KR20020092469A - Pharmaceutical composition comprising a factor viia and a factor xiii - Google Patents

Abstract

The present invention relates to the use of factor VIIa and factor XIII in the treatment or prevention of bleeding events.

Description

PHARMACEUTICAL COMPOSITION COMPRISING A FACTOR VIIA AND A FACTOR XIII}

Hemostasis is initiated by the formation of a complex between a tissue factor (TF) that is exposed to circulating blood after a wound in the vessel wall and FVIIa present in the blood circulation in an amount corresponding to about 1% of the total FVII protein mass. The complex attaches to cells with TF and activates FX to FXa and FIX to FIXa on the cell surface. FXa activates prothrombin as trobin, and thrombin activates FVIII, FV, FXI and FXIII. Moreover, a limited amount of thrombin formed in this early hemostatic stage activates platelets. After the action of thrombin on platelets, platelets change shape and are exposed to charged phospholipids on their surface. This activated platelet surface forms a template for further FX activation and sufficient thrombin production. Further FX activation on the activated platelet surface occurs through the FIXa-FVIIa complex formed on the surface of the activated platelets, which then converts prothrombin to thrombin while FXa remains on the surface. Thrombin then converts the fibrinogen into fibrin, which is insoluble and stabilizes the early platelet plug. This process is compartmentalized, ie localized at the site of TF expression or exposure to minimize the risk of systemic activation of the coagulation system. Moreover, the insoluble fibrin forming the plug is stabilized by FXIII-catalytic crosslinking of the fibrin fiber.

FVIIa is predominantly present in the plasma as single-chain simogen, which is cleaved by FXa into its two-chain active type FVIIa. Recombinant active factor VIIa (rFVIIa) was developed as a pre-hemostatic agent. Administration of rFVIIa provides a fast and highly effective pre-hemostatic response in hemophilic subjects with bleeding that cannot be treated with coagulation factor products due to antibody formation. In addition, bleeding subjects who lack factor VII or subjects with normal coagulation but who experience excessive bleeding can be successfully treated using FVIIa. These studies did not show undesirable side effects of rFVIIa (particularly the development of thromboembolism).

In addition, exogenously administered FVIIa increases the formation of thrombin on activated platelet surfaces. This occurs in hemophilia subjects lacking FIX or FVIII, thereby losing the most potent pathway for sufficient thrombin formation. In addition, extra FVIIa increases thrombin formation in the presence of reduced numbers of platelets or platelets with defective function.

Fibrin stabilizing factor FXIII is a transglutaminase that provides fibrin structures with increased resistance to lysis by plasmin and other proteolytic enzymes by crosslinking fibrin monomers. Factor XIII is also known as "fibrinoligases" and "fibrin stabilizing factors". FXIIa, when activated, may form intermolecular gamma-glutamyl-epsilon-lysine crosslinks between the side chains of the fibrin molecule and between other substrates. FXIII is found in plasma and platelets. The enzyme is a tetrameric simogen (denoted as a2b2) consisting of two alpha-subunits and two beta-subunits in plasma and a simogen (denoted as a2-dimer) consisting of two alpha-subunits. Present in platelets.

Both simogens are activated by thrombin and Ca ²⁺ . Calcium is released from platelets upon aggregation at the wound site. Thrombin cleaves from 1 to 37 N-terminal amino acid residues (of a2-dimer). In the case of a2b2-simogen, the beta-subunit is then dissociated from the activated alpha-subunit. Calcium binds equally well to the simogen and thrombin modified molecules. After thrombin and calcium activation, active center cysteine on the alpha chain is exposed to form a fully activated enzyme. Subjects with severe thrombocytopenia have been found to have low FXIII plasma levels.

It is well known that subjects who excessively bleed in connection with surgery or dangerous trauma and need blood transfusion have more complications than those who have not experienced any bleeding. However, moderate hemorrhagic human viruses (hepatitis, HIV, parvovirus, and other unknown viruses that require the administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation defects, etc.)). May lead to complications related to the risk of transmission. Large bleeding requiring large blood transfusions can lead to the development of multiple organ dysfunction, including impaired lung and kidney function. Once the subject exhibits these serious complications, the cascade of events involving a number of cytokines and inflammatory reactions begins to perform some extremely difficult and unfortunately usually unsuccessful treatments. Therefore, the main goal in the treatment of dangerous tissue damage as well as surgery is to avoid or minimize bleeding.

In order to avoid or minimize such bleeding, it is important to ensure the formation of a stable solid haemostatic plug that is not readily soluble by fibrin lytic enzymes. Moreover, it is important to ensure fast and effective formation of such plugs or blood clots.

Japanese Patent Application No. 2-167234 is characterized by containing fibrinogen, prothrombin, blood coagulation factor VII, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor XIII, antithrombin, proteinase inhibitors and calcium ions. It relates to a bio-tissue adhesion agent.

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

WO 93/12813 (Zimogenetics) relates to the use of FXIII to reduce perioperative blood loss in a subject undergoing surgery. In addition, the composition may comprise aprotinin. FXIII is typically administered to a subject as a bolus injection one day prior to surgery.

European Patent No. 225.160 (Novo Nordisk) relates to a composition of FVIIa and to a method for the treatment of bleeding disorders which are not attributable to a blood clotting factor defect or a blood clotting factor inhibitor.

European Patent No. 82.182 (Bacter Trabenol Lab.) Relates to the composition of Factor VIIa used to counteract the deficiency of blood clotting factors in a subject or the effect of inhibitors on blood clotting factors in a subject.

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

Thrombosis and Haemostasis, 1999 (Suppl), 0951 to Kjalke et al., Relate to the effect of extraneous exogenous FVIIa administration and the formation of thrombin on activated platelet surfaces in a model system that simulates hemophilia A or B status.

There remains a need in the art for improved, reliable and widely available methods to enhance coagulation, to rapidly form stable hemostatic plugs, and to achieve sufficient hemostasis in subjects, particularly those with impaired thrombin generation. There is also a need for a method of lowering the amount of FVIIa needed to achieve sufficient hemostasis.

The present invention relates to pharmaceutical compositions comprising Factor VIIa and Factor XIII. The present invention also relates to the use of a combination of factor VIIa and factor XIII for the manufacture of a medicament for the treatment or prevention of a subject suffering from a bleeding event. The invention also relates to a method of treating a bleeding event in a subject, and to a method of promoting blood clot formation in a subject. The present invention also relates to kits comprising these compounds.

Summary of the Invention

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

It is a second object of the present invention to provide a composition in one dosage form that can be used effectively in the treatment or prevention of bleeding events or as a precoagulant.

It is another object of the present invention to provide a composition, method of treatment or kit that exhibits synergistic effects.

It is a further object of the present invention to provide compositions, methods of treatment or kits that do not exhibit substantial side effects such as high levels of systemic activation of the coagulation system.

Other objects of the present invention will become apparent as the specification is read.

