TWI287452B - Pharmaceutical composition comprising a factor VIIa and a factor XIII - Google Patents

Abstract

The present invention relates to the use of a factor VIIa and a factor XIII in the treatment or prophylaxis of bleeding episodes.

Description

A7 1287452 _ B7___ V. INSTRUCTION DESCRIPTION (I) FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition comprising a factor Vila and a factor XIII. The invention also relates to the use of a combination of factor Vila and factor XIII to produce a therapeutic or related prophylactic agent for a hemorrhagic event in a patient. The invention also relates to a method of treating a hemorrhagic event in a patient and a method of increasing blood clot formation in a patient. The invention also relates to kits comprising these compounds. BACKGROUND OF THE INVENTION The hemostasis process begins with the tissue factor (TF) exposed to circulating blood flow after injury to the vessel wall and the formation of a complex between FVIIa in the blood circulation in an amount equivalent to about 1% of the amount of all FVII protein. This complex is immobilized on cells bearing TF and activates FX on the cell surface to FXa and activates FIX to FIXa. FXa activates prothrombin to thrombin, which activates FVIII, FV, FXI and FXIII. In addition, a limited amount of thrombin produced during the initial steps of this hemostasis process activates platelets. After the thrombin acts on the platelets, these shapes change and the charged phospholipids are exposed to their surface. This activated platelet surface forms a template for further activation of FX and produces intact thrombin. Further FX activation on the surface of activated platelets occurs by forming a FIXa-FVIIIa complex on the surface of the activated platelets, and FXa subsequently converts the prothrombin to thrombin while still on the surface. The thrombin then converts fibrinogen to fibrin, which is insoluble and stabilizes the initial embolization of platelets. This process is distinguished, that is, 3 paper sizes apply to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page)

I n ϋ 11 ϋ ϋ n I One OJfl ϋ n ϋ am— ·ϋ nn I A7 1287452 ___B7 _ V. Invention description (> ) (Please read the note on the back and fill out this page) is limited to TF performance Or the location of exposure, thereby reducing the risk of systemic activation of the coagulation system. In addition, the insoluble fibrin that forms the plug is stabilized by the FXIII-catalyzed cross-linking of fibrin fibers. FVIIa is mainly present in plasma as a single-chain zymogen which is cleaved by FXa into a double strand, i.e., activated FVIIa. The recombinant activator Vila (rFVIIa) has been developed as a pro-hemostatic agent. Administration of rFVIIa provides a rapid and highly effective pro-hemorrhagic response to hemorrhagic conditions in patients with hemorrhagic conditions, which cannot be treated with a clotting factor product due to antibody production. In addition, patients with a deficiency of Factor VII or patients with a normal coagulation system but experiencing excessive bleeding can be successfully treated with FVIIa. In these studies, no adverse side effects (especially thrombotic embolism) with rFVIIa treatment were encountered. Additional exogenous administration of FVIIa increases the formation of thrombin on the surface of activated platelets. This occurs in hemophiliacs who lack FIX and FVin, and thus loses the most efficient route of complete thrombin generation. Similarly, additional FVIIa increases the formation of thrombin in the presence of a reduced number of platelets or platelets with defective function. FXIII, a fibrin stabilizing factor, is a transglutaminase that crosslinks fibrin monomers, thus providing an increased resistance to plasmin and other proteolytic enzymes. Fibrin structure. Factor ΧΙΠ is also known as "fibrin ligase" and, fibrin stabilizing factor. When activated, FXIIIa is capable of forming intermolecular gamma-glutamyl groups between the side chains of fibrin molecules and other substrates. Crosslinking of -ε-lysine. FXIII can be found in plasma and platelets. This enzyme 4 is used in China's 1CN (CNS) A4 specification (210 x 297 public) A7 1287452 B7 - ------ - V. INSTRUCTIONS (4) The tetraplase is present in plasma, which includes two α-subunits and two β-subunits (called a2b2), while the enzyme principle present in platelets includes two hearts. Subunit (called a2-bisome). Both zymogens are activated by thrombin and calcium ions. Calcium ions are released in the agglutinated platelets in the injured part. Coagulation enzyme excision (a^ double body) N-terminal 1-37 amino acid residues. As for the a2b2- zymogen, its β-subunit is then dissociated from the activated α-subunit. Calcium ions are equally bound to the zymogen and thrombin-modified molecules. Activation center cysteine on the α-chain after activation of thrombin and calcium ions (CySte Ine) will expose and form fully activated enzymes. Patients with severe thrombocytopenia (thrombocytopenia) have been found to have reduced plasma concentrations of FXIII. It is well known that patients who require blood transfusion due to excessive bleeding associated with surgery or major trauma will experience less than those who have not experienced Any bleeding patient develops into more complications. However, it is also necessary to administer human blood or blood products (platelets, white blood cells, plasma-derived concentrates to treat coagulation defects, etc.). Moderate bleeding may cause and transfer human viruses. A syndrome associated with the risk of hepatitis virus, HIV, micro-DNA viruses and other unknown viruses. Large-scale bleeding that requires massive blood transfusions may lead to the development of multiple organ failures, including impaired lung and kidney function. When patients develop these serious complications, a series of events related to some cytokines and inflammatory reactions will begin, making any treatment very difficult and unfortunately often failing. Therefore, surgery and treatment of important tissue damage The main goal is to avoid or reduce bleeding. To avoid or reduce For such bleeding, it is important to ensure that the fiber is not easily applied to the Chinese National Standard (CNS) A4 specification (210 X 297 public meals 1 ^ ------- L*---rt----- ------Set--------- ίPlease read the phonetic transcription on the back? Please fill out this page again} A7 1287452 _______B7___ V. Invention description (present) Stabilization and solid hemostasis dissolved by protein lysozyme In addition, it is also important to ensure the rapid and effective formation of such embolisms or blood clots. 专利 Patent Application No. 2-167234A relates to a biological tissue adhesive characterized by containing fibrinogen, pre-coagulation Enzyme, Factor VII, Factor IX, Factor X, Factor χπΐ, Antithrombin, a protease inhibitor, and calcium. Japanese Patent Application No. 59-116213A relates to an aerosol composition for use as a tissue adhesive which contains a blood clotting agent as its active ingredient. The coagulant may be selected from the group consisting of factor I, II, III, IV, V, VII, VIII, : [X, X, XI, XII and XIII, prekallikrein, advanced polymer kininogen (kininogen) ) and thrombin. It is best to combine FXIII with thrombin. WO 93/12813 (ZymoGenetics) is about the use of FXIII to reduce the loss of peripheral blood in patients during surgery. Its composition may also include aprotinin. FXIII is administered to patients in large bolus form, usually on the day before surgery. European Patent No. 225_160 (Novo Nordisk) is a treatment for FVIIa compositions with bleeding disorders that are not caused by clotting factor deficiency or clotting factor inhibitors. European Patent No. 82.182 (Baxter Travenol Lab_) relates to the factor Vila composition used to combat the deficiency of a patient's clot factor or the effect of an inhibitor on a clot factor. International Patent Publication No. WO 93/06855 (Novo Noniisk) relates to the topical application of FVIIa. 6 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) —l··—;—-----------------Mmw (please read first) Remarks on the back side of this page) A7 1287452 V. Description of invention (θ)

