MXPA02011042A - tHARMACEUTICAL 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 prophy laxis of bleeding episodes.

Description

PHARMACEUTICAL COMPOSITION COMPRISING A FACTOR Vlla AND A FACTOR XIII FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition comprising a Vlla factor and a factor XIII. The invention also relates to the use of a combination of a factor Vlla with a factor XIII for the manufacture of a medicament for the treatment of individuals suffering from episodes of hemorrhage, or the prevention thereof. The invention also relates to a method for the treatment of bleeding episodes in individuals and to a method for improving clot formation in an individual. The present invention also relates to equipment comprising these compounds.

BACKGROUND OF THE INVENTION Hemostasis is initiated by the formation of a complex between tissue factor (TF) that is exposed to circulating blood after damage to the vessel wall, and FVIIa which is present in the circulation in an amount corresponding to approximately 1% of the Ref: 143182 total FVIII protein mass. This complex is anchored to the cell that has TF and activates FX in FXa and FIX in FlXa on the cell surface. FXa activates prothrombin to thrombin, which activates FVIII, FV, FXI and FXIII. In addition, the limited amount of thrombin formed in this initial step of hemostasis also activates platelets. After the action of thrombin on the platelets, they change shape and expose the charged phospholipids on their surface. This activated platelet surface forms the template for the subsequent activation of FX and the generation of complete thrombin. Further activation of FX on the surface of activated platelets occurs via a FIXa-FVIIIa complex formed on the surface of the activated platelet, and FXa then converts prothrombin to thrombin while still on the surface. Thrombin then converts fibrinogen into fibrin which is insoluble, and which stabilizes the initial platelet plug. This process is compartmentalized, for example, located at the site of TF expression or exposure, thereby minimizing the risk of a systemic activation of the coagulation system. Insoluble fibrin, which forms the plug, is further stabilized by the FXIII-catalyzed cross-linking of the fibrin fibers. FVIIa exists in the plasma mainly as a single chain zymogen, which is cleaved by FXa in its activated form, of two chains, FVIIa. The activated, recombinant Vlla factor (rFVIIa) has been developed as a pro-hemostatic agent. The administration of rFVIIa offers a fast and highly effective pro-haemostatic response in hemophilic individuals with hemorrhages, who can not be treated with coagulation factor products, due to the formation of antibodies. Also, individuals with hemorrhage with a factor VII deficiency or individuals who have a normal coagulation system but experience excessive bleeding can be successfully treated with FVIIa. In these studies, no unfavorable side effects of rFVIIa have been found (particularly the onset of thromboembolism). FVIIa exogenously administered, extra, increases the formation of thrombin on the surface of activated platelets. This occurs in hemophilic individuals who lack FIX or FVIII and therefore lack the most potent pathway for complete thrombin formation. Also, in the presence of a decreased number of platelets or platelets with defective function, extra FVIIa increases thrombin formation. FXIII, the fibrin stabilizing factor, is a transglutamxnasa that crosslinks the monomers of fibrin with which a fibrin structure is provided with increased resistance to dissolution by plasmin and other proteolytic enzymes. Factor XIII is also known as "fibrinoligase" and "fibrin stabilizing factor". When activated, FXIIIa is capable of forming intermolecular gamma-glutamyl-epsilon-lysine crosslinks between the side chains of fibrin molecules and between other substrates. FXIII is found in plasma and platelets. The enzyme exists in plasma as a tetrameric zymogen consisting of two alpha subunits and two beta subunits (designated a2b2) and in platelets as a zymogen consisting of two alpha subunits (designated dimer a2). Both zymogens are activated by thrombin and Ca2 +. Calcium is being released from platelets after aggregation at the site of damage. Thrombin breaks the N-terminal amino acid residues 1-37 (from a dimer a2) · In the case of zymogen a2b2, the beta subunits are then dissociated from the activated alpha subunits. Calcium binds equally well to the zymogen and to the molecule modified with thrombin. After the activation of thrombin and calcium, the active center cysteine on the alpha chain is exposed and the fully activated enzyme is formed. Individuals with severe thrombocytopenia have been found as possessors of low plasma levels of FXIII. It is well known that subjects who bleed excessively in association with surgery or major trauma, and need blood transfusions develop more complications than those who do not experience any bleeding. However, also moderate bleeding requiring the administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation defects, etc.), can lead to complications associated with the risk of transfer of human viruses (hepatitis, HIV, parvovirus, and others, for now unknown viruses). Large hemorrhages requiring massive blood transfusions can lead to the development of multiple organ failure, including impaired lung function and kidney function. Once an individual has developed these serious complications, a cascade of events involving a number of cytokines and inflammatory reactions begins, making any treatment extremely difficult and unfortunately often not successful. Therefore, a major goal in surgery, as well as in the treatment of major damage to tissues, is to avoid or minimize bleeding. To avoid or minimize such bleeding, it is It is important to ensure the formation of stable and solid hemostatic clots that are not easily dissolved by fibrinolytic enzymes. In addition, it is important to ensure the rapid and effective formation of such plugs or clots. Japanese Patent Application No. 2-1672234 A refers to an adhesive for biotech characterized in that it contains fibrinogen, prothrombin, blood coagulation factor VII, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor. XIII, antithrombin, a proteinase inhibitor, and calcium ion. Japanese Patent Application No. 59-116213A relates to an aerosol composition for use as a tissue glue containing a blood coagulant as an active component. The blood coagulant can be selected from the blood coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII and XIII, prekallikrein, highly polymeric kininogen and thrombin. A combination of factor XIII and thrombin is preferred. W093 / 12813 (ZymoGenetics) refers to the use of factor XIII for the reduction of preoperative blood loss in a subject undergoing surgery. The composition may also comprise aprotinin. The FXIII it is administered to the subject as a bolus injection, typically one day before surgery. European Patent No. 225,160 (Novo Nordisk) refers to FVIIa compositions and methods for the treatment of coagulation disorders not caused by defects in the coagulation factor or inhibitors of the coagulation factor. European Patent No. 82,182 (Baxter Travenol Lab.) Refers to a Vlla factor composition for use in counteracting the deficiencies of blood coagulation factors or the effects of inhibitors for blood coagulation factors in a subject . International Patent Publication No. WO93 / 06855 (Novo Nordisk.) Refers to the topical application of factor Vlla. Kjalke et al., Thrombosis and Haemostasis, 1999 (Suppl), 095 1 refers to the administration of the exogenous factor Vlla, extra, and the effect on the formation of thrombin on the surface of activated platelets in a model system that mimics the conditions of hemophilia A or B. There remains a need in the art for an improved, reliable and widely applicable method of improving coagulation, rapidly forming stable hemostatic plugs and achieving hemostasis complete in subjects, particularly in subjects who have a deteriorated thrombin generation. There is also a need for a method where the amount of FVIIa, necessary to achieve complete hemostasis, is decreased.

BRIEF DESCRIPTION OF THE INVENTION An objective of the present invention is to provide compositions, which can be effectively used in the treatment or prophylaxis of bleeding episodes and coagulation disorders. A second objective of the present invention is to provide compositions in a dosage form, which can be effectively used in the treatment or prophylaxis of episodes of bleeding or bleeding or as a procoagulant. Still another object of the present invention is to provide compositions, methods of treatment or equipment that exhibit a synergistic effect. A further objective of the present invention is to provide compositions, methods of treatment or equipment that do not show substantial side effects, such as a high level of systemic activation of the coagulation system.

