of wild-type factor VIIa produced in the same cell type when tested in one or more of the methods Including those exhibiting at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130%.

  A Factor VII-related polypeptide containing a variant and having substantially reduced biological activity compared to wild-type Factor VIIa is one of the coagulation assays, proteolytic assays, or TF binding experiments as described above. Or at most about 25%, preferably at most about 10%, more preferably at most about 5%, most preferably most of the specific activity of wild-type factor VIIa produced in the same cell type when tested in multiple Both show about 1%. Factor VII variants with substantially modified biological activity compared to wild-type factor VII bind to TF and TF variants that exhibit TF-independent factor X proteolytic activity, but factor X However, the present invention is not limited to these.

  In some embodiments, a Factor VII polypeptide is a Factor VII polypeptide, particularly when tested in an “in vitro hydrolysis assay” (see “Assays” below). And the activity of native human Factor Vila (wild type FVIIa) is at least about 1.25; in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about It is a variant that is 4.0.

  In some embodiments of the invention, the Factor VII polypeptide is a Factor VII related polypeptide, particularly when tested in an “in vitro hydrolysis assay” (see “Assays” below). The ratio between the activity of the factor polypeptide and the activity of native human Factor VIIa (wild type FVIIa) is at least about 1.25; in other embodiments, the ratio is at least about 2.0; A variant that is at least about 4.0; in a further embodiment, the ratio is a variant that is at least about 8.0.

  In some embodiments, the Factor VII polypeptide is human Factor VII, for example, as disclosed in US Pat. No. 4,784,950 (wild type Factor VII). In some embodiments, the Factor VII polypeptide is human Factor VIIa. In a series of embodiments, the Factor VII polypeptide exhibits at least about 10%, more preferably at least about 50%, preferably at least about 30%, and most preferably at least about 70% of the biospecific activity of human Factor VIIa. A factor-related polypeptide. In some embodiments, a Factor VII polypeptide has an amino acid sequence that differs from that of wild-type Factor VII by one or more amino acid insertions, deletions, or substitutions.

  Non-limiting examples of variants of factor VII that have substantially the same or superior biological activity compared to wild type factor VIIa are Danish patent application numbers PA2000 00734 and PA2000 01360 (corresponding to WO01 / 83725) And those described in PA2000 01361 (corresponding to WO02 / 22776), but are not limited thereto. Non-limiting examples of Factor VII variants with biological activity substantially equivalent or improved to wild type Factor VII include S52A-FVII, S60A-FVII (lino et al., Arch. Biochem. Biophys. 352: 182-192 L305V-FVII, L305V / M306D / D309S-FVII, L3051-FVII, L305T-FVII, F374P-FVII, V158T / M298Q-FVII, V158D / E296V / M298Q-FVII, K337A-FVII, M298Q-FVII, V158D / M298Q-FVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVII, V158D / E296V / M298Q / K337A-FVII, V158D / E296V / M298Q / L305V / K337A-FVII, K157A-FVII, E296V- FVII, E296V / M298Q-FVII, V158D / E296V-FVII, V158D / M298K-FVII, and S336G-FVII; FVIIa mutations exhibiting increased proteolytic stability as disclosed in US Pat. No. 5,580,560 Body; Factor VIIa cleaved by proteolysis between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); and the oxidized form of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54, 1999) Mu Non-limiting examples of Factor VII variants with substantially reduced or modified biological activity compared to wild type Factor VII are R152E-FVIIa (Wildgoose et al., Biochem 29: 3413-3420, 1990), S344AFVIIa (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 the Gla domain. Contains the missing factor VIIa (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. No. 5,997,864.

  The biological activity of factor VIIa in blood coagulation is as follows: (i) the ability to bind to tissue factor (TF) and (ii) the proteolytic cleavage of factor IX or factor X to activate Or derived from the ability to produce factor X (factor IXa or factor Xa, respectively).

For the purposes of the present invention, the biological activity of a Factor VII polypeptide ("Factor VII biological activity") is determined using plasma and thromboplastin deficient in Factor VII, for example, as described in US Pat. No. 5,997,864. Alternatively, the preparation may be quantified by measuring its ability to promote blood clotting. In this assay, biological activity is manifested as a decrease in clotting time compared to the subject sample and is converted to “factor VII units” by comparison to a pooled human serum standard containing 1 unit / ml factor VII activity. . Alternatively, factor VIIa biological activity is:
(I) measuring the ability of factor VIIa or a factor VIIa-related polypeptide to produce activated factor X (factor Xa) in a system comprising TF embedded in a lipid membrane and factor X (Persson et al., J. Biol. Chem. 272: 19919-19924,1997);
(Ii) measuring hydrolysis of factor X in an aqueous system (see “In Vitro Proteolysis Assay”, see below);
(Iii) measuring the physical binding of factor VIIa or a factor VIIa related polypeptide to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413: 359-363, 1997); and,
(Iv) measuring hydrolysis of the synthetic substrate by Factor VIIa and / or Factor VIIa related polypeptides (see “In Vitro Hydrolysis Assay”, see below);
(V) measurement of thrombin generation in an in vitro system independent of TF,
May be quantified.

  The term “factor VII biological activity” or “factor VII activity” is intended to include the ability to generate thrombin; the term is used in the surface of platelets activated in the absence of tissue factor. Including the ability to generate

  A Factor VIIa preparation that may be used in accordance with the present invention is, but is not limited to, NovoSeven® (Novo Nordisk AIS, Bagsvaerd, Denmark).

Aprotinin polypeptide :
The present invention encompasses the use of aprotinin polypeptides, such as those having the amino acid sequence of, for example, wild-type uxiaprotinin as well as aprotinin analogs such as those described below; Castro MJ. Et al., Biochemistry 1996 Sep 3; 35 (35 ): 11435-46; Stassen et al., Thromb Haemost. 1995 Aug; 74 (2): 655-96; Stassen et al., Thromb Haemost. 1995 Aug; 74 (2): 646-54; Grzesiak A. et al., J Mol Biol 2000 Aug 4; 301 (1): 205-17; WO 89/10374, U.S. Pat.No. 5591603, U.S. Pat.No. 5618915, U.S. Pat.No. 5610774, European Patent No. 375718, European Patent No. No. 3399428, U.S. Pat.No. 5,730,090, U.S. Pat. No., European Patent No. 621871, German Patent Application PA2001 00859. In practicing the present invention, any aprotinin polypeptide that is effective in preventing or treating bleeding may be used. This includes aprotinin polypeptides derived from bovine tissue or aprotinin polypeptides produced by recombinant means.

  As used herein, “aprotinin polypeptide” includes, but is not limited to, aprotinin as well as aprotinin-related polypeptides. The term “aprotinin” includes polypeptides having the amino acid sequence of wild-type aprotinin derived from native uxiaprotinin (wild-type siaciaprotinin) and other species such as, for example, porcine, dog, mouse, and salmon aprotinin It is contemplated, but not limited to.

  The term “aprotinin” is intended to encompass all naturally occurring aprotinin polypeptides. The term “aprotinin” is also intended to include wild-type aprotinin and polypeptides having the amino acid sequence of wild-type aprotinin from other species such as, for example, porcine, canine, and mouse aprotinin. However, it is not limited to these. It further encompasses natural allelic variations of aprotinin that may exist and occur from one individual to another. Also, the extent and site of glycosylation or other post-translational modifications will vary depending on the host cell chosen and the nature of the host cell environment.

  It further encompasses natural allelic variations of Factor VII that may exist and occur from one individual to another. Also, the extent and site of glycosylation or other post-translational modifications will vary depending on the host cell chosen and the nature of the host cell's environment. The term “aprotinin” is also intended to include aprotinin polypeptides in the form of enzymatic precursors as well as those proteolytically processed to their respective bioactive forms.