Kjalke et al., Thrombosis and Haemostasis, 1999 (Supp.), 095 1 for the effect of administration of additional exogenous FVIIa and thrombin formation on the surface of activated platelets, the model system of which simulates hemophilia A or B. There is still a need in the art for an improved, reliable and widely used method for enhancing blood clotting, rapidly forming a stable thromboembolism and achieving complete hemostasis in a patient, particularly a patient suffering from thrombin production. There is also a need for a method in which the amount of FVIIa required to achieve complete hemostasis is reduced. SUMMARY OF THE INVENTION One object of the present invention is to provide a composition which is effective for treating or preventing hemorrhagic events and clotting disorders. A second object of the present invention is to provide a composition in a single dosage form which is effective for treating or preventing a hemorrhagic event or as a procoagulant. Another object of the invention is to provide a kit of compositions, treatments or performance synergies. A further object of the invention is to provide a composition, a method of treatment or a kit that does not exhibit substantial side effects, such as a systemic activation of a high degree of coagulation system. Other objects of the invention will become more apparent upon reading the description herein. The inventors of the present invention stated that the combination of Factor Vila and Factor XIII is more effective than the Factor Vila or Factor XIII alone in reducing the clot time of normal human plasma. The results also show that the combination of Factor Vila and Factor XIII can increase clot firmness more effectively than Factor Vila or Factor XIII alone. By making the concentration factor Vila, which is no longer observed to increase the firmness of blood clots, a concentration of 7 is also applicable to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) (please read the notes on the back and fill in the form) This page) --------- order --------- A7 1287452 ______B7 _ V. Description of the invention (L) No longer observed the increase in clot firmness factor XIII combined with it, unexpectedly displayed A further increase in the firmness of the blood clot is obtained. The results also showed that the binding factor Vila and Factor XIII prolonged the dissolution time of normal human plasma in vitro blood clots, which was more effective than the presence of Factor Vila or Factor XIII inhibitor alone. Therefore, by improving blood coagulation, patients with bleeding can get more effective treatment. In addition, patients are treated with a relatively low concentration of factor Vila, thus reducing the high cost traditionally treated with the factor Vila alone. In a first aspect, the invention relates to a pharmaceutical composition comprising a factor Vila and a factor XIII, and a pharmaceutically acceptable carrier. In another aspect, the invention relates to a pharmaceutical composition comprising a factor Vila and Factor XIII as the sole activator, and a pharmaceutically acceptable carrier. In another aspect, the invention relates to a pharmaceutical composition formulated for intravenous administration comprising a factor Vila and Factor XIII, and a pharmaceutically acceptable carrier. In another aspect, the invention relates to a pharmaceutical composition formulated for intravenous administration comprising a factor Vila and Factor XIII as the sole activator, and a pharmaceutically acceptable carrier. In a specific embodiment, the factor Vila is the recombinant factor Vila and the factor XIII is the recombinant factor XIII. In a specific embodiment of the invention, the factor Vila is the recombination factor Vila. In another specific embodiment, the factor Vila is the recombinant human factor Vila. In another specific embodiment, the factor Vila is a factor Vila variant. 8 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------r.------------- order-------- - (Please read the notes on the back and fill out this page) A7 1287452 ^___B7__ V. INSTRUCTIONS (1) In one embodiment, the Factor Vila variant is a variant of the amino acid sequence compared to the wild type factor Vila ( That is, a multi-peptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950) is substituted, deleted or inserted in no more than 20 amino acids. In another embodiment, the Factor Vila variant has no more than 15 amino acids replaced, deleted or inserted; in another embodiment, the Factor Vila variant has no more than 10 amino acids replaced, deleted or inserted; In another embodiment, the Factor Vila variant has no more than 8 amino acids replaced, deleted or inserted; in another embodiment, the Factor Vila variant has no more than 6 amino acids replaced, deleted or inserted; In another embodiment, the Factor Vila variant has no more than 5 amino acids replaced, deleted or inserted; in another embodiment, the Factor Vila variant has no more than 3 amino acids replaced, deleted or inserted. In a specific embodiment, the Factor Vila variant is selected from the group consisting of [1^305¥]-FVIIa, [L305V/M306D/D309S]-FVIIa, [L305I]-FVIIa, [L305T]-FVIIa, [F374P]-FVIIa, [V158T/M298Q]-FVIIa, [V158D/E296V/M298Q]-FVIIa and [K337A]-FVIIa. In a specific embodiment, the Factor XIII is FXIII a2b2. In another embodiment, the factor ΧΙΠ is FXIII a2. In another embodiment, Factor XIII is activating factor XIII (FXIIIa). In a specific embodiment, Factor XIII is a Factor XIII variant. In a specific embodiment, the factor XIII is human factor XIII. In a specific embodiment, Factor XIII is recombinant Factor XIII. In a specific embodiment, Factor XIII is recombinant human factor XIII. In one embodiment, Factor XIII is a human a2-bisome. In one aspect, the composition additionally comprises a TFPI inhibitor. In the other 9 paper scales, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) is applied. ------r.---Λ----------- Order---- ----- (Please read the precautions on the back and fill out this page) A7 1287452 V. INSTRUCTIONS (<2>) A viewpoint whose composition additionally contains Factor VIII. In another aspect, the composition additionally comprises Factor VIII and a TFPI inhibitor. In a specific embodiment, the composition additionally comprises a fibrinolytic system inhibitor, for example, aprotinin, epsilon-aminocaproic acid or tranexamic acid. In another aspect, the invention is A kit comprising a therapeutic hemorrhagic event comprising a) an effective amount of a factor of Vila in the first unit dosage form, and a pharmaceutically acceptable carrier; b) in a second unit dosage form There is an effective amount of Factor XIII, and a pharmaceutically acceptable carrier is selected; and c) a container for containing the first and second dosage forms. In one aspect, the kit comprises a) an effective amount of a factor of Vila in the first unit dosage form, and a pharmaceutically acceptable carrier; b) an effective amount in the second unit dosage form. Factor XIII, and optionally a pharmaceutically acceptable carrier; c) an effective amount of a TFPI inhibitor in a third unit dosage form, and optionally a pharmaceutically acceptable carrier; and d) for containing the first, Containers of the second and third dosage forms. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising 'a) an effective amount of a factor of Vila and a TFPI inhibitor in the first unit dosage form, and a selective drug Acceptable carrier; 10 This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page) --------Book---- ----- A7 1287452 _B7_ V. Description of the invention (1) b) an effective amount of factor XIII in the second unit dosage form, and a pharmaceutically acceptable carrier; and C) for containing the A container with a second dosage form. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) an effective amount of a factor Vila in the first unit dosage form, and a pharmaceutically acceptable carrier; Providing an effective amount of Factor XIII and a TFPI inhibitor in a second unit dosage form, and optionally a pharmaceutically acceptable carrier; and c) a container for containing the first and second dosage forms. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) an effective amount of a factor of Vila and Factor XIII in the first unit dosage form, and a pharmaceutically acceptable form a carrier; b) an effective amount of a TFPI inhibitor in a second unit dosage form, and optionally a pharmaceutically acceptable carrier; and c) a container for containing the first and second dosage forms. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) an effective amount of a factor Vila in the first unit dosage form, and a pharmaceutically acceptable carrier; Providing an effective amount of Factor XIII in a second unit dosage form, and optionally a pharmaceutically acceptable carrier;