Other objects of the present invention will become apparent upon reading the present disclosure. The present inventors have shown that a combination of a factor Vlla and a factor XIII can reduce the clotting time of normal human plasma more effectively than factor Vlla or factor XIII alone. It has also been shown that a combination of a factor Vlla and a factor XIII can increase the firmness of the clot, more effectively than factor Vlla or factor XIII alone. By combining a Vlla factor at a concentration where no further increase in coagulum firmness was observed with a factor XIII, also at a concentration where no further increase in coagulum firmness was observed, it was unexpectedly shown that it was obtained an additional increase in the firmness of the clot. It has also been shown that the combination of a factor Vlla and a factor XIII can prolong the lysis time of the clot in vitro, in normal human plasma, more effectively, than the inhibitor of factor Vlla or factor XIII alone. Thus, by improving coagulation, a more effective treatment of hemorrhage in subjects can be obtained. In addition, patients can be treated with relatively lower concentrations of factor Vlla, thus reducing the relatively high costs in connection with conventional treatment with the factor Vlla alone. In a first aspect, the invention relates to a pharmaceutical composition comprising a factor Vlla and a factor XIII and, optionally, a pharmaceutically acceptable carrier. In still another aspect, the invention relates to a pharmaceutical composition comprising a factor Vlla and a factor XIII as the only active agents and, optionally, a pharmaceutically acceptable carrier. In still another aspect, the invention relates to a pharmaceutical composition formulated for intravenous administration, which contains a factor Vlla and a factor XIII, optionally, a pharmaceutically acceptable carrier. In still another aspect, the invention relates to a pharmaceutical composition formulated for intravenous administration, comprising a factor Vlla and a factor XIII, as the only active agents and, optionally, a pharmaceutically acceptable carrier. In one embodiment, the Vlla factor is the recombinant factor Vlla and factor XIII is the recombinant factor XIII. In one embodiment of the invention, the Vlla factor it is the recombinant Vlla factor. In a further embodiment, the factor Vlla is the recombinant human factor Vlla. In an additional mode, the Vlla factor is a variant of the Vlla factor. In one embodiment, Vlla factor variants are variants in the amino acid sequence that have no more than 20 amino acids replaced, deleted or inserted compared to a wild type factor Vlla (eg, a polypeptide having the amino acid sequence described in U.S. Patent No. 4,784,950). In another modality, Vlla factor variants have no more than 15 amino acids replaced, deleted or inserted; in yet another embodiment, the Vlla factor variants have no more than 10 amino acids replaced, deleted or inserted; in another embodiment, the Vlla factor variants have no more than 8 amino acids replaced, deleted or inserted; in yet another embodiment, the Vlla factor variants have no more than 6 amino acids replaced, deleted or inserted; in another embodiment, the Vlla factor variants have no more than 5 amino acids replaced, deleted or inserted; in yet another embodiment, the Vlla factor variants have no more than 3 amino acids replaced, deleted or inserted ared to the wild type factor Vlla. In one embodiment, the variants of the Vlla factor are selected from the list of [L305V] -FVIIa, [L305V / M306D / D309S] -FVIIa, [3051] -FVIIa, [L305T] -FVIIa, [F374P] -FVIIa, [V158T / M298Q] -FVIIa, [V158D / E296V / M298Q] -FVIIa and [K337A] - FVIIa. In one embodiment, factor XIII is FXIII a2b2.

In an additional embodiment, factor XIII is FXIII a2. In an additional embodiment, factor XIII is activated factor XIII (FXIIIa). In a further embodiment, factor XIII is a variant of factor XIII. In one embodiment, factor XIII is a human factor XIII. In one embodiment, factor XIII is recombinant factor XIII. In one embodiment, factor XIII is recombinant human factor XIII. In one embodiment, factor XIII is the human a2 dimer. In one aspect, the composition also contains a TFPI inhibitor. In still another aspect, the composition further contains a factor VIII. In still another aspect, the composition further contains a factor VIII and a TFPI inhibitor. In one embodiment, the composition further comprises an inhibitor of the fibrinolytic system, for example, aprotinin, e-amicaproic acid or tranexamic acid. In still another aspect, the invention relates to an equipment that contains a treatment for bleeding episodes, comprising: a) an effective amount of a Vlla factor and, optionally, a pharmaceutically acceptable carrier in a first unit dose form; b) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; c) container means for containing the first and second dosage forms. In one aspect, the kit comprises: a) an effective amount of a Vlla factor and, optionally, a pharmaceutically acceptable carrier in a first unit dose form; b) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; c) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing the first, second and third dosage forms. In still another aspect, the invention relates to an equipment that contains a treatment for bleeding episodes, comprising: a) an effective amount of a factor Vlla and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; and c) container means for containing the first and second dosage forms. In still another aspect, the invention relates to an equipment containing a treatment for bleeding episodes, comprising: a) an effective amount of a factor Vlla and, optionally, a pharmaceutically acceptable carrier in a first unit dose; b) an effective amount of a factor XIII and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; and c) container means for containing the first and second dosage forms. In still another aspect, the invention relates to an equipment that contains a treatment for bleeding episodes, comprising: a) an effective amount of a factor Vlla and a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing the first and second dosage forms. In still another aspect, the invention relates to an equipment containing a treatment for bleeding episodes, comprising: a) an effective amount of a Vlla factor and, optionally, a pharmaceutically acceptable carrier in a first unit dose form; b) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; and c) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a third unit dosage form; and d) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a fourth unit dose form; and e) container means for containing the first, second, third and fourth dosage forms. In still another aspect, the equipment comprises: a) an effective amount of a Vlla factor and a factor XIII and, optionally, a carrier pharmaceutically acceptable in a first unit dose form; b) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; c) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing the first, second and third dosage forms. In still another aspect, the invention relates to an equipment containing a treatment for hemorrhagic episodes, comprising: a) an effective amount of a factor Vlla and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an amount an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; and c) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing the first one, second and third dosage forms. In still another aspect, the invention relates to an equipment that contains a treatment for bleeding episodes, comprising: a) an effective amount of a factor Vlla and a factor VIII and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; and c) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; And d) container means for containing the first, second and third dose forms. In yet another aspect, the invention relates to a kit containing a treatment for bleeding episodes, comprising: a) an effective amount of a factor VIla and a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a factor VIII and a TFPI inhibitor and, optionally, a carrier pharmaceutically acceptable in a second unit dosage form; and c) container means for containing the first and second dosage forms. In still another aspect, the invention relates to an equipment containing a treatment for bleeding episodes, comprising: a) an effective amount of a factor VIII and a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) an effective amount of a Vlla factor and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; and d) container means for containing the first, second and third dosage forms. In still another aspect, the invention relates to an equipment containing a treatment for bleeding episodes comprising: a) an effective amount of a factor XIII and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; e) an effective amount of a VIla factor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing the first, second and third dosage forms. In still another aspect, the invention relates to an equipment that contains a treatment for bleeding episodes, comprising: a) an effective amount of a TFPI inhibitor and a factor VIII and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a Vlla factor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and f) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dose form; and c) container means for containing the first, second and third dosage forms. In still another aspect, the invention relates to a equipment containing a treatment for bleeding episodes, comprising: a) an effective amount of a TPFI inhibitor and a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first unit dose form; b) an effective amount of a factor VIII and a factor Vlla and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing the first and second dosage forms. In still another aspect, the invention relates to an equipment that contains a treatment for bleeding episodes, comprising: a) an effective amount of a factor VIla and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first form of unit dose; b) an effective amount of a factor VIII and a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing the first, second and third dosage forms.