  An “aprotinin-related polypeptide” is chemically modified compared to siaciaprotinin and / or contains one or more amino acid sequence changes compared to siaciaprotinin (ie, an aprotinin variant), and Including, but not limited to, an aprotinin polypeptide that includes an amino acid sequence that has been cleaved compared to usaprotinin (ie, an aprotinin fragment) and / or any of the Kunitz-type inhibitor polypeptides that have aprotinin activity It is not. Such aprotinin-related polypeptides may exhibit different properties, including stability, phospholipid binding, altered specific activity, etc., compared to oxaprotinin.

  The term “aprotinin-related polypeptide” is intended to include forms of such polypeptide enzyme precursors as well as those proteolytically processed to produce their respective bioactive forms.

  As used herein, an “aprotinin-related polypeptide” is a polypeptide that exhibits substantially the same or improved biological activity as compared to wild-type siasiaprotinin, as well as the activity of wild-type siasiaprotinin. As well as polypeptides with substantially modified or reduced aprotinin bioactivity. These polypeptides include, but are not limited to, chemically modified aprotinin and aprotinin variants into which specific amino acid sequence changes have been introduced to modify or disrupt the physiological activity of the polypeptide.

  A polypeptide having a slightly modified amino acid sequence, for example a modified N-terminal polypeptide containing a deletion or addition of the N-terminal amino acid, and / or chemically modified compared to oxaprotinin It further comprises a polypeptide.

  Aprotinin-related polypeptides, including variants of aprotinin, exhibit substantially the same or better bioactivity as wild-type aprotinin, or alternatively, show modified or reduced bioactivity compared to wild-type aprotinin Or not, including but not limited to, a polypeptide having an amino acid sequence that differs from that of wild-type aprotinin by including one or more amino acid insertions, deletions, or substitutions.

  Aprotinin-related polypeptides, including variants, are at least about 10%, at least about 20% of the specific activity of wild-type aprotinin produced in the same cell type when tested in an aprotinin activity assay as described herein. %, At least about 25%, at least about 30%, at least about 40%, at least about 50%, 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%.

  Aprotinin-related polypeptides, including variants, have substantially the same or improved biological activity compared to wild-type aprotinin and were tested in one or more of the specific aprotinin activity assays as described Sometimes 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 at least about the specific activity of wild-type siasiaprotinin produced in the same cell type. Including those exhibiting about 110%, more preferably at least about 120%, and most preferably at least about 130%. For the purposes of the present invention, the biological activity of aprotinin is determined by aprotinin as described, for example, in Cardian et al., Blood Coagulation & Fibrinolysis 2001; 12: 37-42 (see “Assay Section” of the present invention). Alternatively, it may be quantified by measuring hydrolysis of the synthetic substrate by the aprotinin-related polypeptide.

  An aprotinin-related polypeptide comprising a variant and having substantially the same or improved biological activity compared to wild-type aprotinin when tested in one or more of the specific aprotinin activity assays as described Exhibit at most about 25%, preferably at most about 10%, more preferably at most about 5%, most preferably at most about 1% of the specific activity of wild-type aprotinin produced in the same cell type It is.

  Non-limiting examples of aprotinin equivalents include, for example, WO 89/10374, U.S. Pat.No. 5,591,603, U.S. Pat.No. 5618915, U.S. Pat.No. 5,610,774, European Patent No. 375718, European Patent No. 3399428. , U.S. Patent No. 5373090, U.S. Patent No. 5312736, European Patent No. 487591, U.S. Patent No. 5378614, European Patent No. 551329, European Patent No. 621873, U.S. Patent No. 5618696 An aprotinin equivalent described in European Patent No. 621871, German Patent Application PA2001 00859; in some embodiments, the aprotinin polypeptide has an activity of said aprotinin polypeptide when tested in an aprotinin activity assay. Is an aprotinin equivalent with a ratio of at least about 1.25 to the activity of natural siaciaprotinin (wild-type aprotinin); in other examples, the ratio is low Also from about 2.0; in further embodiments, the ratio is at least about 4.0. In some embodiments, the aprotinin has an aprotinin ratio of at least about 1.25 between the activity of the aprotinin polypeptide and the activity of natural siaciaprotinin (wild type aprotinin) when tested in an “aprotinin assay” In other examples, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0.

  Aprotinin-related polypeptides also include fragments of aprotinin or aprotinin-related polypeptides that retain the hemostasis-related activity of these characteristics. The activity associated with hemostasis of aprotinin or an aprotinin-related polypeptide may be measured, for example, using the aprotinin activity assay described herein.

Definitions Used in the conventional sense, the three-letter or single-letter designations for amino acids were used in their conventional meaning as shown in Table 1. Unless explicitly indicated, the amino acids referred to herein are L amino acids. The first letter represents the amino acid naturally occurring at the position of wild-type factor VIIa, for example in K337, for example [K337A] -FVIIa is represented by the one-letter code K naturally occurring at the indicated position It is well understood that an amino acid represents an FVII-variant substituted with an amino acid represented by the one letter code A.

table 1:
Abbreviations for amino acids:
Amino acid 3 letter code 1 letter code Glycine
Proline Pro P
Alanine Ala A
Valin
Leu L
Isoleucine Ile I
Methionine Met M
Cystine Cys C
Phenylalanine Phe F
Tyrosine Tyr Y
Tryptophan Trp W
Histidine His H
Lysine Lys K
Argin R
Glutamine Gln Q
Asparagine Asn N
Glutamate Glu E
Aspartic acid Asp D

The terms “Factor VIIa” or “FVIIa” may be used interchangeably.

  In this context, a “subject with a disorder of thrombin generation” means a subject that is unable to generate a complete thrombin burst on the activated platelet surface, with a normal amount and function of a clotting factor, Including subjects with less thrombin production than thrombin production in subjects with a fully functioning normal hemostatic system, including platelets and fibrinogen (e.g., of pooled, normal human plasma), Subjects lacking factor VIII; subjects with reduced platelet count or defective platelets (eg, subjects with thrombocytopenia or Granzmann thrombophilia or heavy bleeding); reduced levels of prothrombin, FX or FVII Subjects with reduced levels of some clotting factors (eg trauma or extensive Such as, but not limited to, subjects with reduced plasma concentrations of fibrinogen (eg, subjects with multiple transfusions).

  “Level of thrombin generation” or “normal thrombin generation” means the level of thrombin generation in a patient compared to the level of a healthy person. Levels are shown as a percentage of normal levels. The terms are used interchangeably where appropriate.

  The term “enhancing the hemostatic system” means an enhanced ability to generate thrombin. The term “enhance hemostasis” refers to the same thrombin generation assay when measuring thrombin generation on a test sample comprising a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide It is contemplated to include situations that are prolonged as compared to individual thrombin generation in a subject sample that contains only factor VII or a factor VII-related polypeptide or aprotinin or aprotinin-related polypeptide, respectively, when tested in. Thrombin generation may be assayed as described in the thrombin generation assay described herein (see “Assay Methods”).

  As used herein, the “only” agent or factor is the only hemostatic agent or factor VII or factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide together in a pharmaceutical composition or kit. A situation where it is an active hemostatic agent or coagulation factor, or is the only hemostatic agent or active hemostasis agent or coagulation factor administered to a patient in the course of a particular treatment, such as in the course of a particular bleeding episode Say. It is understood that these situations include those where other hemostatic agents or clotting factors are not present in sufficient amounts or activity as applicable, so as to significantly affect one or more clotting parameters. The

  Clot lysis time, clot strength, fibrin clot formation, and clotting time are clinical parameters used to assess the status of the patient's hemostatic system. Blood samples are drawn from the patient at appropriate intervals, eg Meh et al., Blood Coagulation & Fibrinolysis 2001; 12: 627637; 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) One or more parameters are assayed by thromboelastography.