c) An effective amount of Factor VIII in the form of a third unit dose. 11 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------!·---1 J -----------Book--------- (Please read the notes on the back and then fill out this page) 1287452 V. Invention description (〖0), with selective medicine Receiving a carrier; and d) presenting an effective amount of a TFPI inhibitor in a fourth unit dosage form, optionally with a pharmaceutically acceptable carrier; and c) for containing the first, second, third and fourth A container in the form of a dose. In another aspect, the kit comprises a) an effective amount of a factor Vila and Factor XIII in the first unit dosage form, and a pharmaceutically acceptable carrier; b) in a second unit dosage form An effective amount of Factor VIII, and a pharmaceutically acceptable carrier; c) an effective amount of a TFPI inhibitor in a third unit dosage form, and a pharmaceutically acceptable carrier; and d) The containers of the first, second and third dosage forms. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) an effective amount of a factor of Vila and a TFPI inhibitor in the first unit dosage form, and a selective pharmaceutical Receiving a carrier; b) presenting an effective amount of Factor XIII in a second unit dosage form, and optionally a pharmaceutically acceptable carrier; and c) presenting an effective amount of Factor VIII in a third unit dosage form, and Selectively a pharmaceutically acceptable carrier; and d) a container for holding the first, second and third dosage forms. In another aspect, the present invention is directed to a kit comprising a therapeutic hemorrhagic event comprising 12 paper scales applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------ 1- —: :—————定--------- (Please read the notes on the back and fill out this page) 1287452 A7 ___B7___ V. Invention Description (u) a) First One unit dosage form is one of an effective amount of factor Vila and factor VIII, and a pharmaceutically acceptable carrier is selected; b) an effective amount of factor xin is present in the form of a second unit dose, and a selective drug is available Receiving a carrier; and c) presenting an effective amount of a TFPI inhibitor in a third unit dosage form, and optionally a pharmaceutically acceptable carrier; and d) for containing the first, second and third dosage forms Container. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) an effective amount of a factor of Vila and Factor XIII in the first unit dosage form, and a pharmaceutically acceptable form a carrier; b) an effective amount of Factor VIII and a TFPI inhibitor in a second unit dosage form, and a pharmaceutically acceptable carrier; and c) a container for containing the first and second dosage forms . In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) an effective amount of Factor VIII and Factor XIII in a first unit dosage form, and a pharmaceutically acceptable form a carrier; b) an effective amount of TFPI in a second unit dosage form (formulation, optionally with a pharmaceutically acceptable carrier; and c) an effective amount of a factor of Vila in the third unit dosage form, And optionally a pharmaceutically acceptable carrier; and d) a container for containing the first, second and third dosage forms. In another aspect, the present invention relates to a Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------r.---ί 4-- for the treatment of hemorrhagic 13 paper sizes. ---------Book--------- Μψ (Please read the note on the back and fill out this page) A7 1287452 V. Invention Description (〆) Event set, which contains a Providing an effective amount of Factor XIII and a TFPI inhibitor in the first unit dosage form, and optionally a pharmaceutically acceptable carrier; b) presenting an effective amount of Factor VIII in a second unit dosage form, and selecting a pharmaceutically acceptable carrier; and c) an effective amount of a factor Vila in a third unit dosage form, and optionally a pharmaceutically acceptable carrier; and d) for containing the first, second and third A container in the form of a dose. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) the presence of an effective amount of a TFPI inhibitor and Factor VIII in a first unit dosage form, and the selective medicinal acceptable a carrier; b) an effective amount of a factor Vila in a second unit dosage form, and optionally a pharmaceutically acceptable carrier; and c) an effective amount of a factor in the third unit dosage form, A pharmaceutically acceptable carrier; and d) a container for containing the aforementioned first, second and third dosage forms. In another aspect, the invention relates to a kit comprising a therapeutic hemorrhagic event comprising a) the presence of an effective amount of a TFPI inhibitor and Factor XIII in a first unit dosage form, and optionally a pharmaceutically acceptable form a carrier; b) an effective amount of Factor VIII and Factor Vila in a second unit dosage form, and a pharmaceutically acceptable carrier; and c) a container for containing the first and second dosage forms described above. 14 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------r.---: ±------------Book----- ----Line (please read the note on the back and then fill out this page) A7 1287452 __B7_ V. INSTRUCTION DESCRIPTION (θ) In another aspect, the present invention relates to a kit comprising a therapeutic hemorrhagic event, including a An effective amount of a factor of Vila and a TFPI inhibitor, and a pharmaceutically acceptable carrier, in the first unit dosage form; b) an effective amount of Factor VIII and Factor XIII in a second unit dosage form And optionally a pharmaceutically acceptable carrier; and c) a container for containing the first and second dosage forms. In another aspect, the invention relates to a medicament for treating a hemorrhagic event using a combination of a factor Vila and a factor XIII. In another aspect, the invention relates to the use of a combination of the factor Vila factor XIII to produce a drug that reduces the clotting time of a patient. In another aspect, the present invention relates to the use of a combination of the factor Vila factor XIII to produce a drug that prolongs the blood clot lysis time of a mammalian blood plasma. In another aspect, the invention relates to the use of a combination of the factor Vila factor XIII to produce a drug that increases the clot strength in mammalian plasma. In another aspect, the present invention relates to the use of a combination of the factor Vila factor XIII to produce a drug that promotes blood clots of sputum milk plasma fibrin. In a specific embodiment, the mammalian plasma is human plasma. In another embodiment, the milk plasma is normal plasma; in another embodiment, the plasma is normal human plasma; in one embodiment, the plasma is plasma from a thrombin-producing patient. In a specific embodiment, the plasma is from a patient having a low concentration of fibrinogen. In one embodiment, Factor Vila and Factor XIII prolong the hemolysis time of normal human plasma in vitro. 15 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------!·---- ----------- --- Line zero (please read the note on the back and then fill out this page) A7 1287452 __ B7 V. Invention description (蛑) (Please read the note on the back and then fill out this page) In another point, the present invention is A method for promoting formation of fibrin clots in a patient comprising administering an effective amount of a factor Vila to an effective amount of a factor XIII to the patient. In another aspect, the invention relates to a method of treating a hemorrhagic event in a patient comprising administering to a patient a combination of an effective amount of Vila and an effective amount of a factor of ΧΙΠ. In another aspect, the invention is directed to a method of reducing clotting time in a mammalian plasma comprising contacting the plasma with an effective amount of Factor Vila in combination with an effective amount of Factor XIII. In a specific embodiment, a combination of an effective amount factor Vila and an effective amount factor XIII is administered to a patient in need of such treatment. In another aspect, the invention relates to a method of enhancing the formation of fibrinosis in a patient comprising administering an effective amount of a factor Vila to a patient in combination with an effective amount of factor XIII. In a specific embodiment of the method of the invention, Factor Vila and Factor XIII are the sole activators administered to the patient. In another embodiment of the invention, the pharmaceutical composition comprises the factor Vila as the sole activator and factor XIII. In a specific embodiment of the invention, the Factor Vila and Factor XIII are administered simultaneously and in a single dosage form. In another embodiment, the Factor Vila is administered continuously with Factor XIII. In another embodiment, the factor Vila and Factor XIII are administered within about 1 to 2 hours of each other, for example, within 30 minutes of each other, and if administered within 10 minutes of each other, for example, to include the first Factor in the form of a unit dose of Vila 16 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) A7 1287452 V. Inventive Note (K) and the second unit dosage form of Factor XIII . In a specific embodiment, an effective amount of Factor Vila ranges from 0.05 mg/day to 500 mg/day (70 kg patient). In a specific embodiment, an effective amount of Factor XIII is from 0.05 mg/day to 500 mg/day (70 kg patient). In a particular embodiment of the invention, the pharmaceutical composition (in the form of a single preparation) is mainly composed of the factor Vila and Factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and/or stabilizer, and/or hydrazine. A detergent, and/or a neutral salt, and/or an antioxidant, and/or a preservative, and/or a protease inhibitor. In another embodiment of the invention, the pharmaceutical composition (in a single formulation form) is primarily comprised of the factor Vila and Factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and/or stabilizer, and / Or a detergent, and/or a neutral salt, and/or an antioxidant, and/or a preservative, and/or a protease inhibitor, and/or a TFPI inhibitor. In another embodiment of the invention, the pharmaceutical composition (in a single formulation form) is primarily comprised of the factor Vila and Factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and/or stabilizer, and / Or a detergent, and/or a neutral salt, and/or an antioxidant, and/or a preservative, and/or a protease inhibitor, and/or a TFPI inhibitor, and/or Factor VIII. In another embodiment, the pharmaceutical composition (in the form of a kit) consists of the first unit dosage form, which is primarily comprised of the factor Vila, and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizers, and / or cleaning agents, and / or neutral salts, and / or antioxidants, and / or 17 paper scales apply to the Chinese National Standard (CNS) A4 specifications (210 X 297 mm) ^ ~ (Please read the note on the back and fill out this page) -------- Order — A7 