In still another aspect, the invention relates to the use of a factor Vlla in combination with a factor XIII, for the manufacture of a medicament for the treatment of episodes of hemorrhage. In still another aspect, the invention relates to the use of a Vlla factor in combination with a factor XIII, for the manufacture of a medicament for reducing the clotting time in a subject. In still another aspect, the invention relates to the use of a factor Vlla in combination with a factor XIII, for the manufacture of a medicament for prolonging the time of lysis of the clot in mammalian plasma. In still another aspect, the invention relates to the use of a factor Vlla in combination with a factor XIII, for the manufacture of a medicament for increasing the resistance of the clot in mammalian plasma. In still another aspect, the invention relates to the use of a factor Vlla in combination with a factor XIII, for the manufacture of a medicament for improving the formation of the fibrin clot in mammalian plasma. In one embodiment, the mammalian plasma is human plasma. In yet another embodiment, mammalian plasma is normal plasma; in another embodiment, the plasma is normal human plasma; in one embodiment, the plasma is plasma from a subject that has a generation impaired thrombin. In one embodiment, the plasma is from a subject having a decreased concentration of fibrinogen. In one embodiment, factor Vlla and factor XIII prolong the lysis time of the clot in vitro in normal human plasma. In still another aspect, the invention relates to a method for improving the fibrin clot formation in a subject, which method comprises administering to a subject an effective amount of a factor Vlla in combination with an effective amount of a factor XIII. In still another aspect, the invention relates to a method for the treatment of bleeding episodes in a subject, which comprises administering to a subject an effective amount of a factor Vlla in combination with an effective amount of a factor XIII. In still another aspect, the invention relates to a method for reducing the coagulation time of mammalian plasma, comprising contacting the plasma with an effective amount of a factor Vlla in combination with an effective amount of a factor XIII. In one embodiment, the effective amount of a Vlla factor in combination with an effective amount of a factor XIII, is administered to a subject in need of such treatment. In still another aspect, the invention relates to a method for improving the formation of fibrin in a subject, comprising administering to a subject an effective amount of a factor Vlla in combination with an effective amount of a factor XIII. In one embodiment of the methods of the invention, factor Vlla and factor XIII are the only active agents administered to the subject. In yet another embodiment of the invention, the pharmaceutical composition comprises a factor Vlla and a factor XIII as the only active agents. In one embodiment of the invention, factor Vlla and factor XIII are administered simultaneously in a single-dose form. In another embodiment, the factor Vlla and factor XIII are administered sequentially. In a further embodiment, factor Vlla and factor XIII are administered within approximately 1-2 hours one after the other, for example within 30 minutes one after the other, for example within 10 minutes one after the other, for example , in the form of a kit comprising a Vlla factor in a first unit dose form and a factor XIII in a second unit dose form. In one embodiment, the effective amount of a Vlla factor is 0.05 mg / day up to 500 mg / day (subject of 70 kg). In one modality, the effective amount of a Factor XIII is 0.05 mg / day up to 500 mg / day (subject of 70 kg). In one embodiment of the present invention, the pharmaceutical composition (when in the form of a simple preparation) consists essentially of a factor Vlla and a factor XIII, and, optionally, at least one pharmaceutically acceptable excipient or carrier, and / or a stabilizer, and / or a detergent, and / or a neutral salt, and / or an antioxidant, and / or a preservative, and / or a protease inhibitor. In yet another embodiment of the present invention, the pharmaceutical composition (when in the form of a simple preparation) consists essentially of a factor Vlla and a factor XIII, and, optionally, at least one pharmaceutically acceptable excipient or carrier, and / or a 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 yet another embodiment of the present invention, the pharmaceutical composition (when in the form of a simple preparation) consists essentially of a factor Vlla and a factor XIII, and, optionally, at least one pharmaceutically acceptable excipient or carrier, and / or a stabilizer , and / or a detergent, and / or a neutral salt, and / or an antioxidant, and / or a preservative, and / or an inhibitor of protease and / or a TFPI inhibitor, and / or a factor VIII. In yet another embodiment, the pharmaceutical composition (when in the form of a kit) consists of a first unit dose form consisting essentially of a factor Vlla and, optionally, at least one pharmaceutically acceptable excipient or carrier, and / or a stabilizer , and / or a detergent, and / or a neutral salt, and / or an antioxidant, and / or a preservative, and / or a protease inhibitor, and a second unit dose form consisting essentially of a factor XIII and, optionally, at least one pharmaceutically acceptable excipient or carrier, and / or a stabilizer, and / or a detergent, and / or a neutral salt, and / or an antioxidant, and / or a preservative, and / or a protease inhibitor. In yet another embodiment, the pharmaceutical composition (when in the form of a kit) consists of a first unit dose form consisting essentially of a factor Vlla and, optionally, at least one pharmaceutically acceptable excipient or carrier, and / or a 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 a second unit dosage form consisting essentially of a factor XIII, and, optionally, at least one pharmaceutically acceptable excipient or carrier, and / or a 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 a factor VIII. In an additional mode, the subject is a human; in yet another embodiment, the subject has an impaired generation of thrombin; in one embodiment, the subject has a decreased plasma concentration of fibrinogen (eg, a subject with multiple transfusions). In still another aspect, the composition further contains a factor VIII. In a further embodiment, factor VIII is an activated factor VIII (factor Villa). In a further embodiment, factor VIII is a recombinant Villa factor. In a further embodiment, factor VIII is the recombinant human Villa factor. In a further aspect, the composition further comprises an inhibitor of the fibrinolytic system, for example, aprotinin, e-aminocaproic acid or tranexamic acid.

DESCRIPTION OF THE FIGURES Figure 1 shows that the spontaneous formation of the clot in normal human plasma (NHP), citrated, diluted 1/10 in buffer containing 25 nM HEPES, 150 m sodium chloride, and 5 mM calcium chloride, pH 7.4, in a microtiter well (total volume 250 μ?) was obtained at approximately 2500-3000 seconds. The formation of the fibrin clot was monitored by the increase in optical density at 600 nm in a SpecramaxMR 340. Molecular Devices, Sunny-vale California. Figure 1 shows that the 10 nM recombinant VIla factor (rFVIIa) from Novo Nordisk A / S Bagsvaerd, Denmark, shortened the coagulation time to 1600 seconds (n = 2). Further shortening of the coagulation time was obtained when 30 nM factor XIII (FXIII) was added from American Diagnostica Inc., Greenwich, CT together with 10 nM rFVIIa (n = 3). The clot formed in the presence of FXIII was more transparent (lower maximum DO) than in its absence, indicating that the addition of FXIII resulted in a finer mesh fibrin gel structure, with thinner fibers. Figure 2 shows that supplemental FXIII (30 nM) prolongs the lysis time of the fibrin clot, the clots formed in the presence of rFVIIa and the tissue plasminogen activator (tPA). Clot formation was induced in the presence or absence of 30 nM XIII, by the addition of 25 μ? of NHP at 225 μ? of Hepes 20 nM, 150 mM sodium chloride, 5 mM calcium chloride, pH 7.4, containing 50 nM rFVIIa or 0.5 nM recombinant tPA from Novo Nordisk A / S Bagsvaerd, Denmark. The formation of the clot and the subsequent lysis of the clot, induced by plasminogen activation, mediated by tPA, was monitored by a Spectramax® 340 apparatus at 600 nm as the increase in D0600 nm, followed by reversion of the trace to the basal level. Figure 2 shows that the clot lysis time under these conditions was significantly prolonged by the presence of FXIII. Figure 3 shows the effect of rFVIIA and FXIII on the Maximum Clot Firmness (MCF) as well as the resistance of the clot to lysis mediated by tPA. Before the addition of rFVIIa and / or FXIII, the MCF obtained was 25 mm and the time required for the half of the clot to be lysed was 12.3 minutes (Figure 3). The addition of increasing concentrations of FXIII (0-40 nM) did not alter the MCF; however, a dose-dependent prolongation was observed in the lysis of half a clot, optimal at FXill 30 nm (clot lysis time: 14.3 minutes, Figure 3). Similarly, the addition of rFVIIa (1 nm) resulted in the protection of the clot against t-PA-mediated fibrinolysis (clot half lysis time: 16.4 minutes) without any effect on MCF (Figure 3). However, after the addition of rFVIIa (1 nM) together with FXIII (30 nM) an increase in MCF (29 mm) was observed as well as a deep protection from fibrinolysis (lysis time of coagulum medium; minutes) (Figure 3). Taken together, these results show that the addition of rFVIIa and FXIII to the plasma synergistically improve the mechanical strength of the clot and the resistance to fibrinolysis mediated by t-PA.