  The term "prolonging the clot lysis time" means that when clot lysis time is measured for a test sample comprising a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide, When tested in the same clot lysis assay, the situation is prolonged compared to the individual clot lysis time in a subject sample containing only factor VII or factor VII related polypeptide or aprotinin or aprotinin related polypeptide respectively. It is intended to include. The clot lysis time may be assayed as described above.

  The term “increasing clot strength” is a measure of clot strength, eg mechanical strength, for a test sample comprising a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide. Sometimes increased compared to individual clot lysis time in a subject sample containing only factor VII or factor VII-related polypeptide or aprotinin or aprotinin-related polypeptide, respectively, when tested in the same clot strength assay It is intended to include situations. The clot strength is determined, for example, as described in Carr et al. (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. May be assayed by thromboelastography.

  The term “enhancing fibrin clot formation” refers to the rate or extent of fibrin clot formation for a test sample comprising a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide. When measured, compared to the rate or degree of individual fibrin clot formation in a subject sample containing only factor VII or factor VII-related polypeptide or aprotinin or aprotinin-related polypeptide, respectively, when tested in the same coagulation assay. It is intended to include situations that are increased. Fibrin clot formation may be assayed as described above.

  The term “reducing clotting time” is the measurement of clot formation time (clotting time) for a test sample containing a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide. Include situations that are increased compared to individual clotting times in subject samples each containing only factor VII or factor VII-related polypeptide or aprotinin or aprotinin-related polypeptide when tested in the same coagulation assay. It is contemplated. Clotting time may be assayed by standard Pt or aPTT assay methods known to those skilled in the art.

  The term “reduced platelet count or activity” refers to the number of platelets (thrombocytes) present in a subject's plasma and the activity associated with the biological coagulation of such platelets. Say. The decrease in number may be due, for example, to increased platelet destruction, decreased platelet production, and more pool than the normal fraction of platelets in the spleen. Thrombocytopenia is defined as, for example, a platelet count of less than 150,000 platelets / microliter; generally, the upper limit for normal platelet count is considered to be between 350,000 and 450,000 platelets / microliter. The platelet count may be counted by an automated platelet counter; this is a method well known to those skilled in the art. Syndromes due to reduced platelet count include, but are not limited to, thrombocytopenia and coagulophathy. “Activity” includes, but is not limited to, platelet aggregation, adhesion, and blood clotting activity. The decrease in activity includes, for example, glycoprotein abnormalities, abnormal membrane-cytoskeleton interactions, abnormal platelet granules, abnormal platelet blood clotting activity, abnormal signaling and secretion. Platelet activity, including aggregation, adhesion, and blood clotting activity, may be measured by standard methods known to those skilled in the art, for example, Platelets. A Practical Approach, Ed. SP Watson & K. S. Authi: Clinical Aspects of Platelet Disorders (KJ Clemetson) 15: 299-318, 1996, Oxford University Press; Williams Hematology, Sixth Edition, Eds. Butler, Lichtman, Coller, Kipps & Seligsohn, 2001, McGraw-Hill. Syndromes due to reduced platelet activity include, but are not limited to, Granzman platelet asthenia, Bernard-Sulier syndrome, anticoagulant therapy, and thrombolytic therapy. “Decrease” refers to the number or activity of a sample of test plasma compared to the number or activity of normal pooled plasma as measured in the same assay.

  As used herein, the term “bleeding disorder” reflects any congenital, acquired, or induced defect of cellular or molecular origin that appears during the onset of bleeding. . Examples of bleeding disorders include coagulation factor deficiency (eg, coagulation factor VIII, IX, XI or VII deficiency), coagulation factor inhibitors, platelet function deficiency (eg, Granzmann platelet asthenia, Bernard-Sulier syndrome), platelets Coagulation disorders such as those caused by reduction, von Willebrand disease, and dilution of coagulation proteins, increased fibrinolysis due to bleeding and / or infusion and decreased platelet count (eg, multiple transfusions that have undergone surgery or trauma) But not limited to these).

  Bleeding refers to the extravasation of blood from any component of the circulatory system. The term “onset of bleeding” is associated with surgery, trauma, and other forms of tissue damage that is undesirable, uncontrollable, and often excessive, and desirable in subjects with bleeding disorders Meant to contain no bleeding. The onset of bleeding is basically in subjects with a normal coagulation system but suffering from (temporary) coagulation disorders, as well as in subjects with congenital or acquired coagulopathy or bleeding disorders Will occur. In hemostatic systems in hemophilia, bleeding in subjects with defective platelet function due to lack of or dysfunction of essential coagulation “compounds” (eg, platelets or von Willebrand factor protein) It may be associated with bleeding caused by hemophilia. For example, subjects with extensive tissue damage associated with surgery or enormous trauma are uncontrollable by the immediate hemostatic demands of normal hemostasis mechanisms, and they are essentially ( Excessive bleeding will occur despite normal hemostasis mechanisms (before trauma or surgery). In addition, such frequently transfused subjects may experience (temporary) coagulopathy (ie, clotting protein dilution, fibrinolysis due to bleeding and / or infusion as a result of bleeding and / or infusion. And increase in platelet count). Bleeding may also occur in organs such as the brain, inner ear region, and eyes; in these, the potential for surgical hemostasis is limited and therefore a problem in achieving satisfactory hemostasis There is an area. Similar problems may arise in the process of performing biopsies from various organs (liver, lung, tumor tissue, gastrointestinal tract), as well as in laparoscopic surgery and radical retropubic prostatectomy. Common to all these situations is that even if the bleeding is diffuse (eg hemorrhagic gastritis and massive uterine bleeding), it is difficult to achieve hemostasis with surgical techniques (sutures, clips, etc.) That is. Bleeding will also occur in anticoagulant subjects where hemostatic defects are induced by a given treatment; these bleedings are often acute and massive. Anticoagulant therapy is often performed to prevent thromboembolic diseases. Such treatments include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K-antagonists, and other platelet aggregation inhibitors such as aspirin and eg antibodies or other inhibitors of GPIIb / IIIa activity. I will. Bleeding is also due to so-called thrombolytic therapy, including treatment in combination with antiplatelet agents (eg, acetylsalicylic acid), anticoagulants (eg, heparin), and fibrinolysin (eg, tissue plasminogen activator, tPA). It may be a thing. In addition, bleeding episodes include subjects with acute haemarthroses (joint bleeding), chronic hemophilia arthropathy, hematomas (eg, muscle, retroperitoneum, sublingual, and posterior pharynx). Surgery in subjects with bleeding in other tissues, hematuria (bleeding from the renal tract), cerebral bleeding, surgery (eg, hepatectomy), tooth extraction, and gastrointestinal bleeding (eg, UGI bleeding) or It is intended to include, but is not limited to, bleeding associated with trauma. Hemorrhagic onset is an inhibitor of factor VIII; hemophilia A; hemophilia A and inhibitors; hemophilia B; factor VII deficiency; aprotinin deficiency; thrombocytopenia; Wilbur's disease); severe tissue damage; severe trauma; surgery; laparoscopic surgery; hemorrhagic gastritis; biopsy collection; anticoagulant therapy; upper gastroentestinal bleedings (UGI); or stem cell transplantation May be related to The onset of bleeding may occur in large amounts of uterine bleeding; in organs where the possibility of mechanical hemostasis is limited; in the brain; in the inner ear region; or in the eye. The terms “onset of bleeding” and “bleeding” may be used interchangeably where appropriate.