The inventors have found that the combination of Factor VIIa and Factor XIII can shorten the time to blood clot of normal human plasma more effectively than Factor VIIa or Factor XIII alone. It was also found that the combination of Factor VIIa and Factor XIII could increase blood clotting rigidity more effectively than Factor VIIa or Factor XIII alone. It was found that a further unexpected increase in blood clotting rigidity was obtained by combining a factor VIIa at a concentration for which no further increase in blood clot rigidity was observed and a factor XIII at a concentration for which no further increase in blood clotting rigidity was observed. It has also been found that the combination of Factor VIIa and Factor XIII can prolong blood clot lysis time in normal human plasma in vitro more effectively than Factor VIIa or Factor XIII inhibitors alone. Thus, more effective treatment of bleeding in a subject can be obtained by enhancing coagulation. Moreover, patients can be treated with relatively low concentrations of Factor VIIa, thereby reducing the relatively high costs associated with conventional treatment with Factor VIIa alone.

As a first aspect, the present invention relates to a pharmaceutical composition comprising Factor VIIa and Factor XIII and optionally a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition comprising Factor VIIa and Factor XIII as the only active agents and optionally a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition formulated for intravenous administration comprising Factor VIIa and Factor XIII, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition formulated for intravenous administration comprising Factor VIIa and Factor XIII as the only active agent and optionally a pharmaceutically acceptable carrier.

In one embodiment, factor VIIa is recombinant factor VIIa and factor XIII is recombinant factor XIII.

In one embodiment of the invention the factor VIIa is recombinant factor VIIa. In further embodiments, factor VIIa is recombinant human factor VIIa. In a further embodiment Factor VIIa is Factor VIIa variant.

In one embodiment, the Factor VIIa variant is an amino acid sequence variant having up to 20 amino acids replaced, deleted or inserted as compared to wild type Factor VIIa (ie, a polypeptide having an amino acid sequence disclosed in US Pat. No. 4,784,950). In other embodiments, the Factor VIIa variants have 15 or fewer amino acids replaced, deleted or inserted as compared to wild type Factor VIIa; In other embodiments, the Factor VIIa variants have 10 or fewer amino acids replaced, deleted or inserted; In other embodiments, the Factor VIIa variants have 8 or fewer amino acids replaced, deleted or inserted; In other embodiments, the Factor VIIa variants have up to 6 amino acids replaced, deleted or inserted; In other embodiments, the Factor VIIa variants have 5 or fewer amino acids replaced, deleted or inserted; In other embodiments, the Factor VIIa variants have 3 or fewer amino acids replaced, deleted or inserted.

In one embodiment, the Factor VIIa variant is [L305V] -FVIIa, [L305V / M306D / D309S] -FVIIa, [L3051] -FVIIa, [L305T] -FVIIa, [F374P] -FVIIa, [V158T / M298Q] -FVIIa , [V158D / E296V / M298Q] -FVIIa and [K337A] -FVIIa.

In one embodiment the factor XIII is FXIII a2b2. In further embodiments the factor XIII is FXIII a2. In further embodiments, factor XIII is active form factor XIII (FXIIIa). In one embodiment, Factor XIII is Factor XIII variant. In one embodiment the factor XIII is 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 a human a2-dimer.

In one embodiment, the composition further contains a TFPI inhibitor. In another embodiment, the composition further contains factor VIII. In another embodiment, the composition further contains Factor VIII and a TFPI inhibitor.

In one embodiment, the composition further comprises an inhibitor of the fibrin dissolution system, for example aprotinin, ε-aminocaproic acid or tranexamic acid.

In another aspect, the present invention

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

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

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In one aspect, the kit is

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

b) an effective amount of Factor XIII 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 said first, second and third dosage forms

It includes.

In another aspect, the present invention

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

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

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

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

b) an effective amount of Factor XIII and TFPI inhibitor in a second unit dosage form, and optionally a pharmaceutically acceptable carrier; And

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

a) an effective amount of Factor VIIa and Factor XIII, and optionally a pharmaceutically acceptable carrier, in a first unit dosage form;

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 said first and second dosage forms

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

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

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

c) an effective amount of 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 dosage form; And

e) container means for containing said first, second, third and fourth dosage forms

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another embodiment, the kit is

a) an effective amount of Factor VIIa and Factor XIII, and optionally a pharmaceutically acceptable carrier, in a first unit dosage form;

b) an effective amount of Factor VIII and optionally 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 said first, second and third dosage forms

It includes.

In another aspect, the present invention

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

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

c) an effective amount of Factor VIII and optionally a pharmaceutically acceptable carrier in a third unit dosage form; And

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

a) an effective amount of Factor VIIa and Factor VIII, and optionally a pharmaceutically acceptable carrier, in a first unit dosage form;

b) an effective amount of Factor XIII and optionally a pharmaceutically acceptable carrier in a second unit dosage 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 said first, second and third dosage forms

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

a) an effective amount of Factor VIIa and Factor XIII, and optionally a pharmaceutically acceptable carrier, in a first unit dosage form;

b) an effective amount of Factor VIII and TFPI inhibitor in a second unit dosage form, and optionally a pharmaceutically acceptable carrier; And

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

a) an effective amount of Factor VIII and Factor XIII, and optionally a pharmaceutically acceptable carrier, in a first unit dosage form;

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 Factor VIIa and optionally a pharmaceutically acceptable carrier in a second unit dosage form; And

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

a) an effective amount of Factor XIII and TFPI inhibitor in a first unit dosage form, and optionally a pharmaceutically acceptable carrier;

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

e) an effective amount of Factor VIIa and optionally a pharmaceutically acceptable carrier in a second unit dosage form; And

c) container means for containing said first, second and third dosage forms

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

a) an effective amount of a TFPI inhibitor and Factor VIII, and optionally a pharmaceutically acceptable carrier, in a first unit dosage form;

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

f) an effective amount of Factor XIII and optionally pharmaceutically acceptable carrier in a second unit dosage form; And

c) container means for containing said first, second and third dosage forms

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

a) an effective amount of a TFPI inhibitor and Factor XIII, and optionally a pharmaceutically acceptable carrier, in a first unit dosage form;

b) an effective amount of Factor VIII and Factor VIIa in a second unit dosage form, and optionally a pharmaceutically acceptable carrier; And

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the present invention

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

b) an effective amount of Factor VIII and Factor XIII, and optionally a pharmaceutically acceptable carrier, in a second unit dosage form; And

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

It relates to a kit containing a therapeutic agent for bleeding events comprising a.

In another aspect, the invention relates to the use of Factor VIIa in combination with Factor XIII for the manufacture of a medicament for treating a bleeding event.

In another aspect, the present invention relates to the use of Factor VIIa in combination with Factor XIII for the manufacture of a medicament for shortening the time to blood clot in a subject.

In another aspect, the present invention relates to the use of Factor VIIa in combination with Factor XIII for the manufacture of a medicament for prolonging blood clot lysis time in mammalian plasma.

In another aspect, the present invention relates to the use of Factor VIIa in combination with Factor XIII for the manufacture of a medicament for increasing blood clot strength in mammalian plasma.

In another aspect, the invention relates to the use of Factor VIIa in combination with Factor XIII for the manufacture of a medicament for enhancing fibrin blood clot formation in mammalian plasma.