1287452 _______B7_ ψ V. Invention description (4) (Please read the note on the back and fill in this page) And/or a protease inhibitor; and a second unit dosage form, which is primarily comprised of Factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and/or stabilizer, and/or neat And/or a neutral salt, and/or an antioxidant, and/or a preservative, and/or a protease inhibitor. In another embodiment, the pharmaceutical composition (in the form of a kit) consists of the first unit dosage form, which is primarily comprised of the factor Vila, and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizers, and / or detergents, and / or neutral salts, and / or antioxidants, and / or preservatives, and / or protease inhibitors, and / or TFPI inhibitors; and Two unit dosage forms, which are primarily comprised of factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and/or stabilizer, and/or detergent, and/or neutral salt, and/or An antioxidant, and/or a preservative, and/or a protease inhibitor, and/or a TFPI inhibitor, and/or Factor VIII. In another embodiment, the patient is a human; in another embodiment, the patient has a deficiency in thrombin generation; in one embodiment, the patient has a low plasma fibrinogen (eg, a multiple transfusion patient) . In a further aspect, the composition additionally contains Factor VIII. In a specific embodiment, Factor VIII is an activating factor VIII (factor Villa). In another specific embodiment, Factor VIII is recombinant factor Vma. In another specific embodiment VIII is recombinant human factor Villa. In a further aspect, the composition additionally comprises a fibrinolytic system inhibitor, for example, aprotinin, ε-aminocaproic acid or tranexamic acid. 18 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 1287452 _____B7___ V. Description of invention (0) Figure 3 shows rFVIIa and FXIII for maximum clot firmness (MCF) and clot versus t-PA The effect of resistance to mediation. The MCF obtained before the addition of rFVIIa and/or FXIII was 25 mm, and the time required for the clot to dissolve half was 12.3 minutes (Fig. 3). The addition of increasing concentrations of FXIII (0-40 nM) did not alter MCF; however, a half-dose lysis of the clot was observed to be dose-dependent, preferably at 30 nM FXIII (-half clot lysis time: 14.3 minutes, see image 3). Similarly, the addition of rFVIIa (1 nM) resulted in protection of blood clots from fibrinolysis by t_PA media (half clot lysis time: 16.4 minutes) and had no effect on MCF (Fig. 3). However, when rFVIIa (1 nM) was added with FXIII (30 nM), an increase in MCF (29 mm) and a significant protective effect on fibrinolysis (half clot lysis time: 27.1 minutes) were observed (Fig. 3). Taken together, these results demonstrate that the addition of rFVIIa and FXIII to plasma synergistically improves the physical strength of the blood clot and resists fibrinolytic resistance by t-PA media. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a composition comprising a combination of a factor Vila and a factor XIII. The invention also provides a composition comprising a factor Vila in combination with a factor XIII as the sole activating component. The composition may be in a single composition or may be in the form of a multi-component kit. This composition is effective as a coagulant and fibrin clot stabilizer before disease treatment and prevention, and can rapidly form fibrin clots in mammals, including primates such as humans. Whenever a first or second or third unit dose is mentioned in the specification, this does not mean a preferred order of administration, but only for convenience. 20 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) "" ~~~ ------r---Λ----------- --------- sw (Please read the notes on the back and fill out this page) A7 1287452 V. INSTRUCTIONS (丨 / ) The present invention further provides a treatment (including prophylactic treatment or prevention) Methods of hemorrhagic events, including humans, for example, due to trauma or surgery or lack of blood clotting factor FIX or FVIII or platelet defects. It has now been found that the combination of Factor Vila and Factor Xllla is a beneficial product that ensures the formation of solid, stable and rapid hemostasis embolization. The formation of intact thrombin is required for the formation of a solid, stable thromboembolism. The fibrin structure of this embolization depends on the amount of thrombin formed and the rate at which the initial thrombin is produced. In the presence of damaged thrombin, a highly permeable porous fibrin plug is formed. Fibrinolytic enzymes that normally appear on the surface of fibrin can easily dissolve such fibrin embolism. The formation of stable fibrin embolism also depends on the presence of the factor Xllla, which can be activated by thrombin, and therefore also depends on the formation of intact thrombin. Furthermore, the recently mentioned thrombin-activatable fibrinolysis inhibitor, TAFI, requires a relatively high amount of thrombin to achieve its activation. In the case of an incompletely sufficient formation of thrombin, TAFI may therefore not be activated, resulting in the formation of a hemostatic plug, which is easier than normal fibrinolytic activity to dissolve under normal conditions. By increasing the production of thrombin, the factor Vila provides the basis for the activation of intact factor XIII, which is most important for the formation of a completely stable thromboembolism and sustained hemostasis. In the case of a decrease in the number of platelets, i.e., thrombocytopenia, a faster thrombin generation is initiated too quickly by the additional exogenous factor Vila. However, the production of whole thrombin is not regulated by Factor Vila or even at high concentrations. 21 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page) ---------Book------- -- 1287452 A7 ---------B7 V. INSTRUCTION DESCRIPTION (γ) Complete activation of a factor XIII by binding factor Vila and factor XIII, especially factor XIII α chain (a2-dimer) Can be accelerated, which enhances the hemostatic effect of the factor Vila. In addition, activation of intact factor 不会 does not occur in patients with fibrinogen with reduced plasma concentrations (patients with multiple blood transfusions necessary for multiple trauma or large-scale surgery). Then, by administering the combination of the factor ViIa and the factor XIII, a more effective hemostasis can be obtained. Another way to increase the stability of fibrin to stop thrombus is to ensure the complete performance of the factor Xllla (factor XIII of activation). Patients with thrombocytopenia have impaired thrombin generation and defective fibrin embolization stability, leading to a premature decomposition of the thromboembolism. In addition, patients suffering from major trauma or organ damage, and thus frequent transfusions, often have reduced platelet counts, as well as reduced levels of fibrinogen, factor VIII and other coagulating proteins. These patients experience impaired (or decreased) thrombin generation. Moreover, its reduced amount of fibrinogen negatively interferes with the activation of factor XIII. Therefore, these patients have defective and inefficient hemostasis, which leads to the formation of fibrin embolization easily and prematurely decomposed by proteolytic enzymes. This kind of enzyme will be greatly aggravated in the case of large-scale trauma and organ damage. freed. In order to promote the formation of a completely stable embolization and to have a complete ability to maintain the patient's hemostasis, the composition of the present invention is administered. This composition is particularly effective for patients with a reduced number of platelets and patients with reduced plasma concentrations of fibrinogen and/or other clotting proteins. In the presence of factor XIII, a lower concentration of factor Vila may be sufficient to ensure adequate hemostasis. 22 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page) ---------Book------- -- SW. A7 1287452 ______B7 _ V. INSTRUCTION DESCRIPTION () As mentioned earlier, Factor XIII is present in plasma and is a tetrameric zymogen comprising two alpha units and two beta units (defined as a2b2) However, in other tissues (eg, platelets) it is a 2-dimer. These zymogen forms, or the activated form factor XIII (factor Xllla), can be used in the present invention, and the genetically engineered variant of factor XIII retains its specific cross-linking activity. In a specific embodiment, Factor XIII is human Factor XIII; in another specific embodiment, Factor XIII is a human a2-dimer; in another embodiment, Factor XIII is activating human Factor Xllla. Factor XIII and Factor Vila used in the present invention may be produced by purification or recombinant methods in blood. Significantly, the performance of the methods described herein does not depend on how the purified Factor XIII is derived from the Factor Vila. Therefore, the present invention contemplates the use of any of the Factor XIII and Factor Vila formulations suitable for use herein. Preferred are human factor Vila and human factor XIII. At the same time, the present invention also utilizes genetic engineering variants that retain a factor of special hemostasis-related activity, Vila, and Factor XIII. Fragments of Factor Vila or Factor XIII or a factor that retains a specific hemostatic-related activity, Vila- or Factor XIII-variant, can also be used in the present invention. The hemostatic-related activity of the factor Vila can be measured, for example, by using the factor Vila activity assay described in the present specification. The hemostatic-related activity of Factor XIII, for example, can be measured using Factor XIII activity as described in the specification. Examples of Factor VII variants having substantially the same or better biological activity than the wild-type factor Vila include, but are not limited to, those described in Danish Patent Application No. PA 2000 00734, PA 2000 01360, PA 2000 01361 and PA 2001 00477. Non-limiting examples include 23 paper sizes applicable to China National Standard (CNS) A4 specifications (210 X 297 mm) (please read the notes on the back and fill out this page) -------- --------. A7 1287452 ___B7 V. Description of invention (y>0 (please read the notes on the back and fill out this page) [L305V]-FVIIa, [L305V/M306D/D309S]-FVIIa, [ L305I]·FVIIa, [L305T]-FVIIa, [F374P]-FVIIa, [V158T/M298Q]-FVIIA, [V158D/E296V/M298Q]-FVIIa and [K337A]-FVIIa. In this context, amino acids are in three letters or A letter representation has been used for its conventional meaning, as shown in Table 1. Unless otherwise indicated, the amino acids referred to herein are L-amino acids. It must be understood that, for example, the first in K337 The letters indicate that the amino acid is essentially located at the position shown by wild-type Factor VII, and, for example, [K337A]-FVIIa is represented as a FVII-variant, which is essentially represented by the one-letter code K at the position shown. The amino acid is replaced by an amino acid represented by another letter A. Table 1: Abbreviations for amino acids