  In this context, the term “treatment” prevents, or minimizes, bleeding that has already occurred, such as in surgery, and that has already occurred, such as in trauma. For purposes, it is intended to include both adjusting. The “predicted bleeding” described above may be a bleeding that appears to occur in a specific tissue or organ, or it may be an unknown bleeding. Accordingly, prophylactic administration of a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide is included in the term “treatment”.

  As used herein, the term “subject” is intended to mean any animal, particularly a mammal such as a human, and is used interchangeably with the term “patient” where appropriate. May be. The invention also includes the use of Factor VII or FVII related polypeptides and tPA inhibitors within the veterinary procedures.

  The factor VII or factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide as defined herein may be administered simultaneously or sequentially. The factor may comprise a preparation of factor VII or a factor VII-related polypeptide in a single dosage form containing both coagulation factors in a single dosage form, or as a first unit dosage form in a second unit dosage form. As a kit of parts containing a preparation of aprotinin or an aprotinin-related polypeptide. The first, second, or third, other unit doses are referred to throughout this specification, but this does not indicate the preferred order of administration but is merely for convenience purposes. .

  A “simultaneous” dosing of a factor VII or factor VII-related polypeptide preparation and an aprotinin or aprotinin-related polypeptide preparation is the administration of the coagulation factor in a single unit dosage form, or of the first coagulation factor protein. Following administration, it means that administration of the second clotting factor protein has not been separated by more than 15 minutes, preferably 10 minutes, more preferably 5 minutes, more preferably more than 2 minutes. Either factor may be administered first.

  “Sequential” dosing is followed by administration of the first clotting factor protein followed by administration of the second clotting factor protein for up to 2 hours, preferably 1-2 hours, more preferably up to 1 hour, more preferably 30 It means that it is administered at a time interval of minutes to 1 hour, more preferably up to 30 minutes, more preferably from 15 to 30 minutes. Either two unit dosage forms, or clotting factor protein, are administered first. Preferably, both products are injected by utilizing the same vein.

  One unit of factor VII is defined as the amount of factor VII present in 1 ml of normal plasma, corresponding to about 0.5 μg protein. Once activated, 50 units corresponds to about 1 g of protein.

  “Deficiency” means that the presence or activity of factor VII in plasma is reduced, for example, compared to a normal healthy individual. The term may be used interchangeably with “reduced factor VII level” where appropriate.

  “APTT” or “aPTT” means activated partial thromboplastin time (eg, 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).

  An “aprotinin-responsive syndrome” is any indication, symptom, or disease caused, or present, caused by exogenous aprotinin administered to a subject in need thereof. It refers to a syndrome that will be prevented, cured, or ameliorated. This includes, but is not limited to, syndromes caused by decreased levels of aprotinin, such as bleeding disorders caused by inhibitors to aprotinin. Aprotinin responsive syndrome may be treated with a composition according to the invention.

  “Factor VII-responsive syndrome” is any indication that is predicted, or present, caused by an exogenous factor VII, preferably factor VIIa, administered to a subject in need thereof Means a syndrome whose symptoms or illness will be prevented, cured, or ameliorated, reduced levels of coagulation factor VIII, IX, XI or VII, coagulation factor inhibitors, defective platelet function (eg, Grantsman Thrombocytosis, Bernard-Sulier syndrome), thrombocytopenia, von Willebrand disease, and clotting disorders such as those caused by dilution of clotting proteins, increased fibrinolysis and decreased platelet count due to bleeding and / or infusion Including but not limited to syndromes caused by (for example, those of a subject who has had multiple transfusions who have undergone surgery or trauma). No.

  “Half-life” refers to the time required for the plasma concentration of Factor VII or Factor VII-related polypeptide, or aprotinin or aprotinin-related polypeptide to decrease from a certain value to half that value.

  “Primary hemostasis” is the initial generation of thrombin by FXa and TF: factor VIIa, followed by activation of activated platelets and the formation of an initial loose plug by adhering platelets, which are finally Means not yet stabilized by chemically cross-linked fibrin.

  “Secondary hemostasis” or “maintenance of hemostasis” is a secondary, complete and major thrombin burst or production that occurs on the activated platelet surface and is catalyzed by factor VIIIa and factor VIIIa. Implying subsequent fibrin formation and initial platelet plug stabilization. Stabilization of the plug with fibrin results in complete hemostasis.

  “Complete hemostasis” means the formation of a stable and firm fibrin clot or plug at the site of injury that effectively stops bleeding and is not easily dissolved by the fibrinolytic system. In this context, the term hemostasis is used to describe complete hemostasis as described above.

  The total amount of protein in the preparation may generally be measured by known methods, for example by measuring optical density. The amount of aprotinin coagulation or factor VII protein ("antigen") may be measured by generally known methods such as standard Elisa immunoassay methods. In general terms, such an assay involves contacting a solution of a standard containing, for example, aprotinin protein with an anti-aprotinin antibody bound on an elisa plate, and then immobilizing the immobilized antibody-aprotinin complex. , By contacting with an anti-aprotinin secondary antibody having a marker and measuring the amount in the third step. The amount of each clotting factor may be measured using an appropriate antibody in a similar manner. The total amount of clotting factor protein present in the preparation is determined by adding the amount of individual clotting factor proteins. In one embodiment, the preparation comprises an isolated clotting factor. In another embodiment, the preparation is essentially free of coagulation factor II and coagulation factor IIa (prothrombin and thrombin) and / or X or factor Xa.

  As used herein, the term “isolated” refers to a clotting factor, such as aprotinin or an aprotinin-related polypeptide, that has been separated from the cell in which it was synthesized. Separation of polypeptides from cells of these origins may be accomplished by any method known in the art, removing cell culture media containing the desired product from the culture of adherent cells; Including, but not limited to, centrifugation or filtration to remove the cells. Separation of the polypeptides from the medium in which they are naturally occurring may be accomplished by any method known in the art, for example, affinity chromatography on each column of anti-factor VII or anti-aprotinin antibody; Including hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography; electrophoretic procedures (eg preparative isoelectric focusing (IEF)), solubility differences (eg ammonium sulfate precipitation), or extraction However, it is not limited to these.

  Within the scope of the present invention, an “effective amount” of Factor VII or a Factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide together cure, reduce or partially reduce the disease and its complications. Thus, it is defined as the amount of factor VII or factor VII-related polypeptide, such as FVIIa, and aprotinin or aprotinin-related polypeptide sufficient to prevent or reduce bleeding or blood loss.

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

Abbreviations:
TF tissue factor
FVII Factor VII single chain, inactivated form
Activated form of FVIIa factor VII
rFVIIa Recombinant factor VII activated form
TAFI TAFI enzyme precursor, inactivated

Compound preparation:
Human purified Factor VIIa suitable for use in the present invention is described, for example, in Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986, or European Patent No. 200.421. (ZymoGenetics), preferably made by DNA recombination techniques.

  In addition, factor VII is a method described in Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. Lnvest. 71: 1836-1841, 1983. May be produced. These methods produce Factor VII without a detectable amount of other blood clotting factors. In addition, a purified factor VII preparation may be obtained by including further gel filtration as a final purification step. Factor VII may then be converted to activated factor VIIa by known means, for example by several different plasma proteins such as aprotinin a, IX, or Xa. Alternatively, by passing Factor VII through ion exchange chromatography such as Mono Q® (Pharmacia fine Chemicals) as described by Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565). , May be activated.