DETAILED DESCRIPTION OF THIS INVENTION The present invention provides a composition comprising a combination of a factor Vlla and a factor XIII. The invention also provides a composition comprising a combination of a Vlla factor and a factor XIII as the sole active ingredients. The composition may be in the form of a simple composition, or it may be in the form of a multi-component equipment. The present compositions are useful as a prophylactic or therapeutic pro-coagulant and fibrin clot stabilizing agents, and form fibrin clots rapidly in mammals, including primates such as humans. Whenever a first or second or third, etc. is mentioned. unit dose, throughout this specification, this does not indicate the preferred order of administration, but merely is done for convenience purposes. The present invention also provides a method for treating (including prophylactic treatment or prevention) episodes of bleeding or hemorrhage in a subject, including a human being, for example, due to trauma or surgery, or in subjects lacking or having blood coagulation factors FIX or FVIII or platelets, defective. It has now been found that a combination of a factor Vlla and a factor XlIIa is an advantageous product which ensures the formation of solid, stable and rapid hemostatic plugs. The complete generation of thrombin is necessary to form a stable, solid hemostatic plug. The fibrin structure of such a stopper is dependent on the amount of thrombin formed and the initial generation rate of thrombin. In the presence of a deteriorated thrombin generation, a porous fibrin stopper is being formed, which is highly permeable. The fibrinolytic enzymes normally present on the surface of the fibrin readily dissolve such a fibrin plug. The formation of a stable fibrin plug is also dependent on the presence of factor XlIIa, which is being activated by fibrin, and therefore also dependent on the generation of complete thrombin. In addition, the newly described thrombin-activatable fibrinolytic inhibitor, TAFI, requires rather high amounts of thrombin for activation. In the presence of a not completely adequate thrombin formation, the TAFI may therefore not be activated, resulting in the formation of a hemostatic plug, which is easier to dissolve than is normally dissolved by normal fibrinolytic activity. By increasing the generation of thrombin, the Vlla factor provides the basis for a complete activation of factor XIII, which is of crucial importance for the formation of a fully stabilized hemostatic plug, and therefore for the maintenance of hemostasis. In situations with decreased platelet numbers, thrombocytopenia, a faster thrombin generation is initiated by the administration of the extra Vlla factor, exogenous. However, the generation of total thrombin is not normalized by the Vlla factor even at high concentrations. By the combination of a Vlla factor and a factor XIII, in particular the alpha chain of factor XIII (the dimer a2) is facilitated a complete activation of factor XIII that increases the hemostatic effect of factor Vlla. In addition, in subjects with decreased plasma concentrations of fibrinogen (multitransfused subjects as a consequence of trauma multiple or extensive surgery) complete activation of factor XIII does not occur. A more effective hemostasis is then obtained by administering a combination of a Vlla factor and a factor XIII. Another way to increase the stability of hemostatic fibrin plugs is to ensure the complete presence of factor XlIIa (activated factor XIII). Subjects with thrombocytopenia have an impaired generation of thrombin, as well as a defective stabilization of fibrin plugs, resulting in hemostatic plugs prone to premature dissolution. In addition, subjects subjected to major trauma or organ damage and who, as a consequence, have received frequent blood transfusions, often have decreased platelet counts, as well as decreased levels of fibrinogen, factor VII, and other coagulation proteins. These subjects experience an impaired (or decreased) generation of thrombin. In addition, its decreased fibrinogen level interferes negatively with the activation of factor XIII. These subjects, therefore, have a defective or less efficient haemostasis, which leads to the formation of fibrin plugs that are easily and prematurely dissolved by proteolytic enzymes, such enzymes are also extensively released in situations characterized by extensive trauma and organ damage. In order to facilitate the formation of fully stabilized caps with full capacity to maintain hemostasis in a subject, a composition according to the invention is administered. This composition is especially beneficial in subjects with a decreased number of platelets and in subjects with decreased plasma levels of fibrinogen and / or other coagulation proteins. In the presence of a factor XIII, lower concentrations of factor Vlla may be sufficient to ensure sufficient hemostasis. As stated above, factor XIII exists in the plasma as a tetrameric zymogen consisting of two alpha subunits and two beta subunits (designated a2, b2), but is found in other tissues (e.g., platelets) as an a2 dimer. Any of these forms of zymogen, or activated factor XIII (factor XlIIa), can be used within the present invention, as well as the genetically engineered variants of factor XIII, which retain their characteristic crosslinking activity. In one embodiment, factor XIII is human factor XIII; in another embodiment, factor XIII is the human a2 dimer; in other more mode, factor XIII is activated human factor XlIIa. Factor XIII and factor Vlla used in the present invention can be purified from the blood or produced by recombinant means. It is evident that the practice of the methods described herein is dependent on how purified factor XIII and factor Vlla are derived and, therefore, the present invention is contemplated to cover the use of any factor XIII preparation and factor Vlla, suitable for use in the present. The human factor Vlla and the human factor XIII are preferred. Also variants engineered by genetic factor Vlla and factor XIII that retain their characteristic activity related to hemostasis can be used in the present invention. Fragments of factor Vlla or factor XIII or variants of factor Vlla or factor XIII which retain their characteristic activity related to hemostasis can also be used in the present invention. The activity related to the hemostasis of the Vlla factor can, for example, be measured using the factor Vlla activity assay, described in the present specification. The activity related to hemostasis of a factor XIII can, for example, be measured using the activity assay of factor XIII, described in present specification. Non-limiting examples of factor VII variants that have substantially the same or a better biological activity as compared to the wild type factor Vlla include, but are not limited to those described in the Danish patent applications Nos. PA 2000 00734, PA 2000 01360, PA 2000 01361 and PA 2001 00477. Non-limiting examples include [L305V] -FVIIa, [L305V / M306D / D309S ] -FVIIa, [3051] -FVIIa, [L305T] -FVIIa, [F374P] -FVIIa, [V158T / M298Q] -FVIIa, [V158D / E296V / 298Q] -FVIIa and [K337A] -FVIIa. In the present context, the three-letter or one-letter indications of the amino acids have been used in their conventional meaning, as indicated in Table 1. Unless explicitly indicated, the amino acids mentioned herein are L -amino acids . It is understood that the first letter, for example, in K337, represents the amino acid naturally present in the indicated position of wild type factor VII, and that, for example, [K337A] -FVIIa designates the FVII variant wherein the amino acid represented by the K code of a single letter present naturally in the indicated position, it is represented by the A letter of a single letter.