In one embodiment, the mammalian plasma is human plasma. In other embodiments the mammalian plasma is normal plasma; In other embodiments the plasma is normal human plasma; In one embodiment the plasma is plasma from a subject with impaired thrombin production. In one embodiment the plasma is plasma from a subject having a lower concentration of fibrinogen.

In one embodiment, Factor VIIa and Factor XIII prolong blood clot lysis time in normal human plasma in vitro.

In another aspect, the present invention relates to a method of enhancing fibrin blood clot formation in a subject, the method comprising administering to the subject an effective amount of Factor VIIa in combination with an effective amount of Factor XIII.

In another aspect, the present invention relates to a method of treating a bleeding event in a subject, the method comprising administering to the subject an effective amount of Factor VIIa in combination with an effective amount of Factor XIII.

In another aspect, the present invention is directed to a method of shortening the time to blood clot of mammalian plasma, the method comprising contacting the plasma with an effective amount of Factor VIIa in combination with an effective amount of Factor XIII. In one embodiment, an effective amount of Factor VIIa in combination with an effective amount of Factor XIII is administered to a subject in need of such treatment.

In another aspect, the present invention is directed to a method of enhancing the formation of fibrin in a subject, the method comprising administering to the subject an effective amount of Factor VIIa in combination with an effective amount of Factor XIII.

In one embodiment of the method of the invention, Factor VIIa and Factor XIII are the only active agents administered to the subject. In another embodiment of the invention, the pharmaceutical composition comprises Factor VIIa and Factor XIII as the only active agents.

In one embodiment of the invention, Factor VIIa and Factor XIII are administered in one dosage form simultaneously. In another embodiment, Factor VIIa and Factor XIII are administered sequentially. In further embodiments, Factor VIIa and Factor XIII are in the form of a kit comprising, for example, Factor VIIa in a first unit dosage form and Factor XIII in a second unit dosage form, within about 1 to 2 hours of each other, For example within 30 minutes of each other, for example within 10 minutes of each other.

In one embodiment, the effective amount of Factor VIIa is 0.05 mg / day to 500 mg / day (70 kg subject). In one embodiment, the effective amount of Factor XIII is 0.05 mg / day to 500 mg / day (70 kg subject).

In one embodiment of the invention, the pharmaceutical composition (when in single-form form) consists essentially of Factor VIIa and Factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizer, and And / or detergents, and / or neutral salts, and / or antioxidants, and / or preservatives, and / or protease inhibitors.

In another embodiment of the invention, the pharmaceutical composition (when in single-form form) consists essentially of Factor VIIa and Factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizer, and And / or detergents, and / or neutral salts, and / or antioxidants, and / or preservatives, and / or protease inhibitors, and / or TFPI inhibitors.

In another embodiment of the invention, the pharmaceutical composition (when in single-form form) consists essentially of Factor VIIa and Factor XIII, and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizer, and And / or detergents, and / or neutral salts, and / or antioxidants, and / or preservatives, and / or protease inhibitors, and / or TFPI inhibitors, and / or factor VIII.

In another embodiment, the pharmaceutical composition (when in kit form) consists essentially of Factor VIIa and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizer, and / or detergent, and / or neutral salt. A first unit dosage form consisting of and / or antioxidants, and / or preservatives, and / or protease inhibitors; And essentially Factor XIII and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizer, and / or detergent, and / or neutral salt, and / or antioxidant, and / or preservative, and / Or a second unit dosage form consisting of a protease inhibitor.

In another embodiment, the pharmaceutical composition (when in kit form) consists essentially of Factor VIIa and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizer, and / or detergent, and / or neutral salt. A first unit dosage form consisting of and / or antioxidants, and / or preservatives, and / or protease inhibitors, and / or TFPI inhibitors; And essentially Factor XIII and optionally at least one pharmaceutically acceptable excipient or carrier, and / or stabilizer, and / or detergent, and / or neutral salt, and / or antioxidant, and / or preservative, and / Or a second unit dosage form consisting of a protease inhibitor, and / or a TFPI inhibitor, and / or factor VIII.

In further embodiments the subject is a human; In other embodiments the subject has impaired thrombin production; In one embodiment the subject has a lowered fibrinogen plasma concentration (eg, several transfused patients).

In a further embodiment, the composition further contains factor VIII. In one embodiment, factor VIII is active form factor VIII (factor VIIIa). In further embodiments, factor VIII is recombinant factor VIIIa. In further embodiments, factor VIII is recombinant human factor VIIIa.

In a further embodiment, the composition further comprises an inhibitor of the fibrin dissolution system, for example aprotinin, ε-aminocaproic acid or tranexamic acid.

Brief description of the drawings

1 shows spontaneous blood clots in citrated normal human plasma (NHP) diluted 1/10 in a buffer containing ₂₀ nM Hepes, 150 mM NaCl, and 5 mM CaCl ₂ , pH 7.4 in microwells (250 μl total volume). Indicates that formation was obtained at about 2500 to 3000 seconds. Fibrin blood clot formation was monitored by an increase in optical density at 600 nm on Specramax ^™ 340 (Molecular Devices, Sunnyvale, California). 1 shows that 10 nM recombination factor VIIa (rFVIIa) from Novo Nordisk A / S (Denmark Boxwald) shortened the clotting time by 1600 seconds (n = 2). Further shortening of blood clotting time was obtained when 30 nM factor XIII (FXIII) from American Diagnostica Incorporated (Connecticut Greenwich) was added with 10 nM rFVIIa (n = 3). Blood clots formed in the presence of FXIII were more transparent (lower maximum OD) than in its absence, indicating that the addition of FXIII resulted in a finer mesh fibrin gel structure with thinner fibers.

2 shows that supplemented FXIII (30 nM) prolongs fibrin clot lysis time of clots formed in the presence of rFVIIa and tissue plasminogen activator (tPA). Blood clot formation was achieved by adding 25 μl NHP to 225 μl 20 nM Hepes, 150 mM NaCl, 5 mM CaCl ₂ , pH 7.4 containing 50 nM rFVIIa and 0.5 nM recombinant tPA from Novo Nordisk A / S (Denmark Box-Baert). In the presence or absence of. Blood clot formation and subsequent clot lysis induced by tPA-mediated plasminogen activation was Spectramax The 340 was monitored as an increase from ₆₀₀ nm to OD _{600 nm} followed by a trace back to the base level. 2 shows that the clot lysis time under these conditions was significantly extended by the presence of FXIII.

3 shows the effects of rFVIIa and FXIII on blood clot resistance to tPA-mediated lysis as well as maximal blood clot rigidity (MCF). The MCF obtained before adding rFVIIa and / or FXIII was 25 mm and the time required for half the blood clot to dissolve was 12.3 minutes (FIG. 3). Adding with increasing concentration of FXIII (0-40 nM) did not alter MCF, but dose-dependent prolongation of anti-blood lysis was observed and this was optimal at 30 nM FXIII (anti-blood lysis time: 14.3 min, FIG. 3). ). Similarly, addition of rFVIIa (1 nM) protected blood clots from tPA-mediated fibrin lysis without any effect on MCF (half-blood lysis time: 16.4 min) (FIG. 3). However, addition of rFVIIa (1 nM) with FXIII (30 nM) showed an increase in MCF (29 mm) as well as sufficient protection from fibrin dissolution (semi-blood clotting dissolution time: 27.1 minutes) (FIG. 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 blood clots and resistance to tPA-mediated fibrin dissolution.