  Factor VII related polypeptides were produced by modification of wild type factor VII or by recombinant techniques. Factor VII-related polypeptides that have altered amino acid sequence when compared to wild-type factor VII can be obtained by changing amino acid codons in a nucleic acid encoding native factor VII by well-known means, such as by site-directed mutagenesis. Alternatively, it may be produced by modifying the nucleic acid sequence encoding wild type factor VII by removing some amino acid codons.

  It will be apparent to those skilled in the art that substitutions can be made outside of the region important for the function of the factor VIIa or aprotinin molecule to still produce active polypeptides. Amino acid residues that are essential for the activity of factor VII or a factor VII-related polypeptide, or aprotinin or aprotinin-related polypeptide, and thus preferably do not undergo substitution, are site-directed mutagenesis or alanine scanning mutagenesis (eg, , Cunningham and Wells, 1989, Science 244: 1081-1085), and the like. In the latter technique, mutations are introduced into all positively charged residues in the molecule, and in the resulting mutant molecule, cross-linking activity is determined for the coagulant to identify amino acid residues that are important for the activity of the molecule. Each is tested. Also, substrate-enzyme interaction sites can be detected by nuclear magnetic resonance analysis, crystallography, or photoaffinity labeling (eg, de Vos et al., 1992, Science 255: 306-312; Smith et a /., 1992, Journal of Molecular Biology 224: 899-904; see Wlodaver et al., 1992, FEBS Letters 309: 59-64).

  Introducing a mutation into a nucleic acid sequence to exchange one nucleotide for another may be accomplished by site-directed mutagenesis using any method known in the art. Particularly useful are procedures that utilize a supercoiled double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. Oligonucleotide primers, each complementary to the opposite strand of the vector, were extended during temperature cycling by pfu DNA polymerase. Incorporation of the primer produces a mutant plasmid containing a stagger nick. After temperature cycling, the product is treated with Dpnl, which is specific for methylated and hemimethylated DNA, to digest the parental DNA template and select for synthetic DNA containing the mutation. Other procedures known in the art for creating, identifying, and isolating variants may be used, such as gene mixing techniques or phage display techniques.

  Separation of polypeptides from cells of these origins may be accomplished by any method known in the art, removing cell culture media containing the desired product from the attached cell culture; Including, but not limited to, removing cells by centrifugation or filtration. Optionally, the Factor VII or Factor VII related polypeptide may be further purified. Purification can be accomplished by affinity chromatography, eg, anti-factor VII antibody columns (see, eg, Wakabayashi et al., J. Biol. Chem. 261: 11097,1986; and Thim et al., Biochem. 27: 7785,1988); hydrophobic interactions Chromatography; Ion Exchange Chromatography; Size Exclusion Chromatography; Electrophoretic procedures (such as, but not limited to, preparative isoelectric focusing (IEF), solubility differences (eg ammonium sulfate precipitation) or extraction) It may be achieved using any method known in the art, generally Scopes, Protein Puri-fication, 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 a non-factor VII or non-factor VII related polypeptide derived from the host cell. Less than 10% by weight, more preferably less than about 5%, and most preferably less than about 1%.

  Factor VII or a factor VII-related polypeptide is activated by proteolytic cleavage using factor XIa or other proteases with trypsin-like specificity such as factor IXa, kallikrein, factor Xa, and thrombin Also good. See, for example, Osterud et al., Biochem. 11: 2853 (1972) US Pat. No. 4,456,591; and Hedner et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, factor VII or a factor VII-related polypeptide may be activated by passing it through ion exchange chromatography, such as Mono Q® (Pharmacia fine Chemicals). The resulting activated Factor VII or Factor VII-related polypeptide is then formulated and administered as described below.

  Aprotinin for use within the scope of the present invention may be isolated from plasma according to known methods such as those disclosed in (Bajzar et al., J. Biol. Chem. 270: 14477, 1995). However, it is preferred to use recombinant aprotinin to avoid the use of products derived from blood or tissue at risk of disease transmission. Methods for preparing recombinant aprotinin are known in the art; see, for example, Voffa et al., J. Biol. Chem. 273: 2127, 1998) and Eaton et al., J. Biol. Chem. 266: 21833, See 1991. Activated aprotinin is described, for example, in International Publication No. WO96 / 23064 (Biotechnology General). Aprotinin variants may be made by site-directed mutagenesis as described above.

  Aprotinin-related polypeptides may be produced by modification of wild-type aprotinin or by recombinant techniques. Aprotinin-related polypeptides that have altered amino acids when compared to wild-type aprotinin are nucleic acids that encode natural aprotinin by well-known means such as site-directed mutagenesis, as described in more detail above. May be produced by modifying the nucleic acid sequence encoding wild-type aprotinin, either by changing the amino acid codons of or by removing some of the amino acid codons. Separation of the polypeptide from these source cells may be accomplished by any method known in the art, removing the cell culture medium containing the desired product from the attached cell culture; centrifugation or filtration To remove non-adherent cells; and the like. Optionally, the aprotinin or aprotinin-related polypeptide may be further purified. Purification may be accomplished using any method known to those skilled in the art, for example, affinity chromatography on an anti-aprotinin antibody column; as described in more detail above ;; hydrophobic interaction chromatography; ion exchange Size exclusion chromatography; including but not limited to electrophoretic procedures (eg, preparative isoelectric focusing (IEF)), differences in solubility (eg, ammonium sulfate precipitation), or extraction Absent. Following purification, the preparation comprises less than about 10%, more preferably less than about 5%, and most preferably less than about 1% non-aprotinin or aprotinin-related polypeptide derived from the host cell. The resulting activated aprotinin or aprotinin-related polypeptide is then formulated and administered as described below.

Pharmaceutical compositions and methods of use The preparations of the present invention may be used, for example, to reduce coagulation factor VIII, IX, XI or VII levels, coagulation factor inhibitors, platelet function defects (eg, Grantsmann platelet asthenia, Bernard-Sulier syndrome). ), Thrombocytopenia, von Willebrand disease, and clotting disorders such as those caused by dilution of clotting proteins, increased fibrinolysis and decreased platelet count due to bleeding and / or infusion (eg, undergoing surgery or trauma) May be used to treat any Factor VII-responsive syndrome, including but not limited to those caused by syndromes (such as, but not limited to) those caused by multiple transfused subjects. A pharmaceutical composition comprising a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide according to the present invention is mainly for non-prophylactic and / or therapeutic treatment. Oral administration is contemplated. Preferably, the pharmaceutical composition is administered parenterally, ie intravenously, subcutaneously or intramuscularly; intravenous is most preferred. They may also be administered by continuous or pulsed infusion.

  A pharmaceutical composition or formulation according to the invention is formulated either in a single unit dosage form or in a kit of parts, preferably in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluted. A Factor VII or Factor VII related polypeptide and an aprotinin or aprotinin related polypeptide dissolved in a fluid. Briefly, a pharmaceutical composition suitable for use in accordance with the present invention is a Factor VII or Factor VII related polypeptide, or an aprotinin or aprotinin related polypeptide, or a factor VII or VII in combination with an aprotinin or aprotinin related polypeptide. Factor-related polypeptides are made, preferably in purified form, mixed with a suitable adjuvant and a suitable carrier or diluent. Various water-soluble carriers such as water, buffered water, 0.4% physiological saline, 0.3% glycine may be used. The preparations of the present invention can also be formulated using a non-aqueous carrier, such as in the form of a gel or as a liposome preparation, for delivery or targeting to the site of injury. Liposome preparations are generally described, for example, in US Pat. Nos. 4,837,028, 4,501,728, and 4,975,282. The composition may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solution may be packed for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being mixed with a sterile aqueous solution prior to administration.