Table 1: Abbreviations for amino acids Amino Acid Code of Three Code of a Letter Letters Glycine Gly G Proline Pro P Alanine Wing A Valine Val V Leucine Leu L Isoleucine lie I methionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr And Tryptophan Trp W Histidine His H Lysine Lys K Arginine Arg R Glutamine Gln Q Asparagine Asn N Glutamic Acid Glu E Aspartic Acid Asp D The term "factor Vlla" or "FVIIa" can be used interchangeably. The term factor Vlla includes the zymogous factor VII (single chain factor VII). The term "factor XIII" or "FXIII" can be used interchangeably. The term "factor VIII" or "FVIII" can be used interchangeably. It will be apparent to those skilled in the art that substitutions can be made outside the critical regions for the function of the factor Vlla or factor XIII molecule, and still result in an active polypeptide. The amino acid residues essential for Vlla factor or factor XIII polypeptide activity, and therefore preferably not subject to substitution, can be identified according to procedures known in the art, such as site-directed mutagenesis or Alanine scanning mutagenesis (see, for example, Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced into each positively charged residue in the molecule, and the resulting mutant molecules are tested for coagulant activity, and cross-linked, respectively, to identify the amino acid residues that are critical to the activity of the molecule. The sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure, as determined by techniques such as the analysis of nuclear magnetic resonance, crystallography or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255: 306-312, Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al. ., 1992, FEBS Letters 309: 59-64). The introduction of a mutation within the nucleic acid sequence to exchange one nucleotide for another nucleotide can be achieved by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the method using a double-stranded DNA vector, supercoiled, with an insert of interest, and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to the opposite strands of the vector, extend during the temperature cycles by means of the Pfu DNA polymerase. After incorporation of the primers, a mutated plasmid containing spaced notches is generated. After the temperature cycle, the product is treated with DpnI, which is specific for methylated or hemimetylated DNA, to digest the parent DNA template and to select the synthesized DNA, which contains the mutation. Other methods known in the art for creating, identifying and isolating variants can also be used, such as, for example, the techniques of interspersed with genes or visual representation of phage. The term "factor VIII" or "FVIII" includes activated factor VIII (designated Villa factor), variants and truncated forms of factor VIII which retain their characteristic coagulant activity. In one embodiment, factor VIII is human factor VIII. The term "TFPI inhibitor" means the compounds that inhibit the anticoagulant activity of TPFI (inhibitor of the tissue factor pathway). The term includes compounds such as those described in European Patent No. 558,529, International Patent Application W096 / 28153 and United States Patent No. 5,622,988. "TPFI" and "EPI" (extrinsic pathway inhibitor) can be used interchangeably. Within the present invention an "effective amount" of a factor Vlla and an "effective amount" of a factor XIII is defined as the amount of a factor Vlla and a factor XIII, enough to prevent or reduce bleeding or loss of blood, to cure, relieve, or partially stop the disease and its complications. The amount of a Vlla factor and the amount of a factor XIII administered according to the present invention can vary from a ratio of about 1: 100 to about 100: 1 (μ? Factor Vlla ^ g factor XIII).

In this context "subjects with an impaired generation of thrombin" mean subjects who can not generate a complete burst of thrombin on the surface of activated platelets, and include subjects who have a lower thrombin generation than thrombin generation in subjects who have a normal, fully functioning hemostatic system, including a normal amount and normal function of coagulation factors, platelets and fibrinogen, and include subjects lacking FIX and / or FVIII (hemophilia A and B) ) or having defective FIX and / or FVIII, or having inhibitors against FIX and / or FVIII; subjects who lack FXI; subjects with a decreased number of platelets or platelets with a defective function (eg, thrombocytopenia or Glanzmann's thrombasthenia, or subjects with excessive bleeding); and subjects who have decreased levels of prothrombin, FX or FVII. Subjects with decreased plasma concentrations of fibrinogen (eg, subjects with multiple transfusions as a result of multiple trauma or extensive surgery) also suffer from the formation of looser and unstable fibrin plugs that are easily dissolved. The term "complete hemostasis" means the formation of a stable fibrin plug or clot and solid, at the site of the damage, which effectively stops the hemorrhage and is not easily dissolved by the fibrinolytic system. The term "factor VIla activity" or "factor Vlla activity" means the ability to generate thrombin; the term also includes the ability to generate thrombin on the surface of activated platelets in the absence of tissue factor. The term "normal hemostatic system increase" means an increase in the ability to generate thrombin. As used herein, the term "bleeding disorder or bleeding" reflects any defect, congenital, acquired or induced, of cellular or molecular origin, that is manifested in hemorrhages or bleeding. Examples are deficiencies in coagulation factors (eg, hemophilia A and B or deficiency of coagulation factors XI or VII), inhibitors of coagulation factor, defective platelet function, thrombocytopenia or von Willebrand disease. The term "bleeding or hemorrhage episodes" is understood to include uncontrolled or excessive bleeding that is a major problem in connection with surgery and other forms of tissue damage. Bleeding or Uncontrolled and excessive bleeding can occur in subjects who have a basically normal coagulation system (these subjects do not, however, develop a coagulopathy as a result of bleeding -dilution of the coagulation proteins, increased fibrinolysis and decreased platelets due to an effect of dilution of bleeding) and subjects who have bleeding or bleeding disorders. Deficiencies in coagulation factors (hemophilia A and B, deficiency of coagulation factors XI or VII) or inhibitors of coagulation factors can be the cause of bleeding disorders. Excessive bleeding also occurs in subjects with a normally functioning blood coagulation cascade (no deficiencies in coagulation factors or -inhibitors against any of the coagulation factors) and may be caused by defective platelet function, thrombocytopenia or von Willebrand disease). In such cases, bleeding may be linked to those bleeding caused by hemophilia due to the haemostatic system, such as in hemophilia, which lacks or has essential, abnormal "coagulation" compounds (such as platelets or factor protein). von Willebrand) that causes major bleedings. In subjects who experience extensive tissue damage in association with surgery or trauma roughly, the normal hemostatic mechanism may be overwhelmed or exhausted by the demand for immediate haemostasis and these may develop haemorrhage despite a basically normal hemostatic mechanism (pre-trauma). The achievement of satisfactory hemostasis is also a problem when bleeding occurs in organs such as the brain, the inner region of the ear and eyes, with limited possibility for surgical hemostasis. The same problem may arise in the process of taking biopsies from various organs (liver, lung, tumor tissue, gastrointestinal tract) as well as in laparoscopic surgery. Common for all these situations is the difficulty to provide hemostasis by surgical techniques (sutures, staples, etc.) which is also the case when the bleeding is diffuse (hemorrhagic gastritis and profuse uterine bleeding). Bleeding or acute and profuse bleeding may also occur in subjects after anticoagulant therapy in whom a defective haemostasis has been induced by the therapy given. Such subjects may need surgical interventions in case the anticoagulant effect has to be counterattacked quickly. Radical retropubic prostatectomy is a commonly performed procedure for subjects with localized prostate cancer. The operation is often complicated by significant blood loss and some massive times. Considerable blood loss during prostatectomy is mainly related to a complicated anatomical situation, with several densely vascularized sites that are not easily accessible for surgical haemostasis, and which can result in diffuse bleeding from a large area. Another situation that can cause problems in the case of unsatisfactory haemostasis is when subjects with a normal hemostatic mechanism are administered anticoagulant therapy to prevent thromboembolic disease. Such therapy may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists, as well as aspirin and other inhibitors of platelet aggregation. In one embodiment of the invention, bleeding is associated with hemophilia. In yet another modality, bleeding is associated with hemophilia with acquired inhibitors. In yet another modality, bleeding is associated with thrombocytopenia. In yet another modality, bleeding is associated with von Willebrand's disease. In yet another modality, bleeding is associated with severe tissue damage. In yet another modality, bleeding is associated with severe trauma. In another modality, bleeding is associated with surgery. In yet another modality, bleeding is associated with laparoscopic surgery. In another modality more, the bleeding is associated with hemorrhagic gastritis. In yet another modality, the bleeding is profuse uterine bleeding. In another modality, bleeding is occurring in organs with a limited possibility for mechanical haemostasis. In yet another modality, bleeding is occurring in the brain, in the region of the inner ear or in the eyes. In another modality, bleeding is associated with the process of taking biopsies. In yet another modality, bleeding is associated with anticoagulant therapy. The composition according to the invention may further comprise a TFPI inhibitor. Such a composition should preferably be administered to subjects having hemophilia A or B. The composition according to the invention may further comprise a factor VIII. Such a composition should preferably be administered to subjects who do not have inhibitors for factor VIII. In this context, the term "treatment" is understood to include the prevention of expected bleeding or bleeding, such as, for example, in surgery, and regulation of bleeding that is already occurring, for example, in hemophilia or trauma, for the purpose of inhibiting or minimizing bleeding. The prophylactic administration of a Vlla factor and a factor XIII is from this mode included in the term "treatment". The term "subject" as used herein, is meant to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the term "patient".