Amino acid three letter code one letter code glycine • Gly G proline Pro P alanine Ala A proline Val V leucine Leu L isoleucine lie I methionine Met M cysteine Cys C benzene Alanine Phe F Tyrosine Tyr Y Tryptophan Trp W 24 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 1287452 __B7 V. Description of invention (4)

Histidine His H Lysine Lys K Arginine Arg R Glutamine Gin Q Asparagine Asn N Glutamate Glu E Aspartate Asp D (Please read the back note first and then fill out this page The noun "factor Vila" or "FVIIa" can be used interchangeably. The noun factor Vila contains the enzyme cause VII (single chain factor VII). Noun, factor xm" or "FXIir can be used interchangeably. The noun "factor VIII" or "FVIII" can be used interchangeably. Those skilled in the art will be apparently able to perform substitutions outside of the sites important for the function of Factor VIIa and Factor XIII, and still obtain activated polypeptides. The amino acid residues necessary for the activity of Factor Vila or Factor XIII-polypeptide are, and therefore preferably not, substituted, which can be identified according to procedures well known in the art, such as position-specific mutation or alanine scanning. Mutation methods (see, for example, Cunningham and Wells, 1989, Science 244: 1081-1085). In a later technique, the mutation is introduced into each positively charged residue on the molecule, and the resulting mutant molecule is tested for coagulant activity, with individual cross-linking activities to identify amino acids important for molecular activity. The position of the receptor-enzyme interaction can also be determined by analyzing the three-dimensional structure, as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see eg de Vos et al., 1992, 25 papers). The scale applies to the Chinese National Standard (CNS) A4 specification (210 297 297 mm) A7 1287452 __B7__ V. Description of invention (slip) 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 244: 899-904; Wlodaver Et al., 1992, FEBS Letters 309: 59-64). Introduction of a mutation into a nucleic acid sequence to exchange one nucleotide for another can be accomplished by position specific mutation using any method known in the art. A particularly useful procedure is to utilize a supercoiled, double-stranded DNA vector with the desired insert and two synthetic primers containing the desired mutation. The oligonucleotide primers, each capable of being complementary to the corresponding strand on the vector, are elongated during the temperature cycle with the DNA polymerase. When the primer is inserted, a mutant system containing staggered incisions is formed. After the temperature cycle, the product is π/treated, which is specific for methylated and hemimethylated DNA to cleave the parental DNA template and screen for synthetic DNA containing the mutation. Other conventional procedures in this art can also be used for generating, identifying, and isolating variants, for example, by way of example, gene shuffling or phage display techniques. The designation "Factor VIII" or "FVIII" includes activating factor VIII (determined as Villa), a variant of factor VIII retaining its specific coagulation activity, and truncated forms. In a specific embodiment, Factor VIII is Human Factor VIII. The designation "TFPI inhibitor" means a compound which inhibits the anticoagulant activity of TFPI (tissue factor pathway inhibitor). The term includes compounds such as those disclosed in European Patent Nos. 558 529, WO 96/28153 and US 5,622,988. "TFPI" and "ΕΡΓ (external pathway inhibitor) can be used interchangeably. In the present invention, an "effective amount" factor of Vila and an "effective amount" of 26 paper scales are applicable to the Chinese National Standard (CNS) A4 specification. (210 X 297 mm) (Please read the notes on the back and fill out this page) --------Book --------- A7 1287452 __B7__ V. Invention Description (〆) Factor XIII The amount defined as Factor Vila and Factor XIII is sufficient to prevent or reduce bleeding or blood loss in order to treat, alleviate or partially arrest the disease and its onset. The dosage of Factor Vila and Factor XIII according to the present invention may be approximated by The ratio between 1:100 and about 100:1 (pg factor Vila: gg factor XIII). In this article, "a patient with impaired thrombin generation" means that the patient cannot produce a sudden onset of activated platelets. Coagulase, and includes thrombin-producing patients with less than normal function and normal hemostasis in patients with thrombin production, the normal hemostatic system contains a normal amount and function of coagulation factors, platelets and fibrinogen, and including deficiency FIX and/or FVIII (hemophilia A and B) or defective FIX and/or FVIII or patients with anti-FIX and/or FVIII inhibitors; patients with FXI deficiency; reduced number of platelets or platelet function Patients with defects (for example, patients with thrombocytopenia or Glanzmann's platelet dysfunction or excessive bleeding); and patients with reduced levels of prothrombin, FX or FVII. Fibrinogen with reduced plasma concentrations (eg, Patients with multiple transfusions due to multiple trauma or large-scale surgery also develop a loose and unstable fibrin embolism that is easily broken down. The term "complete hemostasis" means a stable and solid fiber at the wounded site. Vitamin clots or embolizations are effective in stopping bleeding and are not easily broken down by the fibrinolytic system. The term "factor Vila activity" or "factor Vila-activity" means the ability to produce thrombin; the term is also included in the absence of tissue factor The ability to produce thrombin on the surface of activated platelets. 27 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the back first) Note: Please fill out this page) --------Book---------. A7 1287452 V. Description of invention (/b) The term "promotion of normal hemostasis system" means the production of blood clotting The increase in the ability of the enzyme. The term "bleeding disorder" as used herein reflects any defect, innate, acquired or induced cell or molecular origin' which is manifested in a hemorrhagic event. In fact, there is a lack of clot factor (eg, blood A disease type A and type B or lack of factor XI or VII), a clot factor inhibitor, platelet function deficiency, thrombocytopenia or von willebrand's disease. The term "hemorrhagic event" is meant to include uncontrollable and excessive bleeding as a major problem associated with surgical and other forms of tissue damage. Uncontrollable and excessive bleeding may occur in patients with a substantially normal coagulation system (however, these patients develop coagulation due to bleeding - due to dilution of coagulation proteins, increased fibrinolysis, and decreased platelet count) And patients with clotting or bleeding disorders. Clot factor deficiency (hemophilia A and B, deficiency of factor XI or VII) or clot factor inhibitor may be the cause of bleeding disorders. Excessive bleeding also occurs in patients with a normal functional blood clot cascade (no clot factor deficiency or inhibitor of any coagulation factor) and may be due to defective platelet function, thrombocytopenia or von Willebrand Caused by the disease. In these cases, hemorrhagic events can be compared to those caused by hemophilia, because of the lack of or abnormally necessary blood clotting compounds (such as platelets or von Willebrand factor proteins) in hemophilia patients. Major bleeding. In patients experiencing large-scale tissue damage associated with surgery or major trauma, normal hemostatic mechanisms may be suppressed by immediate hemostasis. 28 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). (Please read the note on the back and then fill out this page) —---------------------- 1287452 V. Invention description (yy), and basically (pre-trauma) normal Hemostasis can develop into bleeding. A satisfactory hemostasis is also a problem when hemorrhagic events occur in organs such as the brain, inner ear and eyes, with limited surgical hemostasis. The same problem may occur during the biopsy of different organs (liver, lung, tumor tissue, gastrointestinal tract) and during endoscopic surgery. It is common in all of these situations that it is difficult to provide hemostasis by surgical techniques (sewing, hemostats, etc.), which is also a bleeding spread (hemorrhagic gastritis and excessive uterine bleeding). Acute and excessive bleeding may also occur in patients treated with anticoagulants, whose hemostasis has been triggered by the treatment itself. These patients may require surgical intervention if the anticoagulant effect must be neutralized quickly. Post-pubic prostatectomy is a common surgical procedure for patients with localized prostate cancer. In this procedure, blood loss is often manifested and sometimes excessive. A large amount of blood loss during prostatectomy is mainly related to complex anatomical conditions, which have a variety of dense vascularized sites that are not easily accessible by surgical hemostasis, and may cause large areas of diffuse bleeding. Another problem that may arise in an unsatisfactory hemostasis case is when a patient with a normal hemostasis mechanism is given anticoagulant therapy to prevent thromboembolic disease. These treatments may include heparin, other forms of proteoglycan, warfarin or other forms of vitamin K-antagonists and aspirin, as well as other platelet aggregation inhibitors. In a specific embodiment of the invention, bleeding is associated with hemophilia. In another embodiment, the bleeding is associated with hemophilia with acquired inhibitors. In another embodiment, bleeding is associated with thrombocytopenia. In the other 29 paper scales, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) is applied. -----I.——Λ——--------Set------- -- (Please read the notes on the back and fill out this page) 1287452 A7 __B7____ V. INSTRUCTIONS (>&) In one embodiment, bleeding is associated with von Willebrand's disease. In another embodiment, bleeding is associated with severe tissue damage. In another specific embodiment, bleeding is associated with severe trauma. In another embodiment, the bleeding is associated with surgery. In another embodiment, bleeding is associated with endoscopic surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis. In another embodiment, the bleeding is excessive bleeding in the uterus. In another embodiment, bleeding occurs in an organ with a limited likelihood of mechanical hemostasis. In another embodiment, bleeding occurs in the brain, inner ear area, or eye. In another embodiment, the bleeding is related to the slicing sampling process. In another embodiment, the bleeding is associated with anticoagulant therapy. The composition according to the invention may additionally comprise a TFPI inhibitor. This composition should preferably be administered to patients with hemophilia A or B. The composition according to the invention may additionally comprise factor VIII. This composition should preferably be administered to a patient who does not have a Factor VIII inhibitor. As used herein, the term "treatment" is meant to include the prevention of an expected hemorrhagic event, for example, by way of example, during surgery, and the modulation of a hemorrhagic event, such as, for example, hemophilia or trauma, Its purpose is to inhibit or reduce bleeding. Therefore, the prophylactic administration of the factor Vila and factor XIII is also included in the noun "treatment". The term "patient" as used herein refers to any animal, especially a mammal, such as a human, and if appropriate, can be used interchangeably with the noun "patient." This paper scale applies to the Chinese National Standard (CNS). A4 size (210 X 297 public) (Please read the note on the back and fill out this page) --------tr---------M9. A7 1287452 _______B7 V. Description of invention ( >/) Abbreviation TF Tissue Factor FVII Single-stranded, inactive Factor VII FVIIa Activated Factor VII rFVIIa Activated Recombinant Factor VII FXIII Enzymatic, Inactive Factor XIII FXIIIa Activated Factor XIII rFXIII Recombinant FXIII rFXIIIa Recombinant FXIIIa a2 FXIII or rFXIII oc- or a-chain b2 FXIII or rFXIII β- or b-chain FXIIIa2 Two a-chain dimers FXIII FXIIIa2b2 containing two a-chains and two b-chains FXIII FVIII zymogenic, inactive factor FVIII rFVIII recombinant FVIII FVIIIa activated factor VIII rFVIIIa recombinant FVIIIa TFPI preparation of tissue factor pathway inhibitor compound Human purification factor Vila suitable for use in the present invention This is made by DNA recombination techniques, for example, as described in Hagen et al., Proc. NatL Acad. Sci. USA 83: 2412-2416, 1986, or in European Patent No. 200.421 (ZymoGenetics, Inc.). The factor of technology production ------r---_---------- order--------- (please read the notes on the back and fill out this page) 31 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 1287452 __ B7___ V. Invention description (V)