  The composition comprises a pH adjusted and buffered agent and / or a tonicity adjusted agent such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. It may include, but is not limited to, pharmaceutically acceptable auxiliary substances or adjuvants.

  The formulation may further comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in the form of a liquid, powder, emulsion, sustained release, etc. . One skilled in the art will appreciate that the compositions of the present invention can be approved in a suitable manner and such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990. You may prescribe according to your practice. Thus, a typical pharmaceutical composition for intravenous infusion could be made to contain 250 ml of sterile Ringer's solution and 10 mg of this preparation.

  A composition comprising a preparation of the invention can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the composition is in an amount sufficient to cure, reduce or partially reduce the clinical symptoms of the disease and its complications for a subject already suffering from the disease. And administered as described above. An amount adequate to accomplish this is defined as “therapeutically effective amount”. Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be appreciated that the determination of an appropriate dose will be accomplished using routine testing by building a matrix from the values and testing different points of the matrix.

  Local delivery, eg topical application, of the preparation of the present invention includes, for example, spraying, perfusion, dual balloon catheters, stents, vascular grafts or stents, hydrogels used to coat balloon catheters, or others Well, it may be done by established methods. In any event, the pharmaceutical composition should provide a quantity of preparation sufficient to effectively treat the condition.

  The concentration of factor VII or factor VII-related polypeptide, aprotinin or aprotinin-related polypeptide, or factor VII or factor VII-related polypeptide in combination with aprotinin or aprotinin-related polypeptide in these formulations can vary widely. That is, less than about 0.5 wt.%, Usually at least about 1 wt.% Or more, and on the order of 15 or 20 wt. Administration by injection or infusion, especially injection, is preferred. Thus, factor VII or a factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide are dissolved lyophilized powders or liquid formulations containing both factor VII or factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide. In one dosage form or a dissolved lyophilized powder comprising Factor VII or a Factor VII-related polypeptide or one dosage form of a liquid formulation and a dissolved lyophilized powder comprising aprotinin or an aprotinin-related polypeptide or Prepared in a form suitable for intravenous administration, such as a preparation of one of the liquid dosage forms.

  It is understood that the amount of Factor VII or Factor VII-related polypeptide and the amount of aprotinin or aprotinin-related polypeptide together comprise an amount of aggregate effective to treat the onset of bleeding.

  It must be taken into account that the materials of the present invention are generally used in serious illness or injury, ie in life-threatening or potentially life-threatening situations. In such cases, the therapeutic physician will substantially increase these compositions in view of the minimization of foreign substances and the immunogenicity of factor VIIa in humans and the general lack of aprotinin or aprotinin-related polypeptides. It may be possible and desirable to administer.

  In prophylactic applications, a preparation comprising a preparation of factor VII or a factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide is susceptible to or otherwise at risk for a disease state or injury Administered to a subject to enhance the subject's own clotting ability. Such an amount is defined to be a “prophylactically effective dose”. It is understood that the amount of Factor VII or Factor VII-related polypeptide and the amount of aprotinin or aprotinin-related polypeptide together comprise an amount of aggregate effective to treat the onset of bleeding.

  Single or multiple administrations of the compositions are carried out with dose levels and pattern being selected by the therapist. The composition may be administered one or more times per day or week. An effective amount of such a pharmaceutical composition is an amount that provides a clinically significant effect on the development of bleeding. Such amount will depend, in part, on the particular condition being treated, the age, weight, and general health of the subject, as well as other factors apparent to those of skill in the art.

  The compositions of the invention are generally administered in a single dose prior to the anticipated bleeding or at the beginning of the bleeding. Preferably, however, it may be given repeatedly (in multiple doses) at intervals of 2-4-6-12 hours, depending on the dose given and the subject's symptoms.

  For therapies associated with careful treatment, the factor VII or factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide are typically within about 24 hours prior to treatment and 7 days thereafter. Will be administered for the above. Administration as a coagulant can be by various routes as described herein.

  The composition is in the form of a single preparation (single dosage form) containing both a preparation of an appropriate concentration of Factor VII or a Factor VII-related polypeptide and a preparation of aprotinin or aprotinin-related polypeptide. There may be. The composition may also comprise a preparation of Factor VII or a Factor VII-related polypeptide in a single dosage form containing both coagulation factors in a single dosage form, or as a first unit dosage form. It may be supplied in the form of a kit of parts containing a preparation of aprotinin or aprotinin-related polypeptide as a unit dosage form. In this case, the factor VII or factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide are preferably one after the other, preferably within about 15 minutes of each other, for example within 10 minutes of each other, or more preferably It should be administered and administered within 5 minutes, or more preferably within 2 minutes of each other. Either of the two unit dosage forms can be administered first.

  The kit includes at least two separate pharmaceutical compositions. The kit includes container means for containing separate compositions, such as separate bottles or separate foil packets. Typically, the kit includes, but is not limited to, instructions for administration of the separate components. The kit form is particularly advantageous when the separate components are administered, preferably in different dosage forms, at different dosing intervals, or when titration of a combination of individual components is required by the prescribing physician.

  The amount of Factor VII or Factor VII-related polypeptide and the amount of aprotinin or aprotinin-related polypeptide administered according to the present invention varies in a ratio between about 1: 100 to about 100: 1 (w / w). Also good. Thus, the ratio of Factor VII to aprotinin or aprotinin-related polypeptide is, for example, about 1: 100, or 1:90, or 1:80, or 1:70, or 1:60, or 1:50, or 1: 40, or 1:30, or 1:20, or 1:10, or 1: 5, or 1: 2, or 1: 1, or 2: 1, or 5: 1, or 10: 1, or 20: 1, or 30: 1, or 40: 1, or 50: 1, or 60: 1, or 70: 1, or 80: 1, or 90: 1, or 100: 1; or about 1:90 to about Between 1: 1, between about 1:80 and about 1: 2, or between about 1:70 and about 1: 5, or between about 1:60 and about 1:10, about 1 : Between 50 and about 1:25, between about 1:40 and about 1:30, or between about 90: 1 and about 1: 1, or between about 80: 1 and about 2: 1, or Between about 70: 1 to about 5: 1, or between about 60: 1 to about 10: 1, or between about 50: 1 to about 25: 1, Other about 40: 1 to about 30: may be between 1.

  The dose of Factor VII or Factor VII-related polypeptide is about 0.05 mg to about 0.05 mg for a 70 kg subject as a loading dose or maintenance dose, depending on the subject's weight, symptoms, and severity of symptoms. It varies in a range corresponding to 500 mg / day of wild-type factor VII, such as from about 1 mg to about 200 mg / day, or such as from about 5 mg to about 175 mg / day.

  The dose of aprotinin or aprotinin-related polypeptide is about 0.05 mg to about 500 mg / about 70 kg subject as a loading dose or maintenance dose, depending on the subject's weight, symptoms, and severity of symptoms. Day-to-day wild-type aprotinin varies, for example, in a range corresponding to about 1 mg to about 200 mg / day, or for example about 5 mg to about 175 mg / day.

  The combination of factor VII or a factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide shows a synergistic effect in an in vitro clot firmness assay and a fibrinolysis time assay. In addition, the combination of factor VII or a factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide forms a stable fibrin clot, increases half clot lysis time, increases clot strength, and resistance to fibrinolysis Show a synergistic effect in increasing

  The composition may be in the form of a single preparation comprising an appropriate concentration of Factor VII or Factor VII-related polypeptide and aprotinin or aprotinin-related polypeptide. The present composition may be in the form of a kit comprising a first unit dosage form containing Factor VII or a Factor VII-related polypeptide and a second unit dosage form containing aprotinin or an aprotinin-related polypeptide. . In this case, the Factor VII or Factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide are preferably within about 1-2 hours of each other, for example within 30 minutes of each other, or preferably within 10 minutes, or more preferably They should be administered sequentially within 5 minutes of each other. Either of the two unit dosage forms can be administered first.