Abbreviations TF Factor FVII tissue factor VII in its simple chain, non-activated form FVIIa Factor VII in its activated form rFVIIa Recombinant Factor VII in its activated form FXIII Factor XIII in its zymogenic form not activated FXIIIa Factor XIII in its activated form rFXIII recombinant FXIII rFXIIIa recombinant FXIIIa a2 Alpha chain or a chain of FXIII or rFXIII b2 Beta chain ob of FXIII or rFXIII FXIIIa2 Dimeric form of FXIII containing two chains to FXIIIa2b2 Tetrameric form of FXIII containing two chains a and two chains b FVIII Factor VIII in its non-activated zymogenic form rFVIII recombinant FVIII FVIIIa Factor VIII in its activated form rFVIIIa FVIIIa recombinant TFPI Inhibitor of the tissue factor pathway Preparation of the compounds The purified human factor Vlla suitable for use in the present invention is preferably made by recombinant DNA technology, for example as described by Hagen et al., Proc. Nati Acad. Sci. USA 83: 2412-2416, 1986 or as described in European Patent No. 200, 421 (ZymoGenetics, Inc.). The Vlla factor produced by recombinant technology may be the authentic Vlla factor, or a more or less modified Vlla factor, with the proviso that such Vlla factor has substantially the same biological activity for blood coagulation as the authentic Factor Vlla (factor Vlla of wild type). Such a modified Vlla factor can be produced by modifying the nucleic acid sequence encoding wild-type factor VII either by altering the amino acid codons or by removing some of the amino acid codons in the nucleic acid which codes for the natural factor VII by known means, for example, by site-specific mutagenesis.

Factor VII can also be produced by the methods described by Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. Invest. 71: 1836-1841, 1983. These methods produce factor VII without detectable amounts of other blood coagulation factors. An even more purified factor VII preparation can be obtained by including an additional gel filtration, such as the final purification step. Factor VII is then converted to the Vlla factor activated by known means, for example, by several different plasma proteins, such as factor Xlla, IXa or Xa. Alternatively, as described by Bjoern et al. (Research Disclopsure, 269, September 1986, p.564-565), factor VII can be activated by passing it through a column of ion exchange chromatography, such as Mono Q® (Pharmacia fine Chemicals) or the like. Factor XIII for use within the present invention can be prepared from plasma according to known methods, such as those described by Cooke and Holbrook (Biochem, J. 141: 79-84, 1974) and Curtis and Lorand (Methods Enzymol 45: 177-191, 1976), incorporated by reference herein. The a2 dimer form of factor XIII can be prepared from the placenta as described in the US Patents United Nos. 3,904,751; 3,931,399; 4,597,899 and 4,285,933, incorporated by reference herein. However, it is preferred to use recombinant factor XIII to avoid the use of products derived from blood or tissue that carry a risk of disease transmission. Methods for the preparation of recombinant factor XIII are 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 their entirety. Within one embodiment, the dimer a2 of factor XIII is prepared cytoplasmically in the yeast Saccharomyces cerevisiae as described in copending United States Patent Application Serial No. 07 / 741,263, incorporated by reference herein in its entirety. The cells are harvested and lysed, and a clarified 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 ™ (Pharmacia) or the like. Factor XIII is then precipitated from the eluate of the column, by concentration of the eluate and adjusting the pH to 5.2- . 5, such as by diafiltration against ammonium succinate buffer. 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 factor XIII and factor Vlla proteins, syngeneic with the subject, in order to reduce the risk of inducing an immune response. The preparation and characterization of non-human factor XIII, has been described by Nakamura et al. (J. Biochem. 78: 1247-1266, 1975). The present invention also encompasses the use of such factor XIII and factor Vlla proteins within veterinary procedures.

Administration and pharmaceutical compositions For treatment in connection with deliberate interventions, factor VII and factor XIII will typically be administered within approximately 24 hours before the intervention is performed, and therefore as 7 days or more after this. The administration as a coagulant can be by a variety of routes as described herein. The dose of factor VII is in the range of about 0.05 mg to about 500 mg / day, for example, from about 1 mg to about 200 mg / day, or, for example, from about 10 mg to about 175 mg / day for a 70 kg subject, as a loading dose and maintenance, depending on the weight of the subject, the condition and the severity of the condition. The dose of factor XIII is in the range of from about 0.05 mg to about 500 mg / day, for example, from about 1 mg to about 200 mg / day, or, for example, from about 10 mg to about 175 mg / day to a subject of 70 kg, as loading dose and maintenance, depending on the weight of the subject, the condition and the severity of the condition. The compositions and equipment of the present invention are useful within human and veterinary medicine, such as, for example, in the treatment or prophylaxis of subjects suffering from bleeding episodes or coagulation disorders. For use within the present invention, factor Vlla and factor XIII are formulated, optionally with a pharmaceutically acceptable carrier. Preferably, the pharmaceutical compositions are administered parenterally, for example intravenously, subcutaneously, or intramuscularly, or can be administered by continuous or pulsatile delivery.