Vila can be a true factor Vila or a modified factor Vila, if such a factor Vila has substantially the same coagulation biological activity as the true factor Vila (wild type factor Vila). The modified factor Vila can modify the nucleic acid encoding the wild type factor VII by changing the amino acid sequence of the nucleic acid encoding the natural factor VII or removing the codon of the certain amino acid by a known technique (for example, position-specific mutation). It is made by sequence. Factor VII can also be produced by the method described by Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. Invest. II: 1836-1841, 1983. Factor VII produced by these methods does not have detectable amounts of other coagulation factors. Even the preparation of another purified Factor VII formulation can be accomplished by including an additional gel filtration as the final purification step. Factor VII is subsequently converted to the activating factor Vila, for example, several different plasma proteins, such as the factor Xlla, IXa or Xa. Alternatively, as described by Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565), Factor VII can be activated by passing through an ion exchange chromatography column, such as Mono Q® (Pharmacia Hne Chemicals). String or similar. The factor XIII used in the present invention can be produced from plasma according to a conventional method, for example, by Cooke and Holbrook (Biochem. J. 141: 79-84, 1974) and Curtis and Lorand (Methods EnzymoL 45: 177-191). , 1976), the disclosure of which is incorporated herein by reference. The factor 2 of the a2 singular form can be made from the placenta, as disclosed in U.S. Patent Nos. 3,904,751, 3,931,399, the entire disclosure of which is incorporated herein by reference. However, it is preferable to use the recombinant factor XIII to avoid the use of the 32-paper standard (CNS) A4 specification (210 X 297 public) "" ------r---. ----------Book--------- M9 (Please read the note on the back and fill out this page) 1287452 A7 __B7___ V. Description of invention (force 1) Risk of disease transmission A product derived from blood or tissue. Methods for preparing recombinant factor XIII are well known in the art. See, for example, Davie et al., EP 268,772; Grundmann et al; AU-A-69896/87; Bishop et al; Biochemistry 1990, 29: 1861-1869; Board et al; Thromb. Haemost· 1990 '63: 235- 240; Jagadeeswaran et al; Gene 1990, 86: 279-283; and Broker et al; FEBS Lett-1989, 248: 105_110, which are incorporated by reference herein in its entirety. In a specific embodiment, the Factor XIII a2 dimer is prepared from the brewing yeast (Acc plus mmyea in the cytoplasmic fraction, as described in the commonly-owned U.S. Patent Application Serial No. 07/741,263, the disclosure of which is incorporated herein by reference. The cells are dissolved and a clean lysate is prepared. The lysate is fractionated by anion exchange chromatography at a neutral to slightly alkaline pH using a derivatized agarose column, such as DEAE Fast-Flow. Sepharose TM. (Pharmacia) or the like. The Factor XIII tubing is then precipitated by concentrating the extract and adjusting the pH to 5.2-5.5, for example, with an anti-succinate buffer. (diafiltration). The precipitate is then dissolved and further purified using conventional chromatographic techniques, such as gel filtration and hydrophobic interaction chromatography. As will be appreciated by those skilled in the art, it is preferred to use the same Factor XIII and Factor Vila protein to reduce the risk of inducing immune response. The preparation and characterization of non-human factor XIII has been disclosed by Nakamura et al. (J. Biochem. 78: 1247-1266, 1975). also Contains the use of this factor XIII and Factor Vila protein in veterinary procedures. 33 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) „-------------- --Order -------- IM9 (Please read the note on the back and fill out this page) 1287452 B7 V. INSTRUCTIONS Pharmacy Compositions for Drugs For the treatment of cautious interventional factors, Factor VII and Factor XIII It will usually be administered within about 24 hours prior to the interventional therapy, and 7 days or more thereafter. There are many ways to administer in the form of a coagulant, as shown here. For a 70 kg patient And a sustained dose, the dose of Factor VII is from about 5 mg to about 500 mg per day, for example, from about 1 mg to about 200 mg per day, or, for example, from about 10 mg to about 175 mg per meal. It depends on the patient's weight, condition and severity. For a 70 kg patient to be administered and maintained, the dose of factor 由 ranges from about 0.05 mg to about 500 mg per day, for example, by about every 1 mg to about 1 day 200 mg, or, for example, from about 10 mg to about 175 mg per ounce 'depends on the patient's weight, condition and severity. The compositions and kits of the present invention are quite effective in human and veterinary medicine; for example, For example, in the treatment or prevention of a patient having a hemorrhagic event or a clotting disorder, in the use of the present invention, the factor Vila and the factor XIII are formulated and optionally added to any pharmaceutically acceptable Carrier. Preferably, the pharmaceutical composition is administered parenterally; that is, intravenously, subcutaneously or intramuscularly, or administered as a continuous or pulsed infusion. The formulation may additionally contain one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and may be applied to the Chinese National Standard (CNS) A4 Regulations on liquid, powder, and emulsion standards. 210 X 297 publicly------- (Please read the notes on the back and fill out this page) --------Book --------- A7 1287452 ____B7_ V. Invention Description (;^), controlled release, etc. One of the techniques formulates the compositions of the present invention in a suitable form and is consistent with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990. These parenterally administered pharmaceutical compositions comprise Factor VII and Factor XIII and are preferably soluble in a pharmaceutically acceptable carrier, preferably an aqueous carrier. The carrier may be used, for example, water, aqueous buffer solution, 0.4% saline, 0.3% glycine, and the like. The Factor VII variant of the invention may also be formulated as a liposome preparation for delivery or targeting of the injured site. Liposome preparation For example, U.S. Patent Nos. 4,837,028, 4,501,728 and 4,975,282. A typical pharmaceutical composition for intravenous infusion can be formulated to contain 250 ml of sterile forest format solution with 10 mg of Factor Vila and/or Factor XIII. The actual parenteral administration of compositions is well known or known to those skilled in the art and is described in more detail in, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990. 0 In summary, the pharmaceutical compositions suitable for use in accordance with the invention are prepared by mixing factor Vila, or factor XIII, or factor Vila binding factor XIII, preferably in purified form, with appropriate adjuvants and appropriate A carrier or diluent. Suitable physiologically acceptable carriers or diluents include sterile water and saline. The compositions contain pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, osmotic pressure adjusting agents, and physiological conditions thereof. Analogs, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, chlorinated 35 paper scales applicable to Chinese National Standard (CNS) A 4 specifications (21G X 297 public) " (Please read the note on the back and fill out this page) ---------Book --------- 1287452 A7 ____B7___ V. Invention Description (W) Calcium and so on. Suitable adjuvants also include calcium, proteins (e.g., albumin), or other inert peptides (e.g., glycine glycine) or amino acids (e.g., glycine, or histidine) to stabilize the pure factor Vila and/or factor XIII. The remaining physiologically acceptable adjuvants are non-reducing saccharides, polyols (eg, sorbitol, mannitol or glycerol), polysaccharides such as low molecular weight dextran, detergents (eg polysorbate; polysorbate) With antioxidants (such as bisulfite and ascorbate). Typical concentrations of adjuvants range from 0.001 to 4% w/v. The pharmaceutical composition also includes protease inhibitors such as aprotinin or anti-fibrinolytic acid, as well as preservatives. In addition, the formulation also contains a TFPI inhibitor and/or a factor VIII. The composition may be sterilized by conventional, known sterilization techniques. The final liquid solution is packaged for use or filtered and lyophilized in a sterile environment. The lyophilized formulation must be combined with a sterile aqueous solution prior to use. In these formulations, the concentration of the factor Vila, the factor XIII, or the factor Vila binding factor XIII is quite large, that is, from less than about 0.5% by weight, usually from at least about 1% by weight to at most 15 or 20% by weight, It is mainly selected in the form of liquid volume, viscosity, etc., depending on the particular mode of administration selected. It is preferred to administer by injection or perfusion, especially injection. Thus, the factor Vila and Factor XIII are formulated for intravenous injection, for example, as a preparation of a dissolved lyophilized powder or liquid formulation containing a single dosage form of Factor Vila and Factor XIII, or in a single dosage form containing the factor Vila. A lyophilized powder or liquid formulation, and a dissolved lyophilized powder or liquid formulation containing another dosage form of Factor XIII. 36 This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) " (Please read the note on the back and fill out this page) ---------Book----- ---- A7 1287452 _____Β7__ V. Description of invention (3<) (Please read the note on the back and then fill out this page) Local delivery of factor Vila and factor XIII, for example, for example, local application may be performed, for example, Spraying, perfusion, double balloon catheters, stent stents, vascular grafts or stents, hydrogels for balloon catheters, or other established methods . For ambulatory patients who need to maintain daily levels, the factor Vila and factor ΉΙΙ may be administered continuously using, for example, a hand-held pump system. In any event, the pharmaceutical composition should provide an amount of Factor Vila and Factor XIII that are sufficiently effective to treat the condition. The combination of Factor Vila and Factor XIII showed synergy in the analysis of in vitro clot firmness and fibrinolytic time. In addition, the combination of factor Vila and factor XIII has a synergistic effect on the formation of a stable fibrin clot, an increase in clot lysis for half of the time, an increase in clot strength and an increase in resistance to fibrinolysis. Administration of a composition comprising a factor Vila and a factor XIII can be used for disease prevention and medical treatment. In therapeutic applications, the composition is administered to a patient who is already afflicted, as described above, in an amount sufficient to treat, slow or partially arrest the disease or its onset. One of the quantities sufficient to accomplish this is defined as an "effective amount" or "medical therapeutically effective amount." As will be appreciated by those skilled in the art, the amount effective for this purpose will depend on the severity of the disease or injury and the weight and general condition of the patient. It is important to remember that the materials in this statement are generally used in cases of serious illness or injury, that is, in the case of life threatening or potentially life threatening. In such cases, there is a general lack of immunogenicity in the production of the factor Vila and factor XIII in the human body, and overdose of these compositions may be desirable for the treating physician. 37 The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1287452 A7 __B7___ V. INSTRUCTION DESCRIPTION In the preventive application, the composition containing the factor Vila and factor XIII is administered in a susceptible or otherwise Patients with dangerous or injured disease to increase their own blood coagulation capacity. This dose is defined as "an effective dose for disease prevention." The composition of the single or multiple administrations can be carried out by the dosage range and mode selected by the physician to be treated. The composition may be administered one or more times daily or weekly. An effective amount of such a pharmaceutical composition refers to an amount that provides a clinically significant anti-hemorrhagic event. These amounts are based in part on the patient's specific treatment, age, weight and general health, as well as other factors that are apparent to those skilled in the art. The composition is generally administered in a single dose prior to the expected bleeding or at the onset of bleeding. However, it is also possible to administer in multiple doses, preferably at intervals of 2-4-6-12 hours, depending on the dose administered and the condition of the patient. The composition may be in the form of a single agent formulation containing the appropriate concentration of factor Vila and factor XIII. The composition may also be in the form of a group comprising the first unit dosage form comprising the factor Vila and the second unit dosage form comprising the factor XIII, and optionally one or more other factors VIII and/or The unit dosage form of the TFPI inhibitor consists. In this case, the factor Vila and the factor XIII should be administered continuously, preferably at intervals of about 1-2 hours, for example within 30 minutes of each other, or preferably within 1 minute, or more preferably at intervals. Within 5 minutes. Both unit dosage forms are available as priority donors. Since the present invention relates to the prevention or treatment of hemorrhagic events, or to coagulation therapy which is necessary to be administered separately and in combination with two active ingredients, the present invention also relates to the incorporation of individual pharmaceutical compositions in a set of forms. 38 P-sheet scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ' ' (Please read the phonetic on the back? Please fill out this page) --------Book---- ----- A7 1287452 ___B7__ V. Invention description (^)

This kit includes at least two individual pharmaceutical compositions. This kit contains _ individual container containers, such as sub-bottles or foil packs. Typical kits include instructions for the administration of individual components. When individual components must be administered in a dosage form, the kit format is advantageous, may be administered at different doses, or when the prescribing physician must titrate the individual components of the combination. X-analytical factor Vila activity test A suitable analysis can test the factor Vila activity and thus select the 因子When the Factor Vila variant is performed in a simple ex vivo assay: In vitro hydrolysis analysis of the original (wild-type) factor Vila The specific activity of the factor Vila variant (both of which refers to the factor Vila) was analyzed. Both can also be analyzed in parallel to directly compare their isospecific activities. This analysis was performed with a microtiter plate @ (MaxiSorp, Nunc, Denmark). The chromogenic receptor is dextrorotatory leucine, valine-arginine and nitroaniline (S-2288, Chromogenix, Sweden), with a final concentration of 1 mM, added at 50 mM HEPES, pH 7.4, containing 0·1 Μ Sodium chloride, 5 mM calcium chloride and 1 mg/ml fetal bovine blood albumin factor Vila (final concentration 100 nM) mixed solution. The absorbance at 405 nm was continuously measured on a SpectraMaxTM 340 disc reader (Molecular Devices, USA). After subtracting the absorbance of the enzyme-free blank group, the absorbance enthalpy produced over a period of 20 minutes is used to calculate the activity ratio between the variant and the wild-type Vila: 39 This paper scale applies to the Chinese National Standard (CNS) A4. Specifications (210 X 297 mm) {Please read the notes on the back and fill out this page)

A7 1287452 ____B7___ V. Description of invention (:μ) Ratio = (Α4_Factor Vila variant) / (A4〇5nm factor Vila wild type). According to the above, a factor Vila variant having an activity equivalent to or higher than the original factor Vila can be identified, for example, as shown in Fig. 1, wherein the activity ratio between the variant and the original factor Vila is significantly higher. A variant of 1.0. Factor Vila or Factor Vila variant activity can also be measured using a physiological receptor such as Factor X, suitably at a concentration of 100-1000 nM, wherein the resulting Factor Xa is measured after addition of a suitable chromogenic receptor (eg S-2765). . In addition, the activity assay was performed at physiological temperature. The measurement of the ability of a factor Vila or a factor Vila variant to produce a thrombin may also include all clotting factors and inhibitors associated with physiological concentrations (deducting factor VIII when simulating hemophilia A) and activated platelets ( It is carried out as described in Monroe et al. (1997) Brit. L Haematol. 99, 542-547, p. 543, which is incorporated herein by reference. Testing of Factor XIII Jobs A suitable assay can examine Factor XIII transglutaminase activity and thus select appropriate Factor XIII variants, as described in a simple in vitro assay, for example, in Methods of Enzymology, Vol. 45 (1976), Proteolytic Enzymes, Part B, pages 177-191 (Ed. Lorand, L.) o The invention is further illustrated by the following examples, but is not to be construed as limiting the scope of the invention. The foregoing disclosed features, as well as the following examples, may be used in various combinations and in any combination to understand the various forms of materials of the invention. 40 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page)

1287452 A7 ___B7___ V. Description of the invention (▽) Example 1 Factor XIII increase factor Vila-induced fibrin clot formation Citrate normal human plasma (NHP) is diluted 1/10 on a microtiter plate to contain 20 nM HEPES, 150 mM sodium chloride, 5 mM calcium chloride, pH 7.4 in buffer (total volume 250 μΐ), and fibrin clot formation with SpecramaxTM 340 (Molecular Devices, Sunnyvale CA) at 600 nm optical density Increased detection. Spontaneous clot formation can be seen between about 2500-3000 seconds. Figure 1 shows that the 10 nM recombination factor Vila (rVIIa) (Novo Noniisk A/S Bagsvaerd, Denmark) shortens the clot time to 1600 seconds (n=2). Another shortening of clot time can be seen when 30 nM Factor XIII (FXIII) (American Diagnostica Inc, Greenwich, CT) and 10 nM rFVIIa were added (n=3). The clot formed in the presence of FXIII is clearer than the absence (maximum 〇D値), which indicates that the addition of FXIII results in a finer network of fibrin gels with thinner fibers. Example 2 Factor Vila-induced clot formation during the presence of supplemental factor XIII resulted in an increase in fibrinolytic decomposition compared to a clot containing the same amount of fibrin and arranged in thick fibers or less cross-bonded fibers, A fibrin clot composed of a thin fiber is mechanically tough and difficult to decompose. The experiment shown in Figure 2 illustrates the application of Chinese National Standard (CNS) A4 specification (210 X 297 mm) in the presence of rFVIIa and tissue plasminogen activator (t-PA, American Diagnostica). Read the precautions on the back and fill out this page) --------Book --------- A7 1287452 ___B7___ V. Invention description (Λ*.) Charge FXIII (30 nM) will be extended The fibrin clot dissolution time of the formed clot. Clot formation can be induced by the presence or absence of 30 nM of FXIII by adding 25 μM NHP to 225 μΐ 20 nM HEPES, 150 mM sodium chloride, 5 mM chlorinated with 50 nM rFVIIa and 0.5 nM recombinant t-PA. Calcium, pH 7.4. Clot formation induced by t-PA mediator plasminogen activation followed by clot lysis can be monitored by Specramax® 340 at 600 nm at 〇D_nm and then back to the baseline. Figure 2 shows that in these cases the clot lysis time is significantly prolonged by the presence of FXIII. Example 3 Scorpion Vila binding factor XIII increases clot maximum firmness and increases clot resistance to fibrinolysis Coagulation fiber elasticity curve is measured in citrated normal human plasma, with 6 nM recombinant tissue plasmin added The original activator 屮PA, American Diagnostica), and the effect of adding 1 nM rFVIIa (Novo Nordisk A/S, Bagsvaerd, Denmark) alone or in combination with different concentrations of factor XIII (rFVIIa, Haematologic Technologies, HCXIII-OMO, Lot N1212) was analyzed. Clot initiation was initiated by the addition of Innovin (final concentration 2000 fold, Dade Behring #526945) and calcium (final concentration 15 mM) in a buffer of 20 mM HEPES, 150 mM sodium chloride, pH 7.4. The effect of rFVIIa and FXIII on the maximum clot firmness (MCF) and the resistance of the clot to t-PA media lysis was analyzed using the coagulation enzyme fiber elastic curve measurement. Before the addition of rFVIIa and / or FXIII, the MCF obtained was 25 mm and the time required for the clot to dissolve half was 12.3 minutes (Fig. 3) 42 This paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm). (Please read the notes on the back and fill out this page)

1287452 A7 ______B7___ V. Description of the invention (4丨) (Please read the notes on the back and fill out this page). Adding more and more high concentrations of FXIII (0-40 nM) did not change MCF; however, it was found that half of the clot lysis time was dose-dependently extended, ideally 30 nM FXIII (-half clot lysis time: 14.3) Minutes, see Figure 3). Similarly, the addition of rFVIIa (1 nM) resulted in protection of blood clots from fibrinolysis by t-PA media (half clot lysis time: 16.4 minutes) and had no effect on MCF (Fig. 3). However, when rFVIIa (1 nM) and FXIII (30 nM) were added, an increase in MCF (29 mm) was observed, as well as a significant protective effect on fibrinolysis (half clot lysis time: 27.1 min) (Fig. 3) . Taken together, these results demonstrate that rFVIIa and FXIII are added to the blood paddles in a synergistic manner to improve the resistance of the clot to the fibrinolysis of the t-PA media. 43 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Claims (1)

A8B8C8D8 1287452 Scope of Patent Application 1. A pharmaceutical composition for treating a hemorrhagic event comprising a combination of an effective amount of factor Vila and an effective amount of factor χπΐ. ................------ (Please read the precautions on the back and fill out this page) 2·If you apply for the composition of item 1 of the patent scope, the factor VIIa is a Factor VII variant. 3. The composition of claim 1 wherein the factor VUa is the human factor Vila. 4. The composition of claim 1 or 3, wherein the factor Vila and factor XIII are recombinant human factor VIIa and recombinant human factor XIII. 5. The composition of any one of claims 1 to 3, wherein the factor XIII is an α2-dimer of the factor XIII. 6. The composition of any one of items 1 to 3, wherein the factor XIII is an activation state factor xm. 7. For example, the composition of the patent application, which is used to reduce the clotting time of patients. 8. The composition of claim 1 for use in prolonging the clot lysis time in normal mammalian plasma. 9. The composition of claim 1 of the patent scope for increasing the clot strength in normal mammalian plasma. 10. A pharmaceutical composition for increasing fibrin clot formation comprising a combination of an effective amount of factor Vila and an effective amount of factor 。. 11. If the composition of claim 1 or 1 of the patent application is applied, it is expressed in a single dose. 12. A kit containing a treatment for hemorrhagic events, including the paper width for the Chinese National Standard (CNS) A4 specification (21〇x 297 mm) 1287452 A8 B8 C8 D8 VI. Patent application scope a) One unit dosage form is an effective amount of a factor Vila and a pharmaceutically acceptable carrier; b) an effective amount of Factor XIII in a second unit dosage form and a pharmaceutically acceptable carrier; and c) The container of the first and second dosage forms. ......................0^ —— (Please read the notes on the back and fill out this page) -έ This paper scale applies to the Chinese National Standard (CNS) )A4 size (210 X 297 mm)