  Since the present invention relates to the prevention or treatment of the onset of bleeding or the treatment of coagulation by treating in combination with active ingredients that may be administered separately, the present invention relates to separate pharmaceutical compositions in kit form. Also related to combining. The kit includes at least two separate pharmaceutical compositions. The kit includes container means for containing separate compositions, such as separate bottles or separate foil packets. Typically, the kit includes, but is not limited to, instructions for administration of the separate components. The kit form is particularly advantageous when the separate components are administered, preferably in different dosage forms, at different dosing intervals, or when titration of a combination of individual components is required by the prescribing physician.

Assay method:
Testing for factor VIIa activity:
A suitable assay for selecting the appropriate Factor VIIa variant by testing for Factor VIIa activity can be performed as a single preliminary in vitro test:
In Vitro Hydrolysis Assay Specific activity may be assayed with native (wild-type) Factor VIIa and variant Factor VIIa (both hereinafter referred to as “Factor VIIa”). They may also be assayed in parallel to compare their specific activities directly. The assay is performed in microtiter plates (MaxiSorp, Nunc, Denmark). Final concentration of 1 mM chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), Factor VIIa (final concentration 100 nM) 0.1 M NaCI, 5 mM CaCl ₂ , and 1 mg / Add to 50 mM Hepes, pH 7.4 solution containing ml bovine serum albumin. Absorbance at 405 nm is measured continuously in a SpectraMax ™ 340 plate reader (Molecular Devices, USA). The absorbance generated during the 20 minute incubation is used to calculate the ratio between the mutant and wild type factor VIIa activity after subtracting the absorbance of empty wells without enzyme:
Ratio = (A ₄₀₅ nm VIIa variant) / (A ₄₀₅ nm VIIa wild type).

  Based on the results, a factor VIIa variant with activity comparable to or higher than that of natural factor VIIa is obtained, for example, the ratio between the activity of the variant and the activity of natural factor VII (wild type FVII) , Mutants in the vicinity of more than 1.0 may be identified.

  The activity of factor VIIa or factor VIIa variants may also be measured using a physiological substrate such as factor X, optimally at a concentration of 100-1000 nM, in which case the production of factor Xa is Measure after adding the appropriate chromogenic substrate (eg, S-2765). In addition, activity assays may be performed at physiological temperatures.

In Vitro Proteolytic Assays Native (wild-type) Factor VIIa and variant Factor VIIa (both hereinafter referred to as “Factor VIIa”) are assayed in parallel to directly compare their specific activities . The assay is performed in microtiter plates (MaxiSorp, Nunc, Denmark). A 100 μl solution of 50 mM Hepes, pH 7.4 containing 0.1 M NaCl, 5 mM CaCl ₂ , and 1 mg / ml bovine serum albumin of Factor VIIa (10 nM) and Factor X (0.8 μM) is incubated for 15 minutes. Cleavage of factor X is then stopped by adding 50 μl of 50 mM Hepes, pH 7.4 containing 0.1 M NaCl, 20 mM EDTA, and 1 mg / ml bovine serum albumin. The amount of factor Xa produced is determined by adding the chromogenic substrate ZD-Arg-Gly-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden) (final concentration 0.5). Absorbance at 405 nm is measured continuously in a SpectraMax ™ 340 plate reader (Molecular Devices, USA). The absorbance generated during the 10 minute incubation is used to calculate the ratio between the mutant and wild type factor VIIa activity after subtracting the absorbance of empty wells without enzyme:
Ratio = (A ₄₀₅ nm VIIa variant) / (A ₄₀₅ nm VIIa wild type).

  Based on the results, a factor VIIa variant with activity comparable to or higher than that of natural factor VIIa is obtained, for example, the ratio between the activity of the variant and the activity of natural factor VII (wild type FVII) , Mutants in the vicinity of more than 1.0 may be identified.

Thrombin generation assay:
The ability of factor VII or a factor VII-related polypeptide, or aprotinin or an aprotinin-related polypeptide or aprotinin or an aprotinin-related polypeptide-related polypeptide (eg, a variant) to produce thrombin, all related clotting factors and physiological concentrations (As described on pages 543 of J. Haematol. 99, 542-547, which is incorporated herein by reference).

Testing for aprotinin activity:
A suitable assay for selecting the appropriate aprotinin variant by testing aprotinin activity is as a simple in vitro test as described in the examples herein, or measuring serine protease inhibitors In vitro assays or other assays known to those skilled in the art can be performed. Suitable amydolytic and immunosorbent assays are described, for example, in Cardigan et al., Blood Coagulation & Fibrinolysis 2001; 12: 37-42. (“Aprotinin Assay”).

  The invention is further illustrated by the following examples, which are not to be construed as limiting the scope of protection. In the foregoing and following implementations, the disclosed features, both separately and in any combination, are materials for understanding the various forms of the invention.

Example 1
Methods to improve the stability of hemostatic clots by combining VIIa clotting factor and aprotinin :
Clot lysis assay: Buffer containing Innovin (Dade Behring, 2000-fold dilution), rFVIIa (Novo Nordisk A / S Bagsvaerd, Denmark, various concentrations), and t-PA (American Diagnostica, 8 nm) Normal human plasma diluted 10-fold with 20 mM HEPES, 150 mM NaCl, 5 mM CaCl, pH 7.4) was added to a 96-well ELISA plate and the turbidity at 650 nm was measured over time at room temperature. Where indicated, purified Usia Protinin (Novo Nordisk A / S, Bagsvaerd, Denmark) was included.

  Rotational thromboelastography (roTEG): Measured with normal human plasma with citrate or factor VIII deficient plasma (George King Bio-Medical, Inc. # 0800) supplemented with 5 nM t-PA The effects of adding 1 nM FVIIa alone or in combination with aprotinin were analyzed. Clotting was initiated by the addition of inobin (final concentration 2000 fold dilution, Dade Behring # 526945) and calcium (final concentration 15 mM) 20 mM HEPES, 150 mM NaCl, pH 7.4 buffer.

result:
Clot lysis assay: Addition of FVIIa results in a dose-dependent extension of clot lysis time (Figure 1). This effect was optimal at 10 nM FVIIa. The addition of aprotinin in the presence of 10 nm FVIIa resulted in a further extension of the clot lysis time (FIG. 2). The effect was dose dependent and optimal with 1 μM aprotinin.

Conclusion:
These results demonstrate that adding FVIIa and aprotinin to plasma improves clot fibrinolysis resistance.

Addition of FVIIa (0-15 nM) results in a dose-dependent extension of clot lysis time FIG. 6 illustrates that in the presence of 10 nm FVIIa, addition of aprotinin resulted in further extension of clot lysis time. The effect was dose dependent and optimal with 1 μM aprotinin.

Claims (53)

  A pharmaceutical composition comprising Factor VII or a Factor VII-related polypeptide and aprotinin or an aprotinin-related polypeptide.   2. The composition of claim 1, wherein the Factor VII or Factor VII related polypeptide is a Factor VII related polypeptide.   The composition of claim 2, wherein the Factor VII-related polypeptide is a Factor VII amino acid sequence variant.   4. A composition according to claim 2 or claim 3, wherein the ratio between the activity of the factor VII-related polypeptide and the activity of native human factor VIIa (wild type FVIIa) is described herein. A composition that is at least about 1.25 when tested in the “in vitro hydrolysis assay” as described.   The composition of claim 1, wherein the factor VII or factor VII-related polypeptide is factor VII.   6. The composition of claim 5, wherein the factor VII is human factor VII.   7. A composition according to claim 6, wherein the factor VII is recombinant human factor VII.   The composition according to any one of claims 1 to 7, wherein the factor VII or factor VII-related polypeptide is an active form thereof.   9. A composition according to claim 8, wherein the factor VII is recombinant human factor VIIa.   The composition according to any one of claims 1 to 9, wherein the aprotinin or aprotinin-related polypeptide is an aprotinin-related polypeptide.   11. The composition according to claim 10, wherein the aprotinin-related polypeptide is a Kunitz-type inhibitor domain having aprotinin activity.   11. The composition of claim 10, wherein the aprotinin-related polypeptide is an aprotinin amino acid sequence variant.   The ratio between the activity of the aprotinin-related polypeptide and the activity of native bovine plasma aprotinin (wild type aprotinin) is at least about 1.25 when tested in an “aprotinin assay” as described herein. 12. A composition according to claim 10 or claim 11, wherein:   The composition according to any one of claims 1 to 9, wherein the aprotinin or aprotinin-related polypeptide is an aprotinin polypeptide.   15. The composition of claim 14, wherein the aprotinin is siaprotinin.   16. The composition according to claim 15, wherein the aprotinin is recombinant siasiaprotinin.   17. The composition of any one of claims 1-16, wherein the Factor VII or Factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide are about 100: 1 and about 1: 100 (w / wVII Factor: aprotinin) present in a mass ratio.   18. A composition according to any one of claims 1 to 17, further comprising a pharmaceutically acceptable excipient suitable for injection or infusion, particularly for injection. A kit of parts containing treatment for the development of bleeding,
f) an effective amount of a preparation of the first unit dosage form of Factor VII or a Factor VII-related polypeptide and a pharmaceutically acceptable carrier;
g) an effective amount of a second unit dosage form preparation of aprotinin or an aprotinin-related polypeptide and a pharmaceutically acceptable carrier; and
h) container means for containing said first and second dosage forms;
A kit of parts containing.   20. The kit according to claim 19, wherein the factor VII or factor VII-related polypeptide is a factor VII-related polypeptide.   21. The kit of claim 20, wherein the Factor VII related polypeptide is a Factor VII amino acid sequence variant.   22. A kit according to claim 20 or claim 21, wherein the ratio between the activity of the factor VII-related polypeptide and the activity of native human factor VIIa (wild type FVIIa) is described herein. A kit that is at least about 1.25 when tested in a “in vitro hydrolysis assay” as described.   20. The kit according to claim 19, wherein the factor VII or factor VII-related polypeptide is factor VII.   24. The kit of claim 23, wherein the factor VII is human factor VII.   25. The kit of claim 24, wherein the factor VII polypeptide is recombinant human factor VII.   26. The kit according to any one of claims 19 to 25, wherein the factor VII or factor VII-related polypeptide is an active form thereof.   27. The kit of claim 26, wherein the factor VII is recombinant human factor VIIa.   28. The kit according to any one of claims 19 to 27, wherein the aprotinin or aprotinin-related polypeptide is an aprotinin-related polypeptide.   29. The kit according to claim 28, wherein the aprotinin-related polypeptide is a Kunitz-type inhibitor domain having aprotinin activity.   30. The kit of claim 28, wherein the aprotinin-related polypeptide is an aprotinin amino acid sequence variant.   The ratio between the activity of the aprotinin-related polypeptide and the activity of native bovine plasma aprotinin (wild type aprotinin) is at least about 1.25 when tested in an “aprotinin assay” as described herein. 31. A kit according to claim 28 or claim 29 or claim 30.   28. The kit according to any one of claims 19 to 27, wherein the aprotinin or aprotinin-related polypeptide is aprotinin.   33. The kit of claim 32, wherein the aprotinin is siacia protinin 33.   34. A kit according to claim 33, wherein the aprotinin is recombinant siasiaprotinin.   35. The kit according to any one of claims 19 to 34, wherein the Factor VII or Factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide are about 100: 1 and about 1: 100 (w / kit present in a mass ratio between wVII factor: aprotinin).   Use of factor VII or a factor VII-related polypeptide in combination with aprotinin or an aprotinin-related polypeptide for the manufacture of a medicament for treating the development of bleeding.   Use of a composition according to any one of claims 1 to 18 for the manufacture of a medicament for treating the onset of bleeding.   38. Use according to claim 36 or claim 37, wherein the drug is for reducing clotting time.   38. Use according to claim 36 or claim 37, wherein the drug is for extending clot lysis time.   38. Use according to claim 36 or claim 37, wherein the drug is for increasing clot strength.   41. Use according to any one of claims 36 to 40, wherein the drug is formulated for injection or infusion, in particular for injection.   42. Use according to any one of claims 36 to 41, wherein the onset of bleeding is due to trauma or surgery, or a decrease in platelet count or activity.   43. Use according to any one of claims 36 to 42, wherein the drug is in a single dosage form.   The drug is prepared in the form of a first unit dosage form comprising a preparation of factor VII or a factor VII-related polypeptide and a second unit dosage form comprising a preparation of aprotinin or an aprotinin-related polypeptide. Use of any one of 36-42.   A method for treating the development of bleeding in a subject, said method comprising, for a subject in need thereof, a first amount of a preparation of factor VII or a factor VII-related polypeptide; Administering a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts are both effective for treating bleeding.   A method for reducing clotting time in a subject, said method comprising, for a subject in need thereof, a first amount of a preparation of factor VII or a factor VII-related polypeptide, aprotinin Or a second amount of a preparation of an aprotinin-related polypeptide, wherein the first and second amounts are both effective to reduce clotting time.   A method for enhancing hemostasis in a subject, said method comprising, for a subject in need thereof, a first amount of a preparation of factor VII or a factor VII-related polypeptide and aprotinin or aprotinin-related Administering a second amount of a polypeptide preparation, wherein the first and second amounts are both effective to enhance hemostasis.   A method for prolonging the clot lysis time in a subject, said method comprising, for a subject in need thereof, a first amount of a preparation of factor VII or a factor VII-related polypeptide. And a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts are both effective to prolong clot lysis time.   A method for increasing clot strength in a subject comprising: for a subject in need thereof, a first amount of a preparation of factor VII or a factor VII-related polypeptide; Administering a second amount of a preparation of aprotinin or an aprotinin-related polypeptide, wherein the first and second amounts are both effective to increase clot strength.   50. The method of any one of claims 45 to 49, wherein the Factor VII or Factor VII related polypeptide and the aprotinin or aprotinin related polypeptide are administered in a single dosage form.   50. The method of any one of claims 45-49, wherein the factor VII or factor VII-related polypeptide and the aprotinin or aprotinin-related polypeptide comprise a preparation of a factor VII or factor VII-related polypeptide. A method of administration in the form of a second unit dosage form comprising one unit dosage form and a preparation of aprotinin or an aprotinin related polypeptide.   52. The method of claim 51, wherein the first unit dosage form and the second unit dosage form are not administered more than 15 minutes apart. A kit comprising a therapeutic agent for the onset of bleeding,
a) an effective amount of Factor VII or a Factor VII-related polypeptide and an effective amount of a single unit dosage form of aprotinin or an aprotinin-related polypeptide and a pharmaceutically acceptable carrier; and
b) container means for containing said single unit dosage form;
Including kit.

Images