The formulations may also include one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and can be provided in such forms as liquids, powders, emulsions, controlled release, etc. A person skilled in this art can formulate the compositions of the invention in an appropriate manner, and in accordance with accepted practices, such as those described in Remington's Pharmaceutical Sciences, Gennaro, ed. , Mack Publishing Co., Easton, PA, 1990. Compositions for parenteral administration comprise a factor VII and a factor XIII in combination with, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like. Factor VII variants of the invention can also be formulated in liposomal preparations for distribution or direction to damage sites. Liposomal preparations are generally described in, for example, U.S. Patent No. 4,837,028, U.S. Patent No. 4,501,728 and U.S. Patent No. 4,975,282. A typical pharmaceutical composition for intravenous infusion, could be constituted for contain 250 ml of sterile Ringer's solution and 10 mg of a Vlla factor and / or a factor XIII. Effective methods for the preparation of parenterally administrable compositions will be known or apparent to those of skill in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA ( 1990). In summary, pharmaceutical compositions suitable for use according to the present invention, are elaborated by mixing a factor Vlla, or a factor XIII, or a factor Vlla in combination with a factor XIII, preferably in purified form, with adjuvants adequate and an adequate carrier or diluent. Suitable physiologically acceptable carriers or diluents include sterile water and saline. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as adjusters and buffers, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Suitable adjuvants also include calcium, proteins (e.g., albumins), or other inert peptides (e.g. glycylglycine) or amino acids (e.g., glycine or histidine) to stabilize the purified Vlla factor and / or the purified factor XIII. Other physiologically acceptable adjuvants are non-reducing sugars, polyalcohols (for example sorbitol, mannitol or glycerol), polysaccharides such as low molecular weight dextrins, detergents (for example polysorbate) and antioxidants (for example bisulfite and ascorbate). Adjuvants are generally present at a concentration of 0.001 to 4% w / v. The composition . The pharmaceutical can also contain protease inhibitors, for example aprotinin or tranexamic acid, and preservatives. In addition, the preparation may also contain a TFPI inhibitor and / or factor VIII. The compositions can be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions can be packaged for use or filtered under aseptic and lyophilized conditions, the lyophilized preparation being combined with a sterile aqueous solution before administration. The concentration of a factor Vlla, a factor XIII, or a factor Vlla in combination with a factor XIII in these formulations can vary widely, for example, from less than about 0.5% by weight, usually to at least about 1% by weight up to as much as 15 or % by weight, and will be selected primarily by fluid volumes, viscosities, etc., according to the particular mode of administration selected. Administration by injection or infusion, in particular injection, is preferred. Thus, factor VHa and factor XIII are prepared in a form suitable for intravenous administration, such as a preparation that is either a dissolved lyophilized powder or a liquid formulation containing factor Vlla and factor XIII in a form of dose, or a dissolved lyophilized powder, or a liquid formulation containing factor VIla in a dosage form and a dissolved lyophilized powder, or a liquid formulation containing factor XIII in another dosage form. The local administration of factor Vlla and factor XIII, such as, for example, topical application can be carried out, for example, by means of a spray, perfusion, double-balloon catheters, stents, incorporated in grafts or stents vascular, hydrogels used to coat balloon catheters, or other well-established methods. For outpatients requiring daily maintenance levels, the factor Vlla and factor XIII can be administered by continuous infusion using, for example, a portable pump system. In any case, the compositions Pharmaceuticals must provide an amount of a factor Vlla and a factor XIII sufficient to effectively treat the subject. The combination of a Vlla factor and a factor XIII shows a synergistic effect in an assay of the coagulum firmness and fibrinolysis time. In addition, the combination of a factor Vlla and a factor XIII shows a synergistic effect in the formation of stable fibrin clots, increasing the time of lysis of the clot medium, increasing the resistance of the clot and increasing the resistance to fibrinolysis. The compositions containing a factor VII and a factor XIII can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions are administered to a subject already suffering from a disease as described above, in an amount sufficient to cure, alleviate or partially arrest the disease and its complications. An adequate amount to achieve this is defined as an "effective amount" or "therapeutically effective amount". As will be understood by the person skilled in the art, the effective amounts for this purpose will depend on the severity of the disease or damage, as well as on the weight and general condition of the subject. It must be taken into account that the Materials of the present invention can be used in general in states of illness or serious damage, that is, situations that threaten life or that potentially threaten life. In such cases, in view of the minimization of foreign substances and the general lack of immunogenicity of factor Vlla and factor XIII in humans, it is possible and may be perceived as desirable by the treating physician to administer a substantial excess of these compositions. In prophylactic applications, the compositions containing a factor Vlla and a factor XIII are administered to a subject susceptible of, or otherwise at risk of a state of disease or damage, to improve the coagulant capacity of the subject itself. Such amount is defined as a "prophylactically effective dose". Simple or multiple administrations of the compositions can be carried out, with dose levels and standards that are selected by the treating physician. The compositions can be administered one or more times per day or week. An effective amount of such a pharmaceutical composition is the amount that provides a clinically significant effect against episodes of bleeding or bleeding. Such amounts will depend, in part, on the particular condition, age, weight and general health of the subject, and other factors. obvious to those skilled in the art. The composition is generally administered in a single dose before the expected bleeding, or at the onset of bleeding. However, it can be administered in multiple doses, preferably at intervals of 2-4-6-12 hours, depending on the dose administered and the condition of the subject. The composition may be in the form of a simple preparation comprising a factor Vlla and a factor XIII in suitable concentrations. The composition may also be in the form of a kit consisting of a first unit dose form, comprising a Vlla factor and a second unit dose form comprising a factor XIII and, optionally, one or more additional unit dosage forms. comprising a factor VIII and / or a TPFI inhibitor. In this case, factor Vlla and factor XIII must be administered sequentially, preferably within about 1-2 hours of each other, for example within 30 minutes one after the other or, preferably, within 10 minutes or, more preferably within 5 minutes one after the other. Either of the two dosage unit forms can be administered first. Since the present invention relates to the prevention or treatment of bleeding episodes or Hemorrhages, or for treatment with coagulants by treatment with a combination of active ingredients that can be administered separately, the invention also relates to the combination of the pharmaceutical compositions prepared in the form of equipment. The kit includes at least two separate pharmaceutical compositions. The kit includes container means for containing the separate compositions such as a divided bottle or a divided sheet pack. Typically, the equipment includes instructions for the administration of the separate components. The form of equipment is particularly advantageous when the separate components are preferably administered in different dosage forms, administered at different dose ranges, or when the titration of the individual components of the combination is desired by the prescribing physician.

Essays Test for Vlla factor activity: A suitable test to prove the activity of factor Vlla and with this select the appropriate variants of factor Vlla, can be carried out as a simple preliminary test, in vitro: In Vitro Hydrolysis Assay The native Vlla factor (wild type) and the variant of the Vlla factor (both hereinafter referred to as "factor Vlla") can be evaluated for specific activities. These can also be evaluated in parallel to directly compare their specific activities. The assay is carried out in a microtitre plate (axiSorp, Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration of 1 mm; it is added to factor Vlla (final concentration 100 nM) in 50 mM Hepes, pH 7.4, containing 0.1 M sodium chloride, 5 mM calcium chloride and 1 mg / ml bovine serum albumin. The absorbance at 405 nm is measured continuously in a SpectraMaxMR 340 plate reader (Molecular Devices, USA). The absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank that does not contain an enzyme, is used to calculate the ratio between the variant Vlla factor and the wild-type factor: Proportion = (A405 nm of the Vlla factor variant) / (A405 nm of the wild type Vlla factor).

Based on this, variants of the Vlla factor, with an activity comparable to or higher than the native factor Vlla, can be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of the factor VII native shown in Figure 1 is around versus above 1.0. Vlla factor activity or Vlla factor variants can also be measured using a physiological substrate such as factor X, suitably at a concentration of 100-1000 nM, where the Xa factor generated is measured after the addition of an appropriate chromogenic substrate (for example S-2765). In addition, the activity assay can be run at physiological temperature. The ability of factor Vlla or variants of factor Vlla to generate thrombin can also be measured in a test that includes all the relevant factors of coagulation and inhibitors at physiological concentrations (minus factor VIII when it mimics hemophilia A conditions) and activated platelets (as described on page 543 of Monroe et al (1997) Brit. J. Haematol, 99, 542-547 which is incorporated by reference herein).

Test for factor XIII activity A suitable assay for testing the transglutaminase activity of factor XIII, and thereby selecting suitable variants of factor XIII, can be performed as a simple in vitro test, as described, for example, in ethods of Enzymology, Vol. 45 (1976), Proteolytic Enzymes, Part B, pages 177-191 (Ed. Lorand, L.). The present invention is further illustrated by the following examples, which, however, should not be considered as limiting the scope of protection. The features defined in the foregoing description and in the following examples may, separately and in any combination thereof, be material for realizing the invention in various forms thereof.

EXAMPLES Example 1.

Factor XIII increases the formation of the fibrin clot, induced by factor Vlla Normal human plasma (NHP), citrated, was diluted 1/10 in buffer containing 20 nM HEPES, 150 mM sodium chloride, 5 mM calcium chloride, pH 7.4 in a microtiter well (total volume of 250 μm) and fibrin clot formation was periodically checked the increase in optical density at 600 nm in a SpectramaxMR 340 (Molecular Devices, Sunnyvale California). The spontaneous formation of the clot was obtained approximately at 2500-3000 seconds. Figure 1 shows that the recombinant Vlla factor (rFVIIa) 10 nM (Novo Nordisk A / S Basvaerd, Denmark) shortened the coagulation time to 1600 seconds (n = 2). Further shortening of the clotting time was obtained when factor XIII 30 nM (FXIII) (American Diagnostica Inc., Greenwich, CT) was added together with 10 nM rFVIIa (n = 3). The clot formed in the presence of FXIII was more transparent (lower maximum DO) than in its absence, indicating that the addition of FXIII resulted in a fibrin gel structure in the form of a finer mesh with thinner fibers.

Example 2 The presence of supplementary factor XIII during the formation of the clot induced by factor Vlla, gives as Result increased resistance to fibrinolytic degradation A fibrin clot consisting of thin fibers is mechanically stronger and more difficult to degrade than a clot containing the same amount of accommodated fibrin as thick fibers or less cross-linked fibers. The experiment shown in Figure 2 illustrates that supplemental FXIII (30 nM) prolongs the lysis time of the fibrin clot, the clots formed in the presence of rFVIIa and the tissue plasminogen activator (t-PA, American Diagnostica). Clot formation was induced in the presence or absence of 30 nM FXIII by the addition of 25 μ? of NHP at 225 μ? of HEPES 20 nM, 150 mM sodium chloride, 5 mM calcium chloride, pH 7.4 containing 50 nM rFVIIa and 0.5 nM recombinant t-PA. Clot formation and subsequent lysis of the clot, induced by plasminogen activation, mediated by t-PA, was monitored by a Spectramax® 340 at 600 nm as the increase in D060onmr followed by the reversion of the trace to baseline . Figure 2 shows that the clot lysis time under these conditions was significantly prolonged by the presence of FXIII.

Example 3 The factor Vlla in combination with factor XIII increases the maximum firmness of the clot and increases the resistance of the clot to fibrinolysis Thrombelastographic measurements were conducted on normal human plasma, citrated, added with the 6 nM recombinant tissue plasminogen activator (t-PA, American Diagnostica) and the effect of the addition of 1 nM rFVIIa was analyzed (Novo Nordisk A / S, Bagsvaerd , Denmark) alone or in combination with various concentrations of factor XIII (FXIII, Haematologic Technologies, HCXIII-0160, Lot N1212). Coagulation was initiated by the addition of Innovin (final concentration diluted to 2000 times, Dade Behring # 526945) and calcium (final concentration of 15 mM) in a buffer of 20 mM HEPES, 150 mM sodium chloride, pH 7.4. Thrombelastographic measurements were used to analyze the effect of rFVIIa and FXIII on the Maximum Clot Firmness (MCF) as well as the resistance of the clot to lysis mediated by t-PA. Before the addition of rFVIIa and / or FXIII the MCF obtained was 25 mm, and the time required for the half of the clot to be lysed was 12.3 minutes (Figure 3). The adition of Increasing concentrations of FXIII (0-40 nM) did not alter MCF; however, a prolongation of the lysis of the clot medium was observed, dependent on the dose, optimal at 30 nM FXIII (lysis time of half a clot: 14.3 minutes, Figure 3). Similarly, the addition of rFVIIa (1 nM) resulted in clot protection from t-PA-mediated fibrinolysis (clot half lysis time: 16.4 minutes), with no effect on MCF (Figure 3). However, after the addition of rFVIIa (1 nM) together with FXIII (30 nM), an increase in MCF (290 mm) was observed, as well as a deep protection from fibrinolysis (clot lysis time; 27.1 minutes) (Figure 3). Taken together, these results demonstrate that the addition of rFVIIa and FXIII to the plasma in a synergistic manner improves the mechanical strength of the clot and the resistance to fibrinolysis mediated by t-PA. It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (21)

2. Having described the invention as above, the content of the following claims is claimed as property. A pharmaceutical composition for intravenous administration, characterized in that it comprises a Vlla factor and a factor XIII. 2. The composition according to claim 1, characterized in that the factor VIla is a variant of factor VII.
3. 3. The composition according to claim 1, characterized in that the factor Vlla is the human factor Vlla.
4. 4. The composition according to claim 1 or claim 3, characterized in that the factor Vlla and factor XIII is the recombinant human Factor Vlla and recombinant human factor XIII.
5. 5. A composition according to any of claims 1 to 4, characterized in that factor XIII is a dimer a2 of factor XIII.
6. 6. A composition according to any of claims 1 to 5, characterized in that factor XIII is activated factor XIII.
7. 7. A composition in accordance with any of claims 1 to 6, characterized in that the composition also contains a TFPI inhibitor.
8. 8. A composition according to any of claims 1 to 7, characterized in that the composition also contains a factor VIII.
9. 9. A device for intravenous administration, which contains a treatment for episodes of bleeding or bleeding, characterized in that it comprises: a) an effective amount of a factor Vlla and a pharmaceutically acceptable carrier in a first unit dose form; b) an effective amount of a factor VIII and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing the first and second dosage forms.
10. 10. A kit according to claim 9, characterized in that it further comprises an effective amount of a TFPI inhibitor and a pharmaceutically acceptable carrier in a third unit dose form.
11. 11. A device according to claim 9, characterized in that the first unit dose form or the second unit dose form further comprise a TFPI inhibitor.
12. 12. An equipment according to any of claims 9 to 11, characterized in that it also contains a factor VIII, formulated either in a separate unit dose form, or contained within a unit dose form that also contains one or more of the compounds selected from the list of a Vlla factor, a factor XIII or a TFPI inhibitor.
13. 13. The use of a Vlla factor in combination with a factor XIII, for the manufacture of a medicament for the treatment of hemorrhagic episodes in a subject, the medicament is for intravenous administration. The use according to claim 13, for the manufacture of a medicament for reducing the clotting time in a subject. 15. The use according to claim 13, for the manufacture of a medicament for prolonging the time of lysis of the clot in normal mammalian plasma. 16. The use according to claim 13, for the manufacture of a medicament for increasing the resistance of the clot in normal mammalian plasma. 17. The use according to claim 13, for the manufacture of a medicament for improving the formation of the fibrin clot in normal human plasma. 18. A method for improving the formation of the fibrin clot in a subject, characterized by the method because it comprises the administration to a subject of an effective amount of a factor Vlla in combination with an effective amount of a factor XIII. 19. A method for treating bleeding episodes in a subject, characterized in that it comprises administering to a subject an effective amount of a factor VIla in combination with an effective amount of a factor XIII. 20. The method according to claim 18 or claim 19, characterized in that the factor Vlla and factor XIII are administered in the form of a single dose. 21. The method according to claim 18 or claim 19, characterized in that the factor Vlla and factor XIII are administered sequentially.