Detailed description of the invention

The present invention provides a composition comprising a combination of Factor VIIa and Factor XIII. The present invention also provides compositions comprising a combination of Factor VIIa and Factor XIII as the only active ingredient. The composition may be in the form of a single composition or in the form of a multi-component kit. The compositions are useful as therapeutic and prophylactic precoagulants and fibrin blood-stabilizers and form rapid fibrin blood clots in mammals, including primates such as humans.

Whenever a unit dose such as first or second or third is referred to herein, this dose does not necessarily indicate a desired degree of administration but is referred to as a unit dose for convenience only.

The present invention treats a bleeding event in a subject, including a human, for example due to trauma or surgery, or a bleeding event in a subject lacking or defective in blood coagulation factor FIX or FVIII or platelets (prophylactic treatment or More).

It has been found that the combination of Factor VIIa and Factor XIIIa is an advantageous product that ensures the formation of a solid, stable and fast hemostatic plug.

Sufficient thrombin production is necessary for the formation of a solid stable hemostatic plug. The fibrin structure of such a plug depends on both the amount of thrombin formed and the initial thrombin generation rate. In the presence of impaired thrombin generation, a highly permeable porous fibrin plug is formed. Fibrin lytic enzymes are usually present on the fibrin surface and readily dissolve such fibrin plugs. In addition, the formation of a stable fibrin plug is dependent on the presence of factor XIIIa which is activated by thrombin and thus also dependent on sufficient thrombin generation. Moreover, the recently described thrombin activatable fibrin lysis inhibitor TAFI requires a rather high thrombin amount for its activation. Thus, in the presence of insufficient thrombin formation, TAFI cannot be activated, resulting in the formation of a hemostatic plug that dissolves more easily than normally dissolved by normal fibrin lytic activity.

By increasing thrombin production, Factor VIIa provides the basis for sufficient Factor XIII activation, which is most important for the formation of fully stabilized hemostatic plugs and therefore for maintenance of hemostasis. In thrombocytopenia, a situation where the number of platelets is lowered, faster thrombin production is initiated by exogenous administration of extra factor VIIa. However, total thrombin production is not normalized even by high concentrations of Factor VIIa.

Combining Factor VIIa and Factor XIII, in particular Factor XIII alpha chains (a2-dimer), facilitates sufficient activation of Factor XIII to enhance the hemostatic effect of Factor VIIa.

Moreover, sufficient factor XIII activation does not occur in subjects with reduced plasma levels of fibrinogen (subjects that have been transfused several times as a result of multiple trauma or major surgery). Next, more effective hemostasis is obtained by administration of a combination of factor VIIa and factor XIII.

Another way to increase the stability of the fibrin hemostatic plug is to ensure sufficient presence of factor XIIIa (active factor XIII).

Subjects with thrombocytopenia have not only impaired thrombin generation but also stability deficiencies of the fibrin plug, resulting in hemostatic plugs that tend to dissolve prematurely. Moreover, subjects who are at risk of trauma or organ damage and eventually receive frequent blood transfusions often experience lower platelet counts as well as lower levels of fibrinogen, factor VIII, and other coagulation proteins. These subjects experience impaired (or degraded) thrombin production. In addition, their lowered fibrinogen levels negatively interfere with the activation of Factor XIII. Thus, these subjects have defective or less effective hemostasis leading to the formation of fibrin plugs that are readily and prematurely dissolved by fibrin lytic enzymes, and in addition, such enzymes are widely used in situations characterized by extensive trauma and organ damage. Is released.

To facilitate the formation of a sufficiently stabilized plug having sufficient ability to maintain hemostasis in a subject, a composition according to the invention is administered. This composition is particularly advantageous in subjects with reduced platelet counts and in subjects with reduced plasma levels of fibrinogen and / or other coagulation proteins.

In the presence of factor XIII, lower concentrations of factor VIIa may be sufficient to ensure sufficient hemostasis.

As mentioned above, Factor XIII is present in plasma as tetramer simogen (denoted as a2ba2) consisting of two alpha-subunits and two beta-subunits, but other tissues (eg, platelets as a2-dimers). Also found in). Either of these zymogen forms or active form factor XIII (Factor XIIIa) can be used within the present invention, and genetically engineered variants of Factor XIII having its characteristic crosslinking activity can be used within the scope of the present invention. have. In one embodiment, factor XIII is human factor XIII; In other embodiments, factor XIII is a human a2-dimer; In another embodiment factor XIII is active human factor XIIIa.

Factor XIII and Factor VIIa for use in the present invention may be purified from blood or produced by recombinant means. The implementation of the methods described herein is independent of how purified Factor XIII and Factor VIIa are derived, and therefore it is clear that the present invention is contemplated to cover the use of any Factor XIII and Factor VIIa formulations suitable for use herein. . Preferred are human factor VIIa and human factor XIII. In addition, genetically engineered variants of Factor VIIa and Factor XIII that retain their characteristic hemostatic-related activity can be used in the present invention. In addition, fragments of Factor VIIa or Factor XIII or Factor VIIa- or Factor XIII-variants that retain their characteristic hemostatic-related activity may be used in the present invention. Hemostatic-related activity of factor VIIa can be measured, for example, using the factor VIIa-activity assay described herein. Hemostatic-related activity of factor XIII can be measured using, for example, a factor XIII-activity assay described herein.

Non-limiting examples of Factor VII variants having substantially the same or better biological activity compared to wild type Factor VIIa include those described in Danish patent applications Nos. PA 2000 00734, PA 2000 01360, PA 2000 01361 and PA 2001 00477 However, the present invention is not limited thereto. Non-limiting examples include [L305V] -FVIIa, [L305WM306D / D309S] -FVIIa, [L305I] -FVIIa, [L305T] -FVIIa, [F374P] -FVIIa, [V158T / M298Q] -FVIIa, [V158D / E296V / M298Q] -FVIIa and [K337A] -FVIIa.

Three-letter or one-letter representations of amino acids in this context are used according to their conventional meanings shown in Table 1. Unless expressly indicated otherwise, the amino acids mentioned herein are L-amino acids. For example, it should be understood that the first letter in K337 represents an amino acid naturally present at the indicated position of wild type factor VII. That is, for example, [K337A] -FVIIa is that the amino acid indicated by the one-letter code K, which exists naturally at the indicated position, is replaced by the amino acid indicated by the one-letter code A.

Abbreviation for amino acid amino acid 3-character code 1-character code Glycine Gly G Proline Pro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Histidine His H Lee Sin Lys K Arginine Arg R Glutamine Gln Q Asparagine Asn N Glutamic acid Glu E Aspartic acid Asp D

The terms "factor VIIa" or "FVIIa" may be used interchangeably. The term factor VIIa includes simogen gene VII (single-chain factor VII). The terms "factor XIII" or "FXIII" may be used interchangeably. The terms "factor VIII" or "FVIII" may be used interchangeably.

It will be apparent to those skilled in the art that there may be substitutions outside the region essential for the function of Factor VIIa or Factor XIII-molecules and the results are still active polypeptides. Amino acid residues that are essential for the activity of Factor VIIa or Factor XIII-polypeptides and are therefore preferably unsubstituted can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (eg, , Cu-nningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at all positively charged residues in the molecule, and the resulting mutant molecule is tested for coagulants, each crosslinking activity, to identify amino acid residues that are integral to the activity of the molecule. In addition, sites of substrate-enzyme interactions can be determined by analysis of three-dimensional structures measured by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (eg, 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 mutations into nucleic acid sequences to convert one nucleotide to another can be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the process of using a superannular double stranded DNA vector with the insertion of interest and two synthetic primers containing the desired mutation. Oligonucleotide primers each complementary to the opposite strand of the vector are extended during temperature cycling by Pfu DNA polymerase. Incorporation of the primers results in mutated plasmids containing twisted gaps. After temperature cycling, the product is treated with Dpnl specific for methylated and hemimethylated DNA to digest the parent DNA template and select the synthesized DNA containing the mutation. Other procedures known in the art can also be used to create, identify and isolate variants such as, for example, gene shuffling or phage display techniques.

The term "factor VIII" or "FVIII" includes active factor VIII (denoted factor VIIIa), variants and truncated forms of factor VIII which retain its characteristic coagulant activity. In one embodiment, factor VIII is human factor VIII.

The term "TFPI inhibitor" means a compound that inhibits the anti-coagulant 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 Pat. No. 5,622,988. "TFPI" and "EPI" (foreign pathway inhibitor) can be used interchangeably.

Within the present invention, the "effective amount" of factor VIIa and the "effective amount" of factor XIII are those of factor VIIa and factor XIII sufficient to cure, alleviate or partially stop the disease and its complications by preventing or reducing bleeding or blood loss. It is defined as a quantity.

The amount of Factor VIIa and Factor XIII administered according to the present invention may vary from a ratio of about 1: 100 to a ratio of about 100: 1 (μg Factor VIIa: μg Factor XIII).

In this context, "subject with damaged thrombin generation" refers to a subject that is unable to produce sufficient thrombin release at the activated platelet surface and is fully functional normal hemostasis, including normal amounts and functions of coagulation factors, platelets and fibrinogen Subjects with production of less thrombin than subjects with the system and lack FXI and / or FVIII (hemophilia A and B) or have defective FIX and / or FVIII or FIX and Subjects with inhibitors for FVIII; Subjects lacking FXI; Subjects with a reduced number of platelets or platelets with defective function (eg, thrombocytopenia or thrombocytopenia glanzmann or subjects with excessive bleeding); And subjects with reduced levels of prothrombin, FX or FVII.

In addition, subjects with reduced plasma concentrations of fibrinogen (eg, those transfused several times as a result of a large number of trauma or massive surgery) suffer from the formation of loose and unstable fibrin plugs that dissolve easily.

The term “sufficient hemostasis” refers to the formation of stable solid fibrin blood clots or plugs at the wound site that can effectively stop bleeding and are not readily soluble by the fibrin lysis system.

The term "activity of factor VIIa" or "factor VIIa-activity" means the ability to produce thrombin, which term also includes the ability to produce thrombin at the surface of activated platelets in the absence of tissue factors.

The term "enhancement of normal hemostatic system" means enhancement of the ability to produce thrombin.

The term "bleeding disorder" as used herein reflects any inherent, acquired or induced defect of cellular or molecular origin that becomes evident upon bleeding. Examples are blood clot factor deficiency (eg, hemophilia A and B or deficiency of coagulation factor XI or VII), blood clot factor inhibitors, defective platelet function, thrombocytopenia or von Willebrand disease.

The term "bleeding event" is meant to include uncontrolled excessive bleeding, which is a major problem both involving surgery or some form of tissue damage. Uncontrolled excessive bleeding is basically a subject with a normal coagulation system (but these subjects show coagulopathy as a result of bleeding-dilution of coagulation protein due to the dilution effect of bleeding, increased fibrin lysis and reduced platelets) and coagulation Or in subjects with bleeding disorders. Blood clotting factor deficiency (hemophilia A and B, deficiency of coagulation factor XI or VII) or blood clotting factor inhibitors may be responsible for bleeding disorders. In addition, excessive bleeding occurs in subjects with normally functioning blood clotting cascades (without inhibitors for either clotting factor deficiency or coagulation factor) and are associated with defective platelet function, thrombocytopenia or von Willebrand disease. Can be attributed. In such cases, bleeding may be similar to bleeding due to hemophilia, which lacks abnormal essential hemolytic “compounds” (such as platelets or von Willebrand factor proteins) that the hemostatic system can cause dangerous bleeding, as in hemophilia This is because it has. In subjects who have experienced excessive tissue damage associated with surgery or major trauma, the normal hemostatic mechanism may be overwhelmed by the need for immediate hemostasis, and they may develop bleeding despite the normal (pre-traumatic) normal hemostatic mechanism. have. In addition, achieving satisfactory hemostasis is a problem when bleeding occurs in organs such as the brain, inner ear region, and eyes that have limited potential for surgical hemostasis. The same problem can occur in laparoscopic surgery as well as in the process of taking biopsis from multiple organs (liver, lung, tumor tissue, gastrointestinal tract). Common to all these situations is the difficulty in providing hemostasis by surgical techniques (sutures, clips, etc.), as well as when bleeding spreads (genital bleeding and massive uterine bleeding). In addition, acute and massive bleeding can occur in subjects undergoing anticoagulant therapy, in which defective hemostasis is induced by a given therapy. Such subjects may require surgical intervention if the anticoagulant effect is not quickly offset. Radical prostatectomy is a common procedure for subjects with local prostate cancer. This surgery is often aggravated by a significant amount of massive blood loss. Significant blood loss during prostatectomy is mainly related to the exacerbated anatomical situation with various densely vascularized sites that are not easily accessible for surgical hemostasis, which can lead to the spread of bleeding from large areas. Another situation that can cause problems in the case of unsatisfactory hemostasis is when anticoagulant therapy is provided to subjects with normal hemostatic mechanisms to prevent thromboembolic disorders. Such therapies include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K-antagonists, as well as aspirin and other platelet aggregation inhibitors.

In one embodiment of the invention the bleeding is related to hemophilia. In other embodiments the bleeding is related to hemophilia with acquired inhibitors. In another embodiment the bleeding is associated with thrombocytopenia. In another embodiment the bleeding is related to von Willebrand disease. In other embodiments the bleeding is related to severe tissue damage. In another embodiment the bleeding is related to surgery. In another embodiment the bleeding is related to laparoscopic surgery. In other embodiments the surgery involves genital bleeding. In other embodiments the bleeding is related to massive uterine bleeding. In other embodiments the bleeding occurs in an organ with a limited likelihood of mechanical hemostasis. In other embodiments the bleeding occurs in the brain, inner ear region or eye. In another embodiment the bleeding is related to the process of taking biopsis. In another embodiment the bleeding is related to anticoagulant treatment.

The composition according to the invention may further comprise a TFPI inhibitor. Such compositions should preferably be administered to a subject having hemophilia A or B.

The composition according to the invention may further comprise factor VIII. Such compositions should preferably be administered to subjects who do not have inhibitors against factor VIII.

In the present context, the term "treatment" is intended to include the prevention of anticipated bleeding, for example in surgery, and the control of already occurring bleeding, such as in hemophilia or trauma, in the sense of inhibiting or minimizing bleeding. it means. Thus, prophylactic administration of factor VIIa and factor XIII is included in the term “treatment”.

The term "subject" as used herein is intended to include any animal, especially a mammal, eg a human, and may be used interchangeably with the term "patient" as appropriate.

Abbreviation

TF tissue factor

FVII single-chain inactive factor VII

FVIIa active form factor VII

rFVIIa active recombinant factor VII

Inactive Factor XIII of FXIII Simogen

FXIIIa active form factor XIII

rFXIII recombinant FXIII

rFXIIIa Recombinant FXIIIa

alpha- or a-chain of a2 FXIII or rFXIII

b2 beta- or a-chain of FXIII or rFXIII

FXIIIa2 Dimer form of FXIII containing two a-chains

FXIIIa2b2 Tetrameric form of FXIII containing two a- and two b-chains

Inactive Factor VIII of FVIII Simogen

rFVIII recombinant FVIII

FVIIIa active form factor VIII

rFVIIIa Recombinant FVIIIa

TFPI Tissue Factor Pathway Inhibitors

Preparation of the compound

Purified human factor VIIa suitable for use in the present invention is preferably made by DNA recombination techniques, for example Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986 or in European Patent No. 200.421 (Gemogenetics Incorporated). Factor VIIa produced by recombinant techniques can be factor VIIa with a clear source or more or less modified Factor VIIa, provided that such factor VIIa is substantially equivalent to Factor VIIa (wild-type factor VIIa) with clear source for blood coagulation Have the same biological activity. Such modified factor VIIa is a nucleic acid sequence encoding wild type factor VII by known amino acids, for example by site-directed mutagenesis, by altering the amino acid codons in the nucleic acid encoding native factor VII or by removing a portion of the amino acid codons. Can be produced by modifying.

Factor VII also includes Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. Invest. 71: 1836-1841, 1983. These methods yield Factor VII with no detectable amount of other blood clotting factors. Even more purified Factor VII formulations can be obtained by including additional gel filtration as a final purification step. Factor VII is then converted to active factor VIIa by known means, for example several different plasma proteins such as factor XIIa, IXa or Xa. Or alternatively, factor VII is mono Q as described in Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565). Activated by passing it through an ion-exchange chromatography column such as (Pharmacia Fine Chemicals).

Factor XIII used within the present invention is described in Cooke and Holbrook (Biochem. J. 141: 79-84, 1974) and Curtis and Lorand (Methods Enzymol, 45: 177-191, 1976), which are incorporated herein by reference. It may be prepared from plasma according to known methods such as those. The a2-dimeric form of Factor XIII is disclosed in US Pat. No. 3,904,751; 3,931,399; It can be prepared from the placenta as disclosed in 4,597,899 and 4,285,933. However, it is desirable to use recombinant factor XIII to avoid the use of blood- or tissue-derived products that carry the risk of disease transmission. Methods of making 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. See 1989, 248: 105-110. In one embodiment, Factor XIII a2-dimer is prepared cytoplasmically in East Saccharomyces cerevisiae as disclosed in co-pending US patent application Ser. No. 07 / 741,263, which is incorporated herein by reference in its entirety. The cells are collected and lysed, and a clean cell lysate is prepared. Cell lysates are fractionated by anion exchange chromatography at neutral to slightly alkaline pH using a column of derived agarose, such as DEAE Fast-Flow Sepharose ^™ (Pharmacia). Factor XIII is then precipitated from the column eluate by concentrating the eluate and adjusting the pH to 5.2 to 5.5, for example by diafiltration against ammonium succinate buffer. The precipitate is then dissolved and further purified using conventional chromatography techniques such as gel filtration and hydrophobic interaction chromatography.

As will be appreciated by those in the art, it is preferred to use Factor XIII and Factor VIIa proteins, which are common genes with the subject, in order to reduce the risk of inducing an immune response. Preparation and characterization of non-human factor XIII was disclosed by Nakamura et al. (J. Biochem. 78: 1247-1266, 1975). The present invention also encompasses the use of such Factor XIII and Factor VIIa in veterinary procedures.

Administration and Pharmaceutical Compositions

For the treatment associated with an intentional intervention, Factor VII and Factor XIII will typically be administered within about 24 hours prior to performing the intervention and will then be administered for at least about 7 days. Administration as a coagulant may be by several routes described herein.

Doses of Factor VII range from about 0.05 mg to about 500 mg / day for 70 kg subjects as loading and maintenance doses, for example about 1 mg to about 200 mg / day, or for example about 10 mg to about 175 mg / day, This depends on the subject's weight, condition and severity of condition.

Doses of Factor XIII range from about 0.05 mg to about 500 mg / day for 70 kg subjects as loading and maintenance doses, for example about 1 mg to about 200 mg / day, or for example about 10 mg to about 175 mg / day, This depends on the subject's weight, condition and severity of condition.

The compositions and kits of the present invention are useful within the scope of human and veterinary medicine, for example in the treatment and prevention of subjects suffering from bleeding events or coagulation disorders. For use within the present invention, Factor VIIa and Factor XIII are optionally formulated with a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition may be administered parenterally, ie intravenously, subcutaneously or intramuscularly, or by continuous or pulsatile infusion.

Formulations include one or more diluents, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in the form of liquids, powders, emulsions, controlled release, and the like. One skilled in the art can formulate the compositions of the present invention in a suitable manner and according to accepted techniques such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA 1990. The composition for parenteral administration comprises Factor VII and Factor XIII in combination with a pharmaceutically acceptable carrier, preferably an aqueous carrier, preferably dissolved therein. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like. In addition, Factor VII variants of the invention may be formulated with liposome formulations to deliver to or target the wound site. Liposomal preparations are generally described, for example, in U.S. 4,837,028, U.S. 4,501,728 and U.S. 4,975,282.

Typical pharmaceutical compositions for intravenous infusion can be made to contain 250 ml of sterile Ringer's solution and 10 mg of Factor VIIa and / or Factor XIII. Actual methods of preparing parentally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in Remington's Pharmaceutical Sciences, 18th ed., Mack Pubilshing Company, Easton, PA (1990).

In brief, pharmaceutical compositions suitable for use according to the invention are made by mixing Factor VIIa, or Factor XIII, or Factor VIIa in combination with Factor XIII, in a purified form, with a suitable adjuvant and a suitable carrier or diluent. Suitable physiologically acceptable carriers or diluents include sterile water and saline. The composition may contain pH adjusting and buffering agents, stiffness adjusting agents, and the like, as required, to bring them closer to physiological conditions, such as pharmaceutically acceptable auxiliaries such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like. Can be. In addition, suitable adjuvants include calcium, proteins (eg albumin), or other inactive peptides (eg glycylglycine) or amino acids (eg glycine or) to stabilize purified Factor VIIa and / or Factor XIII. Histidine). Other physiologically acceptable adjuvants include non-reducing sugars, polyalcohols (eg sorbitol, mannitol or glycerol), polysaccharides such as low molecular weight dextrins, detergents (eg polysorbates) and antioxidants (eg Bisulfite and ascorbate). Adjuvants are generally present at a concentration of 0.001-4% w / v. In addition, the pharmaceutical compositions may contain protease inhibitors such as aprotinin or tranexamic acid, and preservatives. Moreover, the formulation may also contain TFPI inhibitors and / or factor VIII.

The composition can be sterilized by conventional well known sterilization techniques. The resulting aqueous solution is packaged for use or filtered and lyophilized in aseptic condition, and the lyophilized formulation is combined with a sterile aqueous solution prior to administration.

The concentration of Factor VIIa, Factor XIII or Factor VIIa in combination with Factor XIII in these preparations can vary widely, ie from less than about 0.5% by weight, usually about 1% or at least 1%, 15 or 20% by weight. Up to%, which will be selected primarily by fluid volume, viscosity, etc., depending on the particular mode of administration chosen.

Administration by injection or infusion is preferred, in particular by infusion. Thus, Factor VIIa and Factor XIII are dissolved lyophilized flours or liquid preparations containing both Factor VIIa and Factor XIII in one dosage form, or dissolved lyophilized flour containing Factor VIIa in one dosage form. Or in a form suitable for intravenous administration, such as a dissolved lyophilized flour or liquid formulation containing factor XIII in liquid formulations and other dosage forms.

Local delivery of Factor VIIa and Factor XIII, such as for example topical application, can be used for example spraying, perfusion, double-ply balloon catheter, stent, binding to vascular grafts or stents, hydrogels used to coat balloon catheter, Or by other well established methods. For walkable subjects requiring a daily maintenance level, Factor VIIa and Factor XIII can be administered by continuous infusion, for example using a portable pump system. In any event, the pharmaceutical composition should provide sufficient amounts of Factor VIIa and Factor XIII to effectively treat the subject.

The combination of Factor VIIa and Factor XIII shows synergistic effects in blood clotting rigidity- and fibrin lysis time-analysis in vitro. Moreover, the combination of Factor VIIa and Factor XIII has a synergistic effect in forming stable fibrin clots, increasing anti-blood clotting time, increasing blood clot strength, and increasing resistance to fibrin lysis.

Compositions containing Factor VII and Factor XIII may be administered for prophylactic and / or therapeutic treatment. In therapeutic use, the composition is 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. Suitable amounts to achieve this are defined as "effective amounts" or "therapeutically effective amounts". As will be appreciated by those skilled in the art, the effective amount for this purpose will depend on the severity of the disease or injury as well as the weight and general condition of the subject. It should be remembered that the materials of the present invention can generally be used in conditions of serious disease or injury, ie life-threatening or potentially life-threatening. In such a case, it is possible and desirable to administer substantially excess of these compositions by therapist in view of minimizing the general lack of immunity to factor VIIa and factor XIII in adventitious agents and humans.

In prophylactic use, compositions containing Factor VIIa and Factor XIII may be administered to a subject suspected of or otherwise at risk of a disease state or injury in order to enhance the subject's own coagulation ability. Such amount is defined as "prophylactically effective dose".

Single or multiple administrations of the composition can be performed using a dose level and pattern selected by the treating physician. The composition may be administered one or more times per day or week. An effective amount of such pharmaceutical composition is an amount that provides a clinically significant effect on the bleeding event. Such amount will depend in part on the particular condition, age, weight and general health of the subject to be treated, and other factors apparent to those skilled in the art.

The composition is generally administered in one single dose prior to expected bleeding or at the start of bleeding. However, depending on the given dose and the condition of the subject, it may be given in multiple doses, preferably at 2-4-6-12 hour intervals.

The composition may be in the form of a single-agent comprising both Factor VIIa and Factor XIII at suitable concentrations. The composition may also be in the form of a kit consisting of a first unit dosage form comprising Factor XIIa and a second unit dosage form comprising Factor XIII, and optionally further unit dosage forms comprising Factor VIII and / or TFPI inhibitor. Can be. In this case Factor VIIa and Factor XIII should be administered continuously, preferably within about 1 to 2 hours of each other, for example within 30 minutes of each other, preferably within 10 minutes of each other, or more preferably within 5 minutes.

Since the present invention relates to the prevention or treatment of bleeding events or coagulation treatment by treating with a combination of active ingredients which can be administered separately, the present invention also relates to combining the individual pharmaceutical compositions in kit form. The kit comprises at least two separate pharmaceutical compositions. The kit includes container means for containing individual compositions such as separate bottles or separate foil packages. Typically, the kit includes instructions for the administration of the individual components. Kit forms are particularly advantageous when the individual components are preferably administered in different dosage forms, when administered at different dosage intervals, or when the titer of the individual components of the combination is desired by the prescriber.

analysis

Factor VIIa Activity Tests:

Suitable assays for testing Factor VIIa activity to select suitable Factor VIIa variants can be performed by simple preliminary in vitro tests.

In vitro hydrolysis analysis

Native (wild-type) Factor VIIa and Factor VIIa variants (hereinafter all referred to as “Factor VIIa”) can be analyzed for specific activity. In addition, they can be analyzed in parallel to directly compare their specific activity. Assays are performed on microtiter plates (MaxiSorp, Nunk, Denmark). 50mM containing 0.1M NaCl, 5mM CaCl ₂ and 1mg / ml bovine serum albumin at a final concentration of 1mM of chromosome substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden) Hepes, added to Factor VIIa (final concentration 100 nM) in pH 7.4. Absorbance at 405 nm is measured continuously in a SpectraMax ^™ 340 plate reader (Mollicul Devices, USA). The absorbance developed during the 20 minute incubation was used to calculate the ratio of the activity of the variant and wild type factor VIIa after subtracting the absorbance of the enzyme-free blank wells:

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

Based on this, factor VIIa variants having a similar or higher activity than autogenous factor VIIa, for example, variants can be identified when the ratio of the activity of the variant shown in FIG. 1 to the activity of autogenous factor VII is near 1.0.

In addition, the activity of Factor VIIa or Factor VIIa variants may suitably be measured using a physiological substrate such as Factor X at a concentration of 100-1000 nM, in which case the resulting Factor Xa is a suitable chromogen substrate (e.g., Measured after addition of S-2765). In addition, activity assays can be performed at physiological temperatures.

In addition, the ability of Factor VIIa or Factor VIIa variants to produce thrombin can be measured in assays involving all relevant coagulation factors (subtract factor VIII if metastatic hemophilia A status) and inhibitors and activated platelets at physiological concentrations. (Described in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547, p 543, which is incorporated herein by reference).

Factor XIII Activity Tests:

Suitable assays for testing Factor XIII transglutaminase to select suitable Factor XIII variants are described, for example, in Mothods of Enxymology, Vol 45 (1976), Proteolytic Enzymes, Part B, pages 177-191 (Ed. Lorand, L.) It may be performed by a simple in vitro test described in.

The invention is further illustrated by the following examples, which however do not limit the scope of protection. The features disclosed in the foregoing specification and the following examples may be materials for realizing the invention in various forms, individually and in any combination thereof.

Example 1

Factor XIII Enhances Factor VIIa-Induced Fibrin Blood clot Formation

Citrated normal human plasma (NHP) is diluted 1/10 in a buffer containing ₂₀ nM Hepes, 150 mM NaCl, 5 mM CaCl ₂ , pH 7.4 in microtiter wells (250 μl total), and fibrin clot formation is Spectramax Monitored as an increase in optical density at 600 nm on ^TM 340 (Molecular Devices, Sunnyvale, California).

Spontaneous clot formation was obtained at about 2500-3000 seconds. 1 shows that 10 nM recombinant factor VIIa (rFVIIa) (Novo Nordisk A / S, Box Baert, Denmark) shortened the clotting time by 1600 seconds (n = 2). Further shortening of blood clotting time was obtained when 30 nM factor XIII (FXIII) (American Diagnostica Inc., Connecticut Greenwich) was added with 10 nM rFVIIa (n = 3). Blood clots formed in the presence of FXIII were more transparent (lower maximum OD) than in its absence, indicating that the addition of FXIII had a finer mesh fibrin gel structure with thinner fibers.

Example 2

The presence of supplemented Factor XIII during factor VIIa-induced clot formation increases resistance to fibrin lytic degradation

Fibrin blood clots composed of thin fibers are mechanically stronger and more difficult to break down than blood clots containing the same amount of fibrin arranged as thick fibers or less crosslinked fibers. The experiments shown in FIG. 2 showed that supplemented FXIII (30 nM) prolonged fibrin clot lysis time of blood clots formed in the presence of rFVIIa and tissue plasminogen activator (tPA, American Diagnostica). Blood clot formation was induced in the presence or absence of 30 nM FXIII by adding 25 μl of NHP to 20 nM Hepes, 150 mM NaCl, 5 mM CaCl ₂ , pH 7.4, 225 μl containing 50 nM rFVIIa and 0.5 nM recombinant tPA. Spectramax at 600 nm for clot formation and subsequent clot lysis induced by tPA-mediated plasminogen activation The increase in OD _600nm by 340 was followed by a return to trace levels of trace levels. 2 shows that under these conditions, the clot lysis time was significantly extended by the presence of FXIII.

Example 3

Factor VIIa in combination with Factor XIII increases maximal clot stiffness and increases blood clot resistance to fibrin lysis

Trombolastograph measurements are performed on citrated normal human plasma added 6nM recombinant tissue-type plasminogen activator (tPA, American Diagnostika) and at various concentrations of Factor XIII (FXIII, Haematologic Technologies, HCXIII- 0160, Lot N1212) was analyzed for the effect of 1 nM rFVIIa (Novo Nordisk A / S, Box Baerhard, Denmark) added in combination or alone. Clotting was initiated by the addition of Innovin (final concentration 2000 fold, Dade Behring # 526945) and calcium (final concentration 15 mM) in 20 mM Hepes, 150 mM NaCl, pH 7.4 buffer.

Trombolastograph measurements were used to analyze the effects of rFVIIa and FXIII on blood clot resistance to tPA mediated lysis as well as maximal blood clot rigidity (MCF). The MCF obtained before adding rFVIIa and / or FXIII was 25 mm and the time required for half the blood clot to dissolve was 12.3 minutes (FIG. 3). Addition with increasing concentration of FXIII (0-40 nM) did not alter MCF, but a dose-dependent prolongation of anti-blood lysis was observed and it was optimal at 30 nM FXIII (anti-blood lysis time: 14.3 min, FIG. 3). ). Similarly, addition of rFVIIa (1 nM) protected blood clots from tPA-mediated fibrin lysis without any effect on MCF (half-blood lysis time: 16.4 min) (FIG. 3). However, addition of rFVIIa (1 nM) with FXIII (30 nM) increased MCF (29 mm) as well as sufficient protection from fibrin lysis (anti-blood clotting time; 27.1 minutes) (FIG. 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 blood clots and resistance to tPA-mediated fibrin dissolution.

Claims (22)

A pharmaceutical composition comprising Factor VIIa and Factor XIII. The composition of claim 1, wherein Factor VIIa is Factor VII variant. The composition of claim 1, wherein factor VIIa is human factor VIIa. 4. The composition of claim 1 or 3, wherein factor VIIa and factor XIII are recombinant human factor VIIa and recombinant human factor XIII. 5. The composition of claim 1, wherein factor XIII is an a2-dimer of factor XIII. 6. The composition according to any one of claims 1 to 5, wherein factor XIII is active form factor XIII. The composition of any one of claims 1 to 6, wherein the composition further contains a TFPI inhibitor. 8. The composition of any one of the preceding claims, wherein the composition further contains factor VIII. a) an effective amount of Factor VIIa and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of Factor XIII and a pharmaceutically acceptable carrier in a second unit dosage form; And c) container means for containing said first and second dosage forms Kit containing a therapeutic agent for bleeding events comprising a. a) an effective amount of Factor VIIa and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of Factor XIII and a pharmaceutically acceptable carrier in a second unit dosage form; c) an effective amount of a TFPI inhibitor and a pharmaceutically acceptable carrier in a third unit dosage form; And d) container means for containing said first, second and third dosage forms Kit according to claim 9 comprising. a) an effective amount of Factor VIIa and TFPI inhibitor in a first unit dosage form, and a pharmaceutically acceptable carrier; b) an effective amount of Factor XIII and a pharmaceutically acceptable carrier in a second unit dosage form; And c) container means for containing said first and second dosage forms Kit containing a therapeutic agent for bleeding events comprising a. a) an effective amount of Factor VIIa and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of Factor XIII and TFPI inhibitor in a second unit dosage form, and a pharmaceutically acceptable carrier; And c) container means for containing said first and second dosage forms Kit containing a therapeutic agent for bleeding events comprising a. The method according to any one of claims 9 to 12, which is formulated in a separate unit dosage form or contained in a unit dosage form that also contains one or more of compounds selected from the list of Factor VIIa, Factor XIII or TFPI inhibitors. And further comprising factor VIII. Use of Factor VIIa in combination with Factor XIII for the manufacture of a medicament for treating a bleeding event in a subject. Use of Factor VIIa in Combination with Factor XIII for the manufacture of a medicament for shortening blood clotting time in a subject. Use of factor VIIa in combination with factor XIII for the manufacture of a medicament for prolonging blood clot lysis time in normal mammalian plasma. Use of Factor VIIa in combination with Factor XIII for the manufacture of a medicament for increasing blood clot strength in normal mammalian plasma. Use of factor VIIa in combination with factor XIII for the manufacture of a medicament for enhancing fibrin blood clot in normal human plasma. A method of enhancing fibrin blood clot formation in a subject comprising administering to the subject an effective amount of Factor VIIa in combination with an effective amount of Factor XIII. A method of treating a bleeding event in a subject comprising administering to the subject an effective amount of Factor VIIa in combination with an effective amount of Factor XIII. 21. The method of claim 19 or 20, wherein factor VIIa and factor XIII are administered in one dosage form. 21. The method of claim 19 or 20, wherein Factor VIIa and Factor XIII are administered sequentially.