AU2002340779B2 - Pharmaceutical composition comprising factor VII polypeptides and factor V polypeptides - Google Patents

Description

I- -I PHARMACEUTICAL COMPOSITION COMPRISING FACTOR VII POLYPEPTIDES AND FACTOR V POLYPEPTIDES tf FIELD OF THIS INVENTION The present invention relates to a pharmaceutical composition comprising factor VII or a factor VII-related polypeptide and factor V or a factor V-related polypeptide. The invention also relates to the use of a combination of factor VII or a factor VII-related Spolypeptide, and a factor V or a factor V-related polypeptide for the manufacture of a CNi medicament for treatment of subjects suffering from bleeding episodes, or prevention thereof. The invention also relates to a method for treatment of bleeding episodes in subjects and to a method for enhancing clot formation in a subject. The present invention also relates to kits comprising these compounds.

BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Haemostasis is initiated by the formation of a complex between tissue factor (TF) being exposed to the circulating blood following an injury to the vessel wall, and FVIIa which is present in the circulation in an amount corresponding to about 1% of the total FVII protein mass. This complex is anchored to the TF-bearing cell and activates FX into FXa and FIX into FIXa on the cell surface. FXa activates prothrombin to thrombin, which activates FVIII, FV, FXI and FXIII. Furthermore, the limited amount of thrombin formed in this initial step of haemostasis also activates the platelets. Following the action of thrombin on the platelets these change shape and expose charged phospholipids on their surface. This activated platelet surface forms the template for the further FX activation and the full thrombin generation. The further FX activation on the activated platelet surface occurs via a FIXa-FVIIIa complex formed on the surface of the activated platelet, and FXa then converts prothrombin into thrombin while still on the surface.

Thrombin then converts fibrinogen into fibrin which is insoluble and which stabilises the initial platelet plug. This process is compartmentalized, localised to the site of TF expression or exposure, thereby minimising the risk of a systematic activation of the lacoagulation system. The insoluble fibrin forming the plug is furthermore stabilised by FXIII-catalysed cross-linking of the fibrin fibres.

FVIIa exists in plasma mainly as a single-chain zymogen, which is cleaved by FXa into its two-chain, activated form, FVIIa. Recombinant activated factor Vila (rFVIIa) has been developed as a pro-haemostatic agent. The administration of rFVIIa offers a rapid and highly effective pro-haemostatic response in haemophilic subjects with bleedings that cannot be treated with coagulation factor products due to antibody formation. Also bleeding subjects with factor VII deficiency or subjects having a normal coagulation system but experiencing excessive bleeding can be treated successfully with FVIIa. In these studies, no unfavourable side effects of rFVIIa (in particular the occurrence of thromboembolism) have been encountered WO 03/039584 PCT/DK02/00736 2 Extra exogenously administered FVIla increases the formation of thrombin on the activated platelet surface. This occurs in haemophiliac subjects lacking FIX or FVIII and therefore missing the most potent pathway for full thrombin formation. Also in the presence of a lowered number of platelets or platelets with a defect function, extra FVIIla increases the thrombin formation.

Commercial preparations of recombinant human FVIla are sold as NovoSeven® (Novo Nordisk A/S,Denmark). Novoseven® is indicated for treatment of bleeding episodes in haemophilia A and B patients. Novoseven® is the only recombinant FVIIla available on the market for effective and reliable treatment of bleeding episodes.

Factor V is a large glycoprotein synthesized as a single chain molecule and circulates in blood as an inactive cofactor at a concentration of 30 nM. Approximately 25 of Factor V in blood is present in the a granules of platelets, whereas the rest is present in plasma. In the coagulation cascade Factor V acts as a cofactor for the serine protease activated factor X. Before it can act effectively as a cofactor, Factor V must be activated by limited proteolysis. Both factor Ila and Xa are able to do so. The first Factor V cDNA sequence was published by Kane et al. (PNAS 83:6800, 1986) but the complete genomic sequence is still not known. Factor V-deficiency is a rare recessive inherited disorder associated with a bleeding tendency due to loss-of-function mutations in the Factor V gene (Guasch et al., Thromb. Haemost. 77:252, 1997). Some discussion remains whether complete Factor V-deficiency is compatible with life. Published studies claim relatively mild bleeding tendencies in affected individuals. At the same time, the Factor V knockout mouse display a severe phenotype and approximately one-half do not make it past midgestation, whereas the remaining embryos come to term normally but die within hours after birth from excessive hemorrhage. In some cases, Factor V-deficiency co-inherits with factor VIIIdeficiency (Seligsohn et al., NEJM 307:1191, 1982). The most common genetic defect in Factor V is the so-called factor Vleiden mutation, which is associated with an increased thrombotic risk due to activated protein c resistance. This genetic abnormality is prevalent in approximately 3% of Caucasians, and numerous studies have shown factor Vleiden to be the most common genetic risk factor for thrombosis.

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 bleedings requiring the administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation defects, etc.) may lead to complications associated with the risk of transferring human viruses (hepatitis, HIV, parvovirus, and other, by now unknown viruses). Extensive bleedings requiring massive blood transfusions may lead to the development of multiple organ failure including impaired lung and kidney function. Once a subject has developed these serious complications a cascade of events involving a number of cytokines and inflammatory reactions is started WO 03/039584 PCT/DK02/00736 3 making any treatment extremely difficult and unfortunately often unsuccessful. Therefore a major goal in surgery as well as in the treatment of major tissue damage is to avoid or minimise the bleeding. To avoid or minimise such bleeding it is of importance to ensure the formation of stable and solid haemostatic plugs that are not easily dissolved by fibrinolytic enzymes. Furthermore, it is of importance to ensure quick and effective formation of such plugs or clots.

Today, subjects experiencing bleeding episodes, including trauma victims and subjects bleeding in association with surgery, are often treated with several injections or infusions of FVlla since the short half-life of FVIla (2.5 hours) may require more than one administration to maintain a certain level of haemostatic ability. A faster arrest of bleedings would be an important benefit to such subjects. So would a reduction in the number of administrations needed to stop bleeding and maintain haemostasis.

European Patent No. 225.160 (Novo Nordisk) concerns compositions of FVIIlla and methods for the treatment of bleeding disorders not caused by clotting factor defects or clotting factor inhibitors.

European Patent No. 82.182 (Baxter Travenol Lab.) concerns a composition of factor Vila for use in counteracting deficiencies of blood clotting factors or the effects of inhibitors to blood clotting factors in a subject.

International Patent Publication No. WO 93/06855 (Novo Nordisk) concerns the topical application of FVlla.

Cripe et al. (Biochemistry 31:3777, 1992), Jenny et al. (PNAS 84: 4846, 1987), Kane et al.

(Biochemistry 26: 6508, 1987), and Kane Davie (PNAS 83: 6800, 1986) concern the structure, amino acid sequence, and DNA encoding Factor V.

There is still a need in the art for improved treatment of subjects experiencing bleeding episodes, including subjects where the bleeding episodes are due to surgery, trauma, or other forms of tissue damage; induced coagulophathy, including coagulopathy in multi-transfused subjects; congenital or acquired coagulation or bleeding disorders, including diminished liver function ("liver disease"); defective platelet function or decreased platelet number; lacking or abnormal essential clotting "compounds" platelets or von Willebrand factor protein); increased fibrinolysis; anticoagulant therapy or thrombolytic therapy; or stem cell transplantation.

There remains a need in the art for an improved, reliable and widely applicable method of enhancing coagulation, enhancing or ensuring formation of stable haemostatic plugs, or enhancing convenience for the treated subject, or achieving full haemostasis in subjects, in particular in subjects having an impaired thrombin generation. There is also a need for methods wherein the time to bleeding arrest is shortened.

SUMMARY OF THE INVENTION The present invention relates to compositions, which can effectively be used in the treatment or prophylaxis of bleeding episodes and coagulation disorders.

The present invention also relates to compositions in single-unit dosage form, which can effectively be used in the treatment or prophylaxis of bleeding episodes or as a procoagulant. The present invention also relates to compositions, methods of treatment or kits exhibiting a synergistic effect.

The present invention further relates to compositions, methods of treatment or kits exhibiting no substantial side effects, such as a high level of systemic activation of the coagulation system.

l0 It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative In a first aspect the present invention provides a pharmaceutical composition comprising factor VII or a factor VII-related polypeptide, and factor V or a factor Vrelated polypeptide.

According to the second aspect the present invention provides a kit of parts when used in the treatment of bleeding episodes comprising; a) an effective amount of a preparation of factor VII or a factor VII-related polypeptide and a pharmaceutically acceptable carrier in a first-unit dosage form; b) an effective amount of a preparation of factor V or a factor V-related polypeptide and a pharmaceutically acceptable carrier in a second-unit dosage form; and c) container means for containing said first- and second-dosage form.

In one embodiment, the invention provides a kit of parts containing a treatment for bleeding episodes comprising; a) an effective amount of a preparation of factor VII or a factor VII-related polypeptide and a pharmaceutically acceptable carrier in a first-unit dosage form; b) an effective amount ofa preparation of factor V or a factor V-related polypeptide and a pharmaceutically acceptable carrier in a second-unit dosage form; and c) container means for containing said first- and second-dosage form.

In a third aspect, the invention provides the use of factor VII or a factor VII-related polypeptide in combination with a factor V or a factor V-related polypeptide for the manufacture of a medicament for treating bleeding episodes.

In a fourth aspect, the invention provides the use of a composition as described in any one of claims 1 to 19, for the manufacture of a medicament for treating bleeding episodes in a subject.

In different embodiments thereof, the medicaments are for reducing time needed to obtain full haemostasis, reducing time needed to maintain haemostasis, reducing clotting time, prolonging the clot lysis time, and increasing clot strength.

In different embodiments, the medicaments are for the treatment of subjects experiencing bleeding episodes due to surgery, trauma, or other forms of tissue damage; coagulophathy, including coagulopathy in multi-transfused subjects; congenital or acquired coagulation or bleeding disorders, including decreased liver function ("liver disease"); defective platelet function or decreased platelet number; lacking or abnormal essential clotting "compounds" platelets or von Willebrand factor protein); increased fibrinolysis; anticoagulation therapy or thrombolytic therapy; stem cell transplantation. In one series of embodiments, the bleedings occur in organs such as the brain, inner ear region, eyes, liver, lung, tumour tissue, gastrointestinal tract; in another series of embodiments, it is diffuse bleeding, such as haemorrhagic gastritis and profuse uterine bleeding. In another series of embodiments, the bleeding episodes are bleeding in connection with surgery or trauma in subjects having acute haemarthroses (bleedings in joints), chronic haemophilic arthropathy, haematomas, muscular, retroperitoneal, sublingual and retroharyngeal), bleedings in other tissue, haematuria (bleeding form the renal tract), cerebral haemorrhage, surgery hepatectomy), dental extraction, and gastrointestinal bleedings UGI bleeds). In one embodiment, the medicament is for treating bleeding episodes due to trauma, or surgery, or lowered count or activity of platelets, in a subject.

In a fifth aspect, the invention provides a method for treating bleeding episodes in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation.of factor VII or a factor VII-related polypeptide, and a second amount of preparation of factor V or a factor V-related polypeptide, wherein the first and second amount together are effective to treat bleedings.

-6- In a sixth aspect, the invention provides a method for reducing clotting time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide, and a second amount of a preparation of factor V or a factor V-related polypeptide where the first and second amount together are effective to reduce clotting time.

In a seventh aspect, the invention provides a method to enhance haemostasis in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide, and a second amount of preparation of factor V or a factor V-related polypeptide wherein the first and second amount together are effective to enhance haemostasis.

In an eighth aspect, the invention provides a method for prolonging the clot lysis time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide, and a second amount of preparation of factor V or a factor V-related polypeptide wherein the first and second amount together are effective to prolong the clot lysis time.

In a ninth aspect, the invention provides a method for increasing clot strength in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide, and a second amount of preparation of factor V or a factor V-related polypeptide wherein the first and second amount together are effective to increase clot strength.

In one series of embodiments of the methods, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are administered in singleunit dosage form.

In another series of embodiments the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are administered in the form of a firstunit dosage form comprising a preparation of factor VII or a factor VII-related polypeptide and a second-unit dosage form comprising a preparation of factor V or a factor V-related polypeptide. In a series of embodiments thereof, the first-unit dosage form and the second-unit dosage form are administered with a time separation of no more than 15 mins.

In a tenth aspect, the invention provides a kit containing a treatment for bleeding episodes comprising 6aa) An effective amount of factor VII or a factor VII-related polypeptide, and an effective amount of factor V or a factor V-related polypeptide, and a pharmaceutically acceptable carrier in a single-unit dosage form; and b) Container means for containing said single-unit dosage form.

According to the eleventh aspect, the present invention provides a method of treating bleeding episodes comprising the step of administering to a subject in need a composition according to the first aspect.

In one series of embodiments of the invention, the factor VII or factor VII-related polypeptide is a factor VII-related polypeptide. In one series of embodiments of the invention the factor VII-related polypeptide is a factor VII amino acid sequence variant.

In one embodiment the ratio between the activity of the factor VII-related polypeptide and the activity of the native human factor VIIa (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay" as described in the present description.

In one series of embodiments of the invention the factor VII or factor VII-related polypeptide is factor VII. In one embodiment said factor VII is human factor VII. In one embodiment the factor VII is bovine, porcine, canine, equine, murine or salmon factor VII. In another embodiment the factor VII is recombinantly made. In another embodiment the factor VII is derived from plasma. In a preferred embodiment the factor VII is recombinant human factor VII. In one series of embodiments of the invention the factor VII or factor VII-related polypeptide is in its activated form. In one preferred embodiment of the invention the factor VII is recombinant human factor VIIa.

In one series of embodiment the factor V or factor V-related polypeptide is a factor V-related polypeptide. In one embodiment the factor V-related polypeptide is a factor V amino acid-sequence variant. In one embodiment the ratio between the activity of said factor V-related polypeptide and the activity of native human plasma factor V (wild-type factor V) is at least about 1.25 when tested in the "factor V assay" as described in the present description. In one embodiment the factor V or factor V-related polypeptide is a factor V polypeptide. In one embodiment the factor V is human factor V. In one embodiment the factor V is bovine, porcine, canine, equine, murine, rat or salmon factor V. In a preferred embodiment the factor V is recombinantly made. In another embodiment the factor V is derived from plasma. In another embodiment the factor V is derived from platelets. In a preferred embodiment the factor V is recombi- WO 03/039584 PCT/DK02/00736 7 nant human plasma factor V. In another embodiment the factor V is human activated factor V (FVa). In one embodiment the factor V-related polypeptide is a fragment of factor V. In one embodiment the factor V-related polypeptide is a hybrid factor V polypeptide, a porcine/human hybrid.

In one embodiment the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII:factor V).

In one embodiment, the factor VII-related polypeptides are amino acid sequence variants having no more than 20 amino acids replaced, deleted or inserted compared to wildtype factor VII a polypeptide having the amino acid sequence disclosed in U.S. Patent No.

4,784,950), In another embodiment, the factor VII variants have no more than 15 amino acids replaced, deleted or inserted; in other embodiments, the factor VII variants have no more than amino acids, such as 8, 6, 5, or 3 amino acids, replaced, deleted or inserted compared to wildtype factor VII. In one embodiment, the factor VII variants are selected from the list of L305V- FVIIa, L305V/M306D/D309S-FVIla, L3051-FVIla, L305T-FVIla, F374P-FVIla, V158T/M298Q-FVIIa, V158D/E296V/M298Q-FVIla, K337A-FVIla, M298Q-FVIla, V158D/M298Q-FVIla, L305V/K337A-FVlla, V158D/E296V/M298Q/L305V-FVIla, V158D/E296V/M298Q/K337A-FVIla, V158D/E296VIM298Q/L305V/K337A-FVlla, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII In a further embodiment, the factor VII-related polypeptides have increased tissue factor-independent activity compared to native human coagulation factor Vlla. In another embodiment, the increased activity is not accompanied by changes in the substrate specificity. In another embodiment of the invention, the binding of the factor VII-related polypeptides to tissue factor are not impaired and the factor VII-related polypeptides have at least the activity of wild-type factor Vila when bound to tissue factor.

In a preferred embodiment, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are recombinant human factor Vila and recombinant human factor V. In another preferred embodiment, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are recombinant human factor Vila and recombinant human factor Va.

In one embodiment, the clotting time is reduced in mammalian blood. In another embodiment the haemostasis is enhanced in mammalian blood. In another embodiment the clot lysis time is prolonged in mammalian blood. In another embodiment the clot strength is increased in mammalian blood. In one embodiment, the mammalian blood is human blood. In another embodiment, the mammalian blood is normal human blood; in one embodiment, the blood is blood from a subject having an impaired thrombin generation. In one embodiment, the blood is blood from a subject h.aving a deficiency of one or more coagulation factors; in another WO 03/039584 PCT/DK02/00736 8 embodiment, the blood is blood from a subject having inhibitors against one or more coagulation factors; in one embodiment, the blood is from a subject having a lowered concentration of fibrinogen; in one embodiment, the blood is factor V-deficient human blood. In one series of embodiments, the blood is plasma.

In one embodiment of the invention, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are the sole haemostatic agents contained in the composition. In another embodiment, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are the sole active haemostatic agents contained in the composition. In another embodiment, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are the sole coagulation factors administered to the subject. In one embodiment of the invention, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are the sole active agents administered to the patient.

In one embodiment, the composition is substantially free of thrombin or prothrombin; in another embodiment, the composition is substantially free of FX; in another embodiment, the composition is substantially free of FXa.

In another embodiment, the pharmaceutical composition is formulated for intravenous administration, preferably injection or infusion, in particular injection. In one embodiment, the composition contains at least one pharmaceutical acceptable excipients or carrier.

In one embodiment of the invention, the composition is in single-unit dosage form wherein the single-unit dosage form contains both coagulation factors. In one embodiment of the invention, the composition is in the form of a kit-of-parts comprising a preparation of factor VII or a factor VII-related polypeptide as a first-unit dosage form and a preparation of factor V or a factor V-related polypeptide as a second-unit dosage form, and comprising container means for containing said first and second unit dosage forms. In one embodiment the composition or kit, as applicable, further contains directions for the administration of the composition or separate components, respectively.

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

In one embodiment of the invention, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are administered simultaneously. In another embodiment, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are administered sequentially. In one embodiment, the factor VII or factor VIIrelated polypeptide and the factor V or factor V-related polypeptide are administered with a WO 03/039584 PCT/DK02/00736 9 time separation of no more than 15 minutes, preferably 10, more preferred 5, more preferred 2 minutes. In one embodiment, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are administered with a time separation of up to 2 hours, preferably from 1 to 2 hours, more preferred up to 1 hour, more preferred from 30 minutes to 1 hour, more preferred up to 30 minutes, more preferred from 15 to 30 minutes.

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

In one embodiment the factor VII or factor VII-related polypeptide and factor V or factor V-related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII:factor V) In one embodiment of the present invention, the pharmaceutical composition is in single-unit dosage form and consists essentially of a preparation of factor VII or a factor VIIrelated polypeptide, and a preparation of factor V or a factor V-related polypeptide, and one or more of the components selected from the list of pharmaceutical acceptable carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and protease inhibitors.

In another embodiment of the present invention, the pharmaceutical composition is in the form of a kit-of-parts with the first-unit dosage form consisting essentially of a preparation of factor VII or a factor VII-related polypeptide, and one or more of the components selected from the list of pharmaceutical acceptable carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and protease inhibitors; and with the second-unit dosage form consisting essentially of a preparation of factor V or a factor V-related polypeptide and one or more of the components selected from the list of pharmaceutical acceptable carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives, and protease inhibitors.

In a further embodiment, the subject is a human; in another embodiment, the subject has an impaired thrombin generation; in one embodiment, the subject has a lowered plasma concentration of fibrinogen a multi-transfused subject); in one embodiment, the subject has a lowered plasma concentration of factor VIII or FIX.

In another aspect, the invention concerns a method to enhance haemostasis in a subject suffering from a factor VII responsive syndrome compared to when the subject is treated with factor VII as the only coagulation protein, the method comprising administering to the subject in need thereof a first amount of a preparation of factor VII or a factor Vll-related polypeptide, and a second amount of a preparation of factor V or a factor V-related polypeptide, wherein the first and second amounts together are effective to enhance haemostasis.

In another aspect, the invention concerns a method to enhance formation of thrombin in a subject, the method comprising administering to the subject in need thereof a first amount of A preparation of factor VII or a factor VII-related polypeptide and a second amount of a SA preparation of factor VI or a factor VI-related polypeptide, wherein he first and second amount of a Ctl ipreparation of factor V or a factor V-related polypeptide, wherein the first and second Samounts together are effective to enhance formation of thrombin.

SIn another aspect, the invention concerns a method to enhance formation of thrombin in a subject suffering from a factor VII responsive syndrome compared to when the subject is treated with factor VII as the only coagulation protein, the method comprising administering to the subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of rC factor V or a factor V-related polypeptide, wherein the first and second amounts together are effective to enhance formation of thrombin.

In another aspect, the invention concerns a method for reducing the number of administrations of coagulation factor protein needed to accomplish haemostasis in a subject suffering from a factor VII responsive syndrome compared to the number of administrations needed when factor VII is administered to the subject as the only coagulation factor protein, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of factor V or a factor V-related polypeptide, wherein the first and second amounts together are effective to reduce the number of administrations of coagulation factor protein.

In another aspect, the invention concerns a method of treating bleedings in a subject suffering from a factor VII responsive syndrome, the method comprising administering to the subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of factor V or a factor V-related polypeptide, wherein the first and second amounts together are effective in treating bleedings.

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

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

In another aspect, the invention relates to a method of enhancing fibrin clot formation in a subject, which method comprises administering to a subject in need lOathereof a first amount of a preparation of factor VII or a factor VII- related polypeptide and a second amount of a preparation of factor V or a factor V-related polypeptide, wherein the first and second amounts together are effective in treating bleedings.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

LIST OF FIGURES Figure 1: Addition of FVIIa results in a significant shortening of the clotting time, further shortened by the addition of FV. Addition of FV alone did not reduce the clotting time. WO 03/039584 PCT/DK02/00736 11 DETAILED DESCRIPTION OF THIS INVENTION Subjects, who bleed excessively in association with surgery or major trauma thus needing blood transfusions, develop more complications than those who do not experience any bleeding. However, also moderate bleedings may lead to complications if they require the administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation defects, etc.) because this is associated with the risk of transferring human viruses hepatitis, HIV, parvovirus, or other, by now unknown viruses) as well as non-viral pathogens. Extensive bleedings requiring massive blood transfusions may lead to the development of multiple organ failure including impaired lung and kidney function. Once a subject has developed these serious complications a cascade of events involving a number of cytokines and inflammatory reactions is started making any treatment extremely difficult and unfortunately often unsuccessful. A patient experiencing a major loss of blood becomes clinically unstable. Such patients are in risk of experiencing atrial fibrillation, which may lead to a fatal stop of cardiac activity; impaired renal function; or fluid extravasations in lungs (so-called "wet lungs" or ARDS). Therefore, a major goal in surgery as well as in the treatment of major tissue damage is to avoid or minimise the bleeding. To avoid or minimize such unwanted bleedings it is important to ensure formation of stable and solid haemostatic plugs that are not readily dissolved by fibrinolytic enzymes. Furthermore, it is of importance to ensure quick and effective formation of such plugs or clots.

Subjects with thrombocytopenia (lowered count or activity of platelets) also have an impaired thrombin generation as well as a defective stabilization of the fibrin plugs resulting in haemostatic plugs prone to premature dissolution. Furthermore, subjects subjected to major trauma or organ damage and who, as a consequence, have obtained frequent blood transfusions often have lowered platelet counts as well as lowered levels of fibrinogen, factor VIII, and other coagulation proteins. These subjects experience an impaired (or lowered) thrombin generation.

These subjects, therefore, have a defective, or less efficient, haemostasis leading to the formation of fibrin plugs that are easily and prematurely dissolved by proteolytic enzymes, such enzymes in addition being extensively released in situations characterized by extensive trauma and organ damage.

Bleedings in tissues may also lead to the formation of haematomas. The sizes of (in particular intercranial and spinal) haematomas are closely correlated to the extent of loss of neurological function, rehabilitation difficulties, and/or the severity and degree of permanent impairments of neurological function following rehabilitation. The most severe consequences of haematomas are seen when they are located in the brain where they may even lead to the death of the patient.

Thus, major objectives in treatment of bleedings are to obtain haemostasis in a minimum of time, thus keeping the blood loss at a minimum.

WO 03/039584 PCT/DK02/00736 12 The present invention thus provides beneficial compositions, uses and methods of treatment for treatment of bleeding episodes in subjects in need of such treatment. The compositions, uses and methods may be associated with beneficial effects such as less blood loss before haemostasis is obtained, less blood needed during surgery, blood pressure kept at an acceptable level until haemostasis is obtained, faster stabilisation of blood pressure, shorter recovery time for the treated patient, shorter rehabilitation time for the treated patient, diminished formation of haematomas or formation of smaller haematomas, including haematomas in the brain, faster arrest of bleedings, reduction in the number of administrations needed to stop bleeding and maintain haemostasis.

The administration of a preparation of factor VII or a factor VII-related polypeptide, factor Vila, in combination with a preparation of factor V or a factor V-related polypeptide provides a shortened clotting time, a firmer clot and an increased resistance to fibrinolysis compared to the clotting time, clot firmness and resistance when either factor Vila or factor V is administered alone.

The administration of a preparation of factor VII or a factor VII-related polypeptide, factor Vila, in combination with a preparation of factor V or a factor V-related polypeptide also provides for a reduced time to obtain bleeding arrest and a reduced number of administrations to maintain haemostasis compared to the situation when either factor Vila or factor V is administered alone. The present invention provides a beneficial effect of simultaneous or sequential dosing of a preparation of factor V or a factor V-related polypeptide and a preparation of factor VII or a factor VII-related polypeptide. The pharmaceutical composition according to the present invention may be in the form of a single composition or it may be in the form of a multi-component kit (kit-of-parts). The composition according to the present invention is useful as a therapeutic and prophylactic procoagulant in mammals, including primates such as humans.

The present invention further provides a method for treating (including prophylactically treating or preventing) bleeding episodes in a subject, including a human being.

Whenever, a first or second or third, etc., unit dose is mentioned throughout this specification this does not indicate the preferred order of administration, but is merely done for convenience purposes.

A combination of a preparation of factor VII or a factor VII-related polypeptide and a preparation of factor V or a factor V-related polypeptide is an advantageous product ensuring short clotting times, rapid formation of haemostatic plugs, and formation of stable haemostatic plugs. It has been found by the present inventor that a combination of factor VII or a factor VIIrelated polypeptide and a factor V or a factor V-related polypeptide is an advantageous product ensuring the formation of solid, stable and quickly formed haemostatic plugs.

The present inventors have shown that a combination of factor Vila and factor V can increase the firmness of the clot more effectively than either factor Vila or factor V alone. It has WO 03/039584 PCT/DK02/00736 13 also been shown that combination of factor VII or a factor VII-related polypeptide and a factor V can prolong the in vitro clot lysis time in normal human plasma more effectively than either factor Vila or factor V alone. It has also been shown that combination of factor VII or a factor VIIrelated polypeptide and a factor V can prolong the half-clot lysis time in normal human plasma more effectively than either factor Vila or factor V alone. It has also been shown that combination of factor VII or a factor VII-related polypeptide and a factor V can protect the clot from fibrinolysis, in particular tPA-mediated fibrinolysis, in normal human plasma more effectively than either factor Vila or factor V alone. Thus, by enhancing coagulation a more effective treatment of bleeding in subjects can be obtained.

Without wishing to be bound by theory, it is believed that the full thrombin generation is necessary for a solid, stabile haemostatic plug to be formed, and thereby for the maintenance of haemostasis. The fibrin structure of such a plug is dependent on both the amount of thrombin formed and the rate of the initial thrombin generation. In the presence of an impaired thrombin generation a porous fibrin plug, which is highly permeable, is being formed. The fibrinolytic enzymes normally present on the fibrin surface easily dissolve such a fibrin plug. The formation of a stable fibrin plug is also dependent on the presence of factor Xllla, which is being activated by thrombin and therefore also dependent on the full thrombin generation. Furthermore, the recently described thrombin activatable fibrinolytic inhibitor, TAFI, requires rather high thrombin amounts for its activation. In the presence of a not fully adequate thrombin formation the TAFI may therefore not be activated resulting in the formation of a haemostatic plug, which is easier than normally dissolved by the normal fibrinolytic activity. In situations with lowered number of platelets, thrombocytopenia, a faster thrombin generation is initiated by the administration of exogenous extra factor Vila. However, the total thrombin generation is not normalised by factor Vila even in high concentrations.

In subjects with lowered plasma concentrations of fibrinogen (multi-transfused subjects as a consequence of multiple trauma or extensive surgery) full thrombin activation does not occur. A more effective haemostasis is then obtained by the administration of a combination of factor VII or a factor Vll-related polypeptide, and a factor V.

Subjects with thrombocytopenia have an impaired thrombin generation as well as a defective stabilization of the fibrin plugs resulting in haemostatic plugs prone to premature dissolution. Furthermore, subjects subjected to major trauma or organ damage and who, as a consequence, have obtained frequent blood transfusions often have lowered platelet counts as well as lowered levels of fibrinogen, factor VIII, and other coagulation proteins. These subjects experience an impaired (or lowered) thrombin generation. In addition, their lowered fibrinogen levels interfere negatively with the activation of factor XIII. These subjects, therefore, have a defective, or less efficient, haemostasis leading to the formation of fibrin plugs which are easily and pre- WO 03/039584 PCT/DK02/00736 14 maturely dissolved by proteolytic enzymes, such enzymes in addition being extensively released in situations characterized by extensive trauma and organ damage.

In order to facilitate the formation of fully stabilized plugs with full capacity to maintain haemostasis in a subject, a composition according to the invention is administered. This composition is especially beneficial in subjects with a lowered number of platelets and in subjects with lowered plasma levels of fibrinogen and/or other coagulation proteins.

Factor VII Polypeptides: In practicing the present invention, any factor VII polypeptide may be used that is effective in preventing or treating bleeding. This includes factor VII polypeptides derived from blood or plasma, or produced by recombinant means.

The present invention encompasses factor VII polypeptides, such as, those having the amino acid sequence disclosed in U.S. Patent No. 4,784,950 (wild-type human factor VII). In some embodiments, the factor VII polypeptide is human factor Vila, as disclosed, in U.S.

Patent No. 4,784,950 (wild-type factor VII). In one series of embodiments, factor VII polypeptides include polypeptides that exhibit at least about 10%, preferably at least about 30%, more preferably at least about 50%, and most preferably at least about 70%, of the specific biological activity of human factor Vila. In one series of embodiments, factor VII polypeptides include polypeptides that exhibit at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least about 140%, and most preferably at least about 160%, of the specific biological activity of human factor Vila. In one series of embodiments, factor VII polypeptides include polypeptides that exhibit at least about 70 preferably at least about more preferably at least about 90 and most preferable at least about 95 of identity with the sequence of wild-type factor VII as disclosed in U.S. Patent No. 4,784,950.

As used herein, "factor VII polypeptide" encompasses, without limitation, factor VII, as well as factor VII-related polypeptides. The term "factor VII" is intended to encompass, without limitation, polypeptides having the amino acid sequence 1-406 of wild-type human factor VII (as disclosed in U.S. Patent No. 4,784,950), as well as wild-type factor VII derived from other species, such as, bovine, porcine, canine, murine, and salmon factor VII, said factor VII derived from blood or plasma, or produced by recombinant means. It further encompasses natural allelic variations of factor VII that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment. The term "factor VII" is also intended to encompass factor VII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated factor Vila. Typically, factor VII is cleaved between residues 152 and 153 to yield factor Vila.

WO 03/039584 PCT/DK02/00736 "Factor VII-related polypeptides" include, without limitation, factor VII polypeptides that have either been chemically modified relative to human factor VII and/or contain one or more amino acid sequence alterations relative to human factor VII factor VII variants), and/or contain truncated amino acid sequences relative to human factor VII factor VII fragments). Such factor VII-related polypeptides may exhibit different properties relative to human factor VII, including stability, phospholipid binding, altered specific activity, and the like.

The term "factor VII-related polypeptides" are intended to encompass such polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated "factor Vila-related polypeptides" or "activated factor VII-related polypeptides" As used herein, "factor VII-related polypeptides" encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wildtype human factor VII, as well as polypeptides in which the factor Vila biological activity has been substantially modified or reduced relative to the activity of wild-type human factor Vila.

These polypeptides include, without limitation, factor VII or factor Vila that has been chemically modified and factor VII variants into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.

It further encompasses polypeptides with a slightly modified amino acid sequence, for instance, polypeptides having a modified N-terminal end including N-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified relative to human factor Vila.

Factor VII-related polypeptides, including variants of factor VII, whether exhibiting substantially the same or better bioactivity than wild-type factor VII, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type factor VII, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wildtype factor VII by insertion, deletion, or substitution of one or more amino acids.

Factor VII-related polypeptides, including variants, encompass those that exhibit at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, or at least about 130%, of the specific activity of wild-type factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.

Factor VII-related polypeptides, including variants, having substantially the same or improved biological activity relative to wild-type factor Vila encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, WO 03/039584 PCT/DK02/00736 16 and most preferably at least about 130% of the specific activity of wild-type factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.

Factor VII-related polypeptides, including variants, having substantially reduced biological activity relative to wild-type factor Vila are those that exhibit less than about preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the specific activity of wild-type factor Vila that has been produced in the same cell type when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above, factor VII variants having a substantially modified biological activity relative to wild-type factor VII include, without limitation, factor VII variants that exhibit TFindependent factor X proteolytic activity and those that bind TF but do not cleave factor X.

In some embodiments the factor VII polypeptides are factor VII-related polypeptides, in particular variants, wherein the ratio between the activity of said factor VII polypeptide and the activity of native human factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay" (see "Assays", below); in other embodiments, the ratio is at least about in further embodiments, the ratio is at least about 4.0. In some embodiments of the invention, the factor VII polypeptides are factor VII-related polypeptides, in particular variants, wherein the ratio between the activity of said factor VII polypeptide and the activity of native human factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Proteolysis Assay" (see "Assays", below); in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0; in further embodiments, the ratio is at least about In some embodiments, the factor VII polypeptide is human factor VII, as disclosed, e.g., in U.S. Patent No. 4,784,950 (wild-type factor VII). In some embodiments, the factor VII polypeptide is human factor Vila. In one series of embodiments, the factor VII polypeptides are factor VII-related polypeptides that exhibits at least about 10%, preferably at least about more preferably at least about 50%, and most preferably at least about 70%, of the specific biological activity of human factor Vila. In some embodiments, the factor VII polypeptides have an amino acid sequence that differs from the sequence of wild-type factor VII by insertion, deletion, or substitution of one or more amino acids.

Non-limiting examples of factor VII variants having substantially the same or better biological activity compared to wild-type factor Vila include, but are not limited to, those described in Danish Patent Applications Nos. PA 2000 00734 and PA 2000 01360 (corresponding to WO 01/83725), and PA 2000 01361 (corresponding to WO 02/22776).Non-limiting examples of factor VII variants having substantially the same or improved biological activity as wild-type factor VII include S52A-FVII, S60A-FVII (lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); L305V-FVII, L305V/M306D/D309S-FVII, L3051-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, WO 03/039584 PCT/DK02/00736 17 V158D/E296V/M298Q-FVI I, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII; FVlla variants exhibiting increased proteolytic stability as disclosed in U.S. Patent No. 5,580,560; factor Vila that has been proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); and oxidized forms of factor Vila (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54, 1999). Non-limiting examples of factor VII variants having substantially reduced or modified biological activity relative to wild-type factor VII include R152E-FVIla (Wildgoose et al., Biochem 29:3413-3420, 1990), S344A-FVIIa (Kazama et al., J. Biol. Chem. 270:66-72, 1995), FFR-FVIla (Hoist et al., Eur. J. Vasc. Endovasc. Surg. 15:515-520, 1998), and factor Vila lacking the Gla domain, (Nicolaisen et al., FEBS Letts. 317:245-249, 1993).

Non-limiting examples of chemically modified factor VII polypeptides and sequence variants are described, in U.S. Patent No. 5,997,864.' The biological activity of factor Vila in blood clotting derives from its ability to bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of factor IX or factor X to produce activated factor IX or X (factor IXa or Xa, respectively).

For purposes of the invention, biological activity of factor VII polypeptides ("factor VII biological activity") may be quantified by measuring the ability of a preparation to promote blood clotting using factor VII-deficient plasma and thromboplastin, as described, in U.S.

Patent No. 5,997,864. In this assay, biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "factor VII units" by comparison with a pooled human serum standard containing 1 unit/ml factor VII activity. Alternatively, factor Vila biological activity may be quantified by Measuring the ability of factor Vila or a factor Vila -related polypeptide to produce activated factor X (factor Xa) in a system comprising TF embedded in a lipid membrane and factor X. (Persson et al., J. Biol. Chem. 272:19919-19924, 1997); (ii) Measuring factor X hydrolysis in an aqueous system ("In Vitro Proteolysis Assay", see below); (iii) Measuring the physical binding of factor Vila or a factor Vila -related polypeptide to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts.

413:359-363, 1997); and (iv) Measuring hydrolysis of a synthetic substrate by factor Vila and/or a factor Vila -related polypeptide ("In Vitro Hydrolysis Assay", see below); and Measuring generation of thrombin in a TF-independent in vitro system.

WO 03/039584 PCT/DK02/00736 18 The term "factor VII biological activity" or "factor VII activity" is intended to include 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.

A factor Vila preparation that may be used according to the invention is, without limitation, NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark).

Factor V polypeptides: The present invention encompasses Factor V polypeptides, such as, those having the amino acid sequence disclosed in Cripe et al. (Biochemistry 31:3777, 1992), Jenny et al. (PNAS 84:4846, 1987), Kane et al. (Biochemistry 26: 6508, 1987), and Kane Davie (PNAS 83: 6800, 1986) (wild-type human Factor V).

In practicing the present invention, any factor V polypeptide may be used that is effective in preventing or treating bleeding. This includes factor V polypeptides derived from blood or plasma, or produced by recombinant means.

As used herein, "factor V polypeptide" encompasses, without limitation, factor V, as well as factor V-related polypeptides. The term "factor V" is intended to encompass, without limitation, polypeptides having the amino acid sequence as described in Cripe et al. (Biochemistry 31:3777, 1992), Jenny et al. (PNAS 84: 4846, 1987), Kane et al. (Biochemistry 26: 6508, 1987), and Kane Davie (PNAS 83: 6800, 1986) (wild-type human factor as well as wild-type factor V derived from other species, such as, bovine, porcine, canine, murine, rat and salmon factor V. It further encompasses natural allelic variations of factor V that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment. The term "factor V" is also intended to encompass factor V polypeptides in their zymogen form, as well as those that have been processed to yield their respective bioactive forms.

"Factor V-related polypeptides" include, without limitation, factor V polypeptides that have either been chemically modified relative to human factor V and/or contain one or more amino acid sequence alterations relative to human factor V factor V variants), and/or contain truncated amino acid sequences relative to human factor V factor V fragments).

Such factor V-related polypeptides may exhibit different properties relative to human factor V, including stability, phospholipid binding, altered specific activity, and the like.

The term "factor V-related polypeptides" are intended to encompass such polypeptides in their zymogen form, as well as those that have been processed to yield their respective bioactive forms.

As used herein, "factor V-related polypeptides" encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild- WO 03/039584 PCT/DK02/00736 19 type human factor V, as well as polypeptides, in which the factor V biological activity has been substantially modified or reduced relative to the activity of wild-type human factor V. These polypeptides include, without limitation, factor V that has been chemically modified and factor V variants into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.

It further encompasses polypeptides with a slightly modified amino acid sequence, for instance, polypeptides having a modified N-terminal end including N-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified relative to human factor V.

Factor V-related polypeptides, including variants of factor V, whether exhibiting substantially the same or better bioactivity than wild-type factor V, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type factor V, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wildtype factor V by insertion, deletion, or substitution of one or more amino acids.

Factor V-related polypeptides, including variants, encompass those that exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, and at least about 130%, of the specific activity of wild-type factor V that has been produced in the same cell type, when tested in the factor V activity assay as described in the present specification.

Factor V-related polypeptides, including variants, having substantially the same or improved biological activity relative to wild-type factor V encompass those that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130% of the specific biological activity of wild-type human factor V that has been produced in the same cell type when tested in one or more of the specific factor V activity assay as described. For purposes of the invention, Factor V biological activity may be quantified by measuring the ability of a preparation to clot plasma, as described, in Thorelli et al., Thromb Haemost 80: 92, 1998. In this assay, biological activity is expressed as the reduction in clotting time relative to a control sample.

Factor V-related polypeptides, including variants, having substantially reduced biological activity relative to wild-type factor V are those that exhibit less than about preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the specific activity of wild-type factor V that has been produced in the same cell type when tested in one or more of the specific factor V activity assays as described above.

Non-limiting examples of Factor V equivalents include plasma-derived human Factor V as described, in Dahlback, J.Clin.lnvest. 66:583-591, 1980; Kane Majerus, J.Biol.Chem.

WO 03/039584 PCT/DK02/00736 256:1002-1007, 1981; or Katzmann et al., USA 78:162-166; recombinant human Factor V as described, in Kane Davie, USA 83:6800-6804, 1986 or Jenny et al., USA 84:4846-4850, 1987; platelet-derived Factor V as described, in Tracy et al., Blood 60:59-63, 1982 or in Tracy et al., J.Biol.Chem. 260:2119-2124, 1985, bovine Factor V as described, e.g. in Nesheim et al., J.Biol.Chem. 254:508-517,1979a or Esmon, J.Biol.Chem. 254:964-973, 1979.

In some embodiments the Factor V are Factor V equivalents wherein the ratio between the activity of said Factor V polypeptide and the activity of native human Factor V (wild-type Factor V) is at least about 1.25 when tested in the "factor V assay" (Thorelli et al, see above); in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about Factor V-related polypeptides also include fragments of factor V or factor V-related polypeptides retaining their characteristic haemostasis-related activity. The haemostasis-related activity of a factor V polypeptide may, for example, be measured using the factor V-activity assay described in the present specification.

Definitions 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 indicated explicitly, the amino acids mentioned herein are L-amino acids. It is to be understood, that the first letter in, for example, K337 represent the amino acid naturally present at the indicated position wild-type factor VII, and that, for example, [K337A]-FVIla designates the FVII-variant wherein the amino acid represented by the one-letter code K naturally present in the indicated position is replaced by the amino acid represented by the one-letter code A.

Table 1: Abbreviations for amino acids: Amino acid Tree-letter code One-letter code Glycine Gly G Proline Pro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine lie I Methionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Histidine His H Lysine Lys K Arginine Arg R Glutamine Gin Q Asparagine Asn N Glutamic Acid Glu E Aspartic Acid Asp D WO 03/039584 PCT/DK02/00736 21 The term "factor Vila" or "FVlla" may be used interchangeably.

In this context, "subjects with an impaired thrombin generation" means subjects who cannot generate a full thrombin burst on the activated platelet surface and includes subjects having a generation of thrombin less that the thrombin-generation in subjects having a fully functioning, normal haemostatic system, including a normal amount and function of coagulation factors, platelets and fibrinogen as in pooled, normal human plasma), and includes, without limitations, subjects lacking factor VIII; subjects having a lowered number of platelets or platelets with a defective function thrombocytopenia or thrombasthenia Glanzmann or subjects with excessive bleeds); subjects having lowered levels of prothrombin, FX or FVII; subjects having a lowered level of several coagulation factors due to exessive bleeding as a consequence of trauma or extensive surgery); and subjects with lowered plasma concentrations of fibrinogen multitransfused subjects).

By "level of thrombin generation" or "normal thrombin generation" is meant the level of the patient's level of thrombin generation compared to the level in healthy subjects. The level is designated as a percentage of the normal level. The terms may, where appropriate, be used interchangeably.

The term "enhancement of the haemostatic system" means an enhancement of the ability to generate thrombin. The term "enhancing haemostasis" is intended to encompass the situations when the measured thrombin generation for a test sample containing a preparation of factor VII or a factor VII-related polypeptide and a preparation of factor V or a factor V-related polypeptide is prolonged relative to the individual thrombin generation of a control sample containing only the factor VII or factor VII-related polypeptide or the factor V or factor V-related polypeptide, respectively, when tested in the same thrombin generation assay. The thrombin generation may be assayed as described in the thrombin generation assay of the present description (see "assay part").

"Sole" agents or factors as used herein refers to situations in which the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide, taken together, are the only haemostatic agents, or active haemostatic agents, or coagulation factors contained in the pharmaceutical composition or kit, or are the only haemostatic agents,or active haemostatic agents, or coagulation factors administered to the patient in the course of a particular treatment, such as, in the course of a particular bleeding episode. It will be understood that these situations encompass those in which other haemostatic agents or coagulation factors, as applicable, are not present in either sufficient quantity or activity so as to significantly influence one or more coagulation parameters.

Clot lysis time, clot strength, fibrin clot formation, and clotting time are clinical parameters used for assaying the status of patient's haemostatic system. Blood samples are drawn from the WO 03/039584 PCT/DK02/00736 22 patient at suitable intervals and one or more of the parameters are assayed by means of, e.g., thromboelastograpy as described by, Meh et al.,Blood Coagulation Fibrinolysis 2001;12:627- 637; Vig et al., Hematology, Vol. 6 pp. 205-213 (2001); Vig et al., Blood coagulation fibrinolysis, Vol. 12 pp. 555-561 (2001) Oct; Glidden et al., Clinical and applied thrombosis/hemostasis, Vol. 6 pp. 226-233 (2000) Oct; McKenzie et al., Cardiology, Vol. 92 pp. 240-247 (1999) Apr; or Davis et al., Journal of the American Society of Nephrology, Vol. 6 pp. 1250-1255 (1995).

The term "prolonging clot lysis time" is intended to encompass the situations when the measured clot lysis time for a test sample containing a preparation of factor VII or a factor VIIrelated polypeptide and a preparation of factor V or a factor V-related polypeptide is prolonged relative to the individual clot lysis time of a control sample containing only the factor VII or factor VII-related polypeptide or the factor V or factor V-related polypeptide, respectively, when tested in the same clot lysis assay. The clot lysis time may be assayed as described above.

The term "increasing clot strength" is intended to encompass the situations when the measured clot strength, mechanical strength, for a test sample containing a preparation of factor VII or a factor VII-related polypeptide and a preparation of factor V or a factor V-related polypeptide is increased relative to the individual clot lysis time of a control sample containing only the factor VII or factor VII-related polypeptide or the factor V or factor V-related polypeptide, respectively, when tested in the same clot strength assay. The clot strength may be assayed as described, e.g. in Carr et al, 1991. (Carr ME, Zekert SL. Measurement of platelet-mediated force development during plasma clot formation. AM J MED SCI 1991; 302: 13-8), or as described above by means of thromboelastography.

The term "enhancing fibrin clot formation" is intended to encompass the situations when the measured rate for or degree of fibrin clot formation for a test sample containing a preparation of factor VII or a factor VII-related polypeptide and a preparation of a preparation of factor V or a factor V-related polypeptide is increased relative to the individual rate for or degree of fibrin clot formation of a control sample containing only the factor VII or factor VII-related polypeptide or the factor V or factor V-related polypeptide, respectively, when tested in the same clotting assay. The fibrin clot formation may be assayed as described above.

The term "shortening clotting time" is intended to encompass the situations when the measured time for clot formation (clotting time) for a test sample containing a preparation of factor VII or a factor VII-related polypeptide and a preparation of a preparation of factor V or a factor V-related polypeptide is increased relative to the individual clotting time of a control sample containing only the factor VII or factor VII-related polypeptide or the factor V or factor V-related polypeptide respectively, when tested in the same clotting assay. The clotting time may be assayed by means of standard PT og aPTT assays, which are known to the general skilled person.

The term "lowered count or activity of platelets" refers to the number of platelets (thrombocytes) present in the subject's plasma and to the biological, coagulation-related activity of such WO 03/039584 PCT/DK02/00736 23 platelets. Lowered counts may be due, to increased platelet destruction, decreased platelet production, and pooling of a larger than normal fraction of platelets in the spleen. Thrombocytopenia, for example, is defined as a platelet count less than 150,000 platelets per microliter; the upper limit of the normal platelet count is generally considered to be between 350,000 and 450,000 platelets per microliter. Platelet count may be measured by automated platelet counters; this is a well known method to the skilled worker. Syndromes due to lowered platelet count include, without limitation, thrombocytopenia, coagulophathy. "Activity" includes, without limitation, aggregation, adhesion, and coagulant activity of the platelets. Decreased activity may be due, to glycoprotein abnormalities, abnormal membrane-cytoskeleton interaction, abnormalities of platelet granules, abnormalities of platelet coagulant activity, abnormalities of signal transduction and secretion. Platelet activity, including aggregation, adhesion, and coagulant activity, are measured by standard methods known to the skilled worker, see e.g.,Platelets. A Practical Approach, Ed. S.P.

Watson K.S. Authi: Clinical Aspects of Platelet Disorders Clemetson) 15:299-318, 1996, Oxford University Press; Williams Hematology, Sixth Edition, Eds. Beutler, Lichtman, Coller, Kipps Seligsohn, 2001, McGraw-Hill. Syndromes due to lowered platelet activity includes, without limitaion, Glanzmann thrombathenis, Bernard-Soulier syndrome, anticoagulant treatment and thrombolytic treatment. "Lowered" refers to the count or activity of a sample of the test plasma compared to the count or activity in a sample of normal pooled plasma when measured in the same assay As used herein the term "bleeding disorder" reflects any defect, congenital, acquired or induced, of cellular or molecular origin that is manifested in bleeding episodes. Examples of bleeding disorders include, but are not limited to, clotting factor deficiencies deficiency of coagulation factors VIII, IX, XI or VII), clotting factor inhibitors, defective platelet function Glanzmann thombasthenia and Bernard-Soulier syndrome), thrombocytopenia, von Willebrand's disease, and coagulophathy such as that caused by a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to bleedings and/or transfusions in multi transfused subjects having been subjected to surgery or trauma).

Bleeding refers to extravasation of blood from any component of the circulatory system.

The term "bleeding episodes" is meant to include unwanted, uncontrolled and often excessive bleeding in connection with surgery, trauma, or other forms of tissue damage, as well as unwanted bleedings in subjects having bleeding disorders. Bleeding episodes may occur in subjects having a basically normal coagulation system but experiencing a (temporary) coagulophathy, as well as in subjects having congenital or acquired coagulation or bleeding disorders. In subjects having a defective platelet function, the bleedings may be likened to bleedings caused by haemophilia because the haemostatic system, as in haemophilia, lacks or has abnormal essential clotting "compounds" platelets or von Willebrand factor protein). In subjects who experience extensive tissue damage, for example in association with surgery or vast trauma, the normal haemostatic mechanism may be overwhelmed by the demand of immediate haemostasis and WO 03/039584 PCT/DK02/00736 24 they may develop excessive bleeding in spite of a basically (pre-trauma or pre-surgery) normal haemostatic mechanism. Such subjects, who further often are multi transfused, develop a (temporary) coagulopathy as a result of the bleeding and/or transfusions a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to the bleeding and/or transfusions). Bleedings may also occur in organs such as the brain, inner ear region and eyes; these are areas with limited possibilities for surgical haemostasis and thus problems with achieving satisfactory haemostasis. Similar problems may arise in the process of taking biopsies from various organs (liver, lung, tumour tissue, gastrointestinal tract) as well as in laparoscopic surgery and radical retropubic prostatectomy. Common for all these situations is the difficulty to provide haemostasis by surgical techniques (sutures, clips, etc.) which also is the case when bleeding is diffuse haemorrhagic gastritis and profuse uterine bleeding). Bleedings may also occur in subjects on anticoagulant therapy in whom a defective haemostasis has been induced by the therapy given; these bleedings are often acute and profuse. Anticoagulant therapy is often given 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 platelet aggregation inhibitors, such as, antibodies or other inhibitors of GP llb/Illa activity. The bleeding may also be due to so-called thrombolytic therapy which comprises combined treatment with an antiplatelet agent acetylsalicylic acid), an anticoagulant heparin), and a fibrinolytic agent tissue plasminogen activator, tPA). Bleeding episodes are also meant to include, without limitation, uncontrolled and excessive bleeding in connection with surgery or trauma in subjects having acute haemarthroses (bleedings in joints), chronic haemophilic arthropathy, haematomas, muscular, retroperitoneal, sublingual and retropharyngeal), bleedings in other tissue, haematuria (bleeding from the renal tract), cerebral haemorrhage, surgery hepatectomy), dental extraction, and gastrointestinal bleedings UGI bleeds).

The bleeding episodes may be associated with inhibitors against factor VIII; haemophilia A; haemophilia A with inhibitors; haemophilia B; deficiency of factor VII; deficiency of factor V; thrombocytopenia; deficiency of von Willebrand factor (von Willebrand's disease); severe tissue damage; severe trauma; surgery; laparoscopic surgery; haemorrhagic gastritis; taking biopsies; anticoagulant therapy; upper gastroentestinal bleedings (UGI); or stem cell transplantation. The bleeding episodes may be profuse uterine bleeding; occurring in organs with a limited possibility for mechanical haemostasis; occurring in the brain; occurring in the inner ear region; or occurring in the eyes. The terms "bleeding episodes" and "bleedings" may, where appropriate, be used interchangeably.

In this context, the term "treatment" is meant to include both prevention of an expected bleeding, such as, for example, in surgery, and regulation of an already occurring bleeding, such as, for example, in trauma, with the purpose of inhibiting or minimising the bleeding.

The above-referenced "expected bleeding" may be a bleeding expected to occur in a particular WO 03/039584 PCT/DK02/00736 tissue or organ, or it may be an unspecified bleeding. Prophylactic administration of a preparation of factor VII or a factor VII-related polypeptide and a preparation of factor V or a factor Vrelated polypeptide is thus included in the term "treatment".

The term "subject" as used herein is intended to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the term "patient".

The present invention also encompasses the use of factor VII or FVII-related polypeptides, and tPA inh'ibitors within veterinary procedures.

The factor VII or factor VII-related polypeptides and factor V or factor V-related polypeptides as defined in the present specification may be administered simultaneously or sequentially. The factors may be supplied in single-dosage form wherein the single-dosage form contains both coagulation factors, or in the form of a kit-of-parts comprising a preparation of factor VII or a factor VII-related polypeptide as a first unit dosage form and a preparation of factor V or a factor V-related polypeptide as a second unit dosage form. Whenever a first or second or third, etc., unit dose is mentioned throughout this specification this does not indicate the preferred order of administration, but is merely done for convenience purposes By "simultaneous" dosing of a preparation of factor VII or a factor VII-related polypeptide and a preparation of factor V or a factor V-related polypeptide is meant administration of the coagulation factor proteins in single-dosage form, or administration of a first coagulation factor protein followed by administration of a second coagulation factor protein with a time separation of no morethan 15 minutes, preferably 10, more preferred 5, more preferred 2 minutes. Either factor may be administered first.

By "sequential" dosing is meant administration of a first coagulation factor protein followed by administration of a second coagulation factor protein with a time separation of up to 2 hours, preferably from 1 to 2 hours, more preferred up to 1 hour, more preferred from minutes to 1 hour, more preferred up to 30 minutes, more preferred from 15 to 30 minutes.

Either of the two unit dosage form, or coagulation factor proteins, may be administered first.

Preferably, both products are injected through the same intravenous access.

By "level of factor V" or "factor V level" is meant the level of the patient's factor V activity compared to the level in healthy subjects. The level is designated as a percentage of the normal level. The terms may, where appropriate, be used interchangeably.

By "reduced level of factor V" or "reduced factor V level" is meant a decrease in the presence or activity of factor V in the blood stream compared to the mean factor V level in a population of subjects having no factor V deficiency or inhibitors to factor V. The level of circulating factor V can be measured by either a coagulant or an immunologic assay. Factor V activity is determined by the ability of the patient's plasma to correct the clotting time of factor Vdeficient plasma an APTT assay, see below; see also "assay part" of the present description).

WO 03/039584 PCT/DK02/00736 26 One unit of factor V has been defined as the amount of factor V present in one millilitre of normal (pooled) human plasma (corresponding to a factor V level of 100 One unit of factor VII is defined as the amount of factor VII present in 1 ml of normal plasma, corresponding to about 0.5 |ig protein. After activation 50 units correspond to about 1 gg protein.

By "deficiency" is meant a decrease in the presence or activity of, factor V in plasma compared to that of normal healthy individuals. The term may, where appropriate, be used interchangeably with "reduced factor V level".

By "APTT" or "aPTT" is meant the activated partial thromboplastin time (described by, Proctor RR, Rapaport SI: The partial thromboplastin time with kaolin; a simple screening test for first-stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212, 1961).

By "factor V-responsive syndrome" is meant a syndrome where exogenous factor V administered to the subject in need thereof may prevent, cure or ameliorate any symptoms, conditions or diseases, expected or present, caused by the syndrome. Included are, without limitation, syndromes caused by a reduced level of factor V, bleeding disorders caused by inhibitors to factor V. A factor V-responsive syndrome may also be treated with a composition according to the present invention.

By "factor VII-responsive syndrome" is meant a syndrome where exogenous factor VII, preferably factor Vila, administered to the subject in need thereof may prevent, cure or ameliorate any symptoms, conditions or diseases, expected or present, caused by the syndrome. Included are, without limitation, syndromes caused by a reduced level of clotting factors VIII, IX, XI or VII, clotting factor inhibitors, defective platelet function Glanzmann thombasthenia and Bernard-Soulier syndrome), thrombocytopenia, von Willebrand's disease, and coagulophathy such as that caused by a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to bleedings and/or transfusions in multi transfused subjects having been subjected to surgery or trauma).

"Half-life" refers to the time required for the plasma concentration of factor VII or a factor VII-related polypeptide, or factor V or a factor V-related polypeptide to decrease from a particular value to half of that value.

By "primary haemostasis" is meant the initial generation of thrombin by FXa and TF:factor Vila, the subsequent activation of platelets and formation of the initial loose plug of activated, adhered platelets which has not yet been stabilized by fibrin and, finally, by crosslinked fibrin. If not stabilized by the fibrin formed during the second step of the haemostatic process (maintained haemostasis), the plug is easily dissolved by the fibrinolytic system.

By "secondary haemostasis" or "maintained haemostasis" is meant the secondary, full, and major, burst or generation of thrombin taking place on the surface of activated platelets WO 03/039584 PCT/DK02/00736 27 and catalysed by factor Villa and factor Villa, the subsequent formation of fibrin and the stabilization of the initial platelet plug. Stabilization of the plug by fibrin leads to full haemostasis.

By "full haemostasis" is meant the formation of a stable and solid fibrin clot or plug at the site of injury which effectively stops the bleeding and which is not readily dissolved by the fibrinolytic system. In this context, the term haemostasis will be used to represent full haemostasis as described above.

The total amount of protein in a preparation may be measured by generally known methods, e.g, by measuring optical density. Amounts of factor V- or factor VII protein ("antigen") may be measured by generally known methods such as standard Elisa immuno assays. In general terms, such assay is conducted by contacting, a solution of the factor V proteincontaining preparation with an anti-thromobomodulin antibody immobilised onto the elisa plate, subsequently contacting the immobilised antibody-factor V complex with a second antifactor V antibody carrying a marker, the amounts of which, in a third step, are measured. The amounts of each coagulation factor may be measured in a similar way using appropriate antibodies. The total amount of coagulation factor protein present in a preparation is determined by adding the amounts of the individual coagulation factor proteins. In one embodiment, the preparation comprises isolated coagulation factor. In another embodiment the preparation is essentially free of coagulation factor II and coagulation factor Ila (prothrombin and thrombin) and/or factor X or Xa.

As used herein, the term "isolated" refers to coagulation factors, factor V or factor V-related polypeptides that have been separated from the cell in which they were synthesized or the medium in which they are found in nature plasma or blood). Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like. Separation of polypeptides from the medium in which they naturally occur may be achieved by any method known in the art, including, without limitation, affinity chromatography, such as, on an anti-factor VII or anti-factor V antibody column, respectively; hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures preparative isoelectric focusing differential solubility ammonium sulfate precipitation), or extraction and the like.

Within the present invention an "effective amount" of factor VII or a factor VII-related polypeptide, and factor V or a factor V-related polypeptide is defined as the amount of factor VII or a factor VII-related polypeptide, FVlla, and factor V or a factor V-related polypeptide, that together suffices to prevent or reduce bleeding or blood loss, so as to cure, alleviate or partially arrest the disease and its complications.

WO 03/039584 PCT/DK02/00736 28 The term "activity of factor Vila" or "factor Vila-activity" includes 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.

Abbreviations TF tissue factor FVII factor VII in its single-chain, unactivated form FVlla factor VII in its activated form rFVlla recombinant factor VII in its activated form TAFI TAFI in its zymogenic, unactivated form Factor V Factor V in its zymogenic, unactivated form Factor Va Factor V in its activated form rFactor V recombinant Factor V rFactor Va recombinant Factor Va Preparation of compounds: Human purified factor Vila suitable for use in the present invention is preferably made by DNA recombinant technology, e.g. as described by Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2412- 2416, 1986, or as described in European Patent No. 200.421 (ZymoGenetics, Inc.).

Factor VII may also be produced by the methods described by Broze and Majerus, J.Biol.Chem. 255 1242-1247, 1980 and Hedner and Kisiel, J.Clin.lnvest. 71: 1836-1841, 1983. These methods yield factor VII without detectable amounts of other blood coagulation factors. An even further purified factor VII preparation may be obtained by including an additional gel filtration as the final purification step. factor VII is then converted into activated factor Vila by known means, e.g. by several different plasma proteins, such as factor Via, IX a or Xa. Alternatively, as described by Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565), factor VII may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia fine Chemicals) or the like.

Factor VII -related polypeptides may produced by modification of wild-type factor VII or by recombinant technology, factor VII -related polypeptides with altered amino acid sequence when compared to wild-type factor VII may be produced by modifying the nucleic acid sequence encoding wild-type factor VII either by altering the amino acid codons or by removal of some of the amino acid codons in the nucleic acid encoding the natural factor VII by known means, e.g. by site-specific mutagenesis.

It will be apparent to those skilled in the art that substitutions can be made outside the regions critical to the function of the factor Vila or factor V-molecule and still result in an active WO 03/039584 PCT/DK02/00736 29 polypeptide. Amino acid residues essential to the activity of the factor VII or factor Vll-related polypeptide or factor V or factor V-related polypeptide, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for coagulant, respectively cross-linking activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the threedimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, de Vos etal., 1992, Science 255: 306-312; Smith et 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure that utilizes a super coiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with Dpnl, which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA. Other procedures known in the art for creating, identifying and isolating variants may also be used, such as, for example, gene shuffling or phage display techniques.

Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove nonadherent cells; and the like.

Optionally, factor VII or factor VII-related polypeptides may be further purified. Purification may be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, on an anti-factor VII antibody column (see, Wakabayashi et al., J. Biol. Chem. 261:11097, 1986; and Thim etal., Biochem. 27:7785, 1988); hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures preparative isoelectric focusing (IEF), differential solubility ammonium sulfate precipitation), or extraction and the like. See, generally, Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, J.C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. Following purification, the preparation preferably contains less than about 10% by weight, more preferably less than about 5% and most preferably WO 03/039584 PCT/DK02/00736 less than about of non-factor VII or factor VII-related polypeptides derived from the host cell.

Factor VII or factor VI I-related polypeptides may be activated by proteolytic cleavage, using factor Via or other proteases having trypsin-like specificity, such as, factor IXa, kallikrein, factor Xa, and thrombin. See, Osterud et al., Biochem. 11:2853 (1972); Thomas, U.S.

Patent No. 4,456,591; and Hedner et al., J. Clin. Invest. 71:1836 (1983). Alternatively, factor VII or factor VII-related polypeptides may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia) or the like. The resulting activated factor VII or factor VII-related polypeptide may then be formulated and administered as described below.

Factor V for use within the present invention may be isolated from plasma according to known methods, such as those disclosed by Katzmann et al., Proc. Natl. Acad. Sci. USA 78: 162, 1981. It is preferred, however, to use recombinant Factor V so as to avoid to the use of blood- or tissue-derived products that carry a risk of disease transmission. Methods for preparing recombinant Factor V are known in the art; see, for example, Kane Davie, Proc. Natl. Acad. Sci. U.S.A.

83: 6800, 1986. Activated Factor V is described in, for example, Esmon, J.Biol.Chem. 254: 964-973, 1979; Nesheim, J.Biol.Chem. 254:1326-1334, 1979; Suzuki et al., J.Biol.Chem. 257:6556-6564,1982; Nesheim et al, J.Biol.Chem. 259:3187-3196, 1984a; and Jenny et al., USA 84:4846- 4850,1987. Factor V variants may be produced by means of site-directed mutagenesis as described above.

Factor V -related polypeptides may produced by modification of wild-type factor V or by recombinant technology, factor V -related polypeptides with altered amino acid sequence when compared to wild-type factor V may be produced by modifying the nucleic acid sequence encoding wild-type factor V either by altering the amino acid codons or by removal of some of the amino acid codons in the nucleic acid encoding the natural factor V by known means, e.g. by site-specific mutagenesis, as described in more detail above. Separation of polypeptides from their cell of origin may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like. Optionally, factor V or factor V-related polypeptides may be further purified. Purification may be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, on an anti-factor V antibody column; hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures preparative isoelectric focusing (IEF), differential solubility ammonium sulfate precipitation), or extraction and the like, as described in more detail above. Following purification, the preparation preferably contains less than about 10% by weight, more preferably less than about 5% and most preferably less than about of non-factor V or factor V-related polypeptides derived from the host cell. The result- WO 03/039584 PCT/DK02/00736 31 ing activated factor V or factor V-related polypeptide may then be formulated and administered as described below.

As will be appreciated by those skilled in the art, it is preferred to use Factor V polypeptides and Factor VII polypeptides syngeneic with the subject in order to reduce the risk of inducing an immune response. Preparation and characterization of non-human Factor V has been disclosed by, for example, Katzmann et al., Proc. Natl. Acad. Sci. USA, 78: 162, 1981. The present invention also encompasses the use of such Factor V and factor Vila proteins within veterinary procedures.

Pharmaceutical Compositions and Methods of Use The preparations of the present invention may be used to treat any factor VII responsive syndrome, such as, bleeding disorders, including, without limitation, syndromes caused by a reduced level of clotting factors VIII, IX, XI or VII, clotting factor inhibitors, defective platelet function Glanzmann thombasthenia and Bernard-Soulier syndrome), thrombocytopenia, von Willebrand's disease, and coagulophathy such as that caused by a dilution of coagulation proteins, increased fibrinolysis and lowered number of platelets due to bleedings and/or transfusions in multi transfused subjects having been subjected to surgery or trauma).

Pharmaceutical compositions comprising a preparation of factor VII or a factor VII-related polypeptide and a preparation of factor V or a factor V-related polypeptide according to the present invention are primarily intended for parenteral administration for prophylactic and/or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenterally, intravenously, subcutaneously, or intramuscularly; intravenously being most preferred. They may also be administered by continuous or pulsatile infusion.

Pharmaceutical compositions or formulations according to the invention comprise a factor VII or a factor VII-related polypeptide, and factor V or a factor V-related polypeptide, either formulated in a single-unit dosage form or in the form of a kit-of parts, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent. Briefly, pharmaceutical compositions suitable for use according to the present invention is made by mixing factor VII or a factor VII-related polypeptide, or a factor V, or factor VII or a factor VII-related polypeptide in combination with a factor V, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent. A variety of aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like. The preparations of the invention can also be formulated using non-aqueous carriers, such as, in the form of a gel or as liposome preparations for delivery or targeting to the sites of injury. Liposome preparations are generally described in, U.S. Patents Nos. 4,837,028, 4,501,728, and 4,975,282. The compositions may be sterilised by conventional, well-known sterilisation techniques. The resulting aqueous solutions may WO 03/039584 PCT/DK02/00736 32 be packaged for use or filtered under aseptic conditions and lyophilised, the lyophilised preparation being combined with a sterile aqueous solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliary substances or adjuvants, including, without limitation, pH adjusting and buffering agents and/or tonicity adjusting agents, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

Formulations may further include one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, controlled release, etc. One skilled in this art may formulate the compositions of the invention an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990.

Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution and 10 mg of the preparation.

The compositions containing the preparations of the present invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, 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 clinical manifestations of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix.

Local delivery of the preparations of the present invention, such as, for example, topical application, may be carried out, by means of a spray, perfusion, double balloon catheters, stent, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods. In any event, the pharmaceutical compositions should provide a quantity of the preparation sufficient to effectively treat the condition.

The concentration of factor VII or factor VII-related polypeptide, factor V or factor Vrelated polypeptide, or factor VII or factor VII-related polypeptide in combination with factor V or factor V-related polypeptide in these formulations can vary widely, from less than about by weight, usually at or at least about 1% by weight to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Administration by injection or infusion, in particular injection, is preferred. Thus, the factor VII or factor VII-related polypeptide and the factor V or factor Vrelated polypeptide 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 both the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide WO 03/039584 PCT/DK02/00736 33 in one dosage form, or a dissolved lyophilized powder or a liquid formulation containing the factor VII or factor VII-related polypeptide in one dosage form and dissolved lyophilized powder or a liquid formulation containing the factor V or factor V-related polypeptide in another dosage form.

It is to be understood that the amount of factor VII or factor VII-related polypeptide and the amount of factor V or factor V-related polypeptide together comprise an aggregate effective amount for treating the bleeding episode.

It must be kept in mind that the materials of the present invention may generally be employed in serious disease or injury states, that is, life threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and general lack of immunogenicity of factor Vila and factor V in humans, it is possible and may be felt desirable by the treating physician to administer a substantial excess of these compositions.

In prophylactic applications, compositions containing a preparation of factor VII or a factor VII-related polypeptide and a preparation of factor V or a factor V-related polypeptide are administered to a subject susceptible to or otherwise at risk of a disease state or injury to enhance the subject's own coagulative capability. Such an amount is defined to be a "prophylactically effective dose." It is to be understood that the amount of factor VII or factor VII-related polypeptide and the amount of factor V or factor V-related polypeptide together comprise an aggregate effective amount for preventing a bleeding episode.

Single or multiple administrations of the compositions can be carried out with dose levels and patterns being selected by the treating physician. The compositions may 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 bleeding episodes.

Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health of the subject, and other factors evident to those skilled in the art.

The composition of the invention is generally administered in a single dose before the expected bleeding or at the start of the bleeding. It may however also be given repeatedly (in multiple doses) preferably with intervals of 2-4-6-12 hour, depending on the dose given and the condition of the subject.

For treatment in connection with deliberate interventions, the factor VII or factor VIIrelated polypeptide and the factor V or factor V-related polypeptide will typically be administered within about 24 hours prior to performing the intervention, and for as much as 7 days or more thereafter. Administration as a coagulant can be by a variety of routes as described herein.

The composition may be in the form of a single preparation (single-dosage form) comprising both a preparation of a preparation of factor VII or a factor VII-related polypeptide and a preparation of a preparation of factor V or a factor V-related polypeptide in suitable concentrations. The composition may also be in the form of a kit-of-parts consisting of a first unit WO 03/039584 PCT/DK02/00736 34 dosage form comprising a preparation of a preparation of factor VII or a factor VII-related polypeptide and a second unit dosage form comprising a preparation of a preparation of factor V or a factor V-related polypeptide. In this case, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide should be administered one after the other, preferably within about 15 minutes of each other, for example within 10 minutes of each other or, preferably, within 5 minutes or, more preferred, within 2 minutes of each other. Either of the two unit dosage forms can be administered first.

The kit includes at least two separate pharmaceutical compositions. The kit includes container means for containing the separate compositions such as a divided bottle or a divided foil packet. Typically the kit includes directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms, are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

The amount of factor VII or factor VII-related polypeptide and the amount of factor V or factor V-related polypeptide administered according to the present invention may vary from a ratio of between about 1:100 to about 100:1 The ratio of factor VII to factor V may thus be, e.g., about 1:100, or 1:90, or 1:80, or 1:70 or 1:60, or 1:50, or 1:40, or 1:30, or 1:20, or 1:10, or 1:5, or 1:2, or 1:1, or 2:1, or 5:1, or 10:1, or 20:1, or 30.1, or 40:1, or 50:1, or 60:1, or 70:1, or 80:1, or 90:1, or 100:1; or between about 1:90 to about 1:1, or between about 1:80 to about 1:2, or between about 1:70 to about 1:5, or between about 1:60 to about 1:10, or between about 1:50 to about 1:25, or between about 1:40 to about 1:30, or between about 90:1 to about 1:1, or between about 80:1 to about 2:1, or between about 70:1 to about 5:1, or between about 60:1 to about 10:1, or between about 50:1 to about 25:1, or between about 40:1 to about 30:1.

The dose of the factor VII or factor VII-related polypeptide ranges from what corresponds to about 0.05 mg to about 500 mg/day of wild-type factor VII, from about 1 mg to about 200 mg/day, or, from about 5 mg to about 175 mg/day for a 70-kg subject as loading and maintenance doses, depending on the weight of the subject, the condition and the severity of the condition.

The dose of the factor V or factor V-related polypeptide ranges from what corresponds to about 0.05 mg to about 500 mg/day of wild-type factor V, from about 1 mg to about 200 mg/day, or, from about 1 mg to about 175 mg/day for a 70-kg subject as loading and maintenance doses, depending on the weight of the subject, the condition and the severity of the condition.

The combination of factor VII or a factor VII-related polypeptide and Factor V or a factor V-related polypeptide shows a synergistic effect in an in vitro clotting time assay.

WO 03/039584 PCT/DK02/00736 The composition may be in the form of a single preparation comprising both factor VII or a factor VII-related polypeptide and Factor V or a factor V-related polypeptide in suitable concentrations. The composition may also be in the form of a kit consisting of a first unit dosage form comprising factor VII or a factor VII-related polypeptide, and a second unit dosage form comprising factor V or a factor V-related polypeptide. In this case, the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide should be administered sequentially, preferably within about 1-2 hours of each other, for example within 30 minutes of each other or, preferably, within 10 minutes or, more preferred, within 5 minutes of each other. Either of the two unit dosage forms can be administered first.

Since the present invention relates to the prevention or treatment of bleeding episodes or for coagulative treatment by treatment with a combination of active ingredients that may be administered separately, the invention also relates to combining separate pharmaceutical compositions in kit form. 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 foil packet. Typically the kit includes directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms, are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician Assays: Test for factor Vila activity: A suitable assay for testing for factor Vila activity and thereby selecting suitable factor Vila variants can be performed as a simple preliminary in vitro test: In Vitro Hydrolysis Assay Native (wild-type) factor Vila and factor Vila variant (both hereafter referred to as "factor Vila") may be assayed for specific activities. They may also be assayed in parallel to directly compare their specific activities. The assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). The chromogenic substrate D-lle-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mM, is added to factor Vila (final concentration 100 nM) in 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI 2 and 1 mg/ml bovine serum albumin. The absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA). The absorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank well containing no enzyme, is used to calculate the ratio between the activities of variant and wild-type factor Vila: Ratio (A 4 05 nm factor Vila variant)/(A 40 nm factor Vila wild-type).

WO 03/039584 PCT/DK02/00736 36 Based thereon, factor Vila variants with an activity comparable to or higher than native factor Vila may be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is around, versus above The activity of factor Vila or factor Vila variants may also be measured using a physiological substrate such as factor X, suitably at a concentration of 100-1000 nM, where the factor Xa generated is measured after the addition of a suitable chromogenic substrate (eg. S-2765). In addition, the activity assay may be run at physiological temperature.

In Vitro Proteolysis Assay Native (wild-type) factor Vila and factor Vila variant (both hereafter referred to as "factor Vila") are assayed in parallel to directly compare their specific activities. The assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). factor Vila (10 nM) and factor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI2 and 1 mg/ml bovine serum albumin, are incubated for 15 min. factor X cleavage is then stopped by the addition of 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1 mg/ml bovine serum albumin. The amount of factor Xa generated is measured by addition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration mM. The absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA). The absorbance developed during 10 minutes, after subtraction of the absorbance in a blank well containing no FVla, is used to calculate the ratio between the proteolytic activities of variant and wild-type factor Vila: Ratio (A405 nm factor Vila variant)/(A405 nm factor Vila wild-type).

Based thereon, factor Vila variants with an activity comparable to or higher than native factor Vila may be identified, such as, for example, variants where the ratio between the activity of the variant and the activity of native factor VII (wild-type FVII) is around, versus above Thrombin generation assay: The ability of factor VII or factor VII-related polypeptides or factor V or factor V-related polypeptides variants) to generate thrombin can be measured in an assay comprising all relevant coagulation factors and inhibitors at physiological concentrations and activated platelets (as described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 which is hereby incorporated as reference).

Test for Factor V activity: WO 03/039584 PCT/DK02/00736 37 Factor V polypeptides useful in accordance with the present invention may be selected by suitable assays that can be performed as simple preliminary in vitro tests, such as, as the clotting assay described by Thorelli et al., Thromb Haemost 80: 92, 1998 (the "factor V assay") The present invention is further illustrated by the following examples, which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

EXAMPLES

Example 1

METHODS:

Clot assay: Aliquots (55 pl) of rFVlla (1 pg/ml) in 50 mM Pipes, 100 mM NaCI, 2 mM EDTA, 1% BSA, pH 7.2, were incubated for 5 min with an 55 pl aliquot containing 100 pM PC/PS vesicles and mM CaCI2 in the same buffer. A 55 pl aliquot of either normal human plasma (NHP) or factor VIII-deficient plasma (FVIII-DP) was then added and clotting followed for 400 seconds in an ACL clotting machine using the standard APTT program. Where indicated, Factor V (30 nM) was included.

RESULTS:

Clot assay: Prior to addition of Factor Vila or Factor V, NHP as well as FVIIl-DP did not clot within the 400 seconds monitoring time. Addition of Factor Vila (1 pg/ml) reduced the clot time to 184.0 S1.1 seconds in NHP and 126.6 3.1 seconds in FVIII-DP (Fig Addition of both Factor Vila (1 pg/ml) and Factor V (30 nM) reduced the clot time to 116.2 0.8 and 109.8 1.41 seconds in NHP and FVIII-DP, respectively (Fig 1).

CONCLUSION:

These results demonstrate that Factor Vila and Factor V addition to plasma in a synergistic fashion shorten the clotting time of normal and FVIII-DP.

Claims (49)

1. A pharmaceutical composition comprising factor VII or a factor VII-related polypeptide, and factor V or a factor V-related polypeptide.

2. A composition according to claim 1, wherein said factor VII or factor VIl-related polypeptide is a factor VII-related polypeptide.

3. A composition according to claim 2, wherein said factor VII-related polypeptide is a factor VII amino acid sequence variant.

4. A composition according to claim 2 or claim 3, wherein the ratio between the activity of said factor VII-related polypeptide and the activity of native human factor Vila (wild-type FVIIa) is at least about 1.25 when tested in the "In Vitro Hydrolysis Assay" as described in the present de- scription. A composition according to claim 1, wherein said factor VII or factor Vll-related polypeptide is factor VII.

6. A composition according to claim 5, wherein said factor VII is human factor VII

7. A composition according to claim 6, wherein said factor VII is recombinant human factor VII.

8. A composition according to any one of claims 1 to 7, wherein said factor VII or factor VII- related polypeptide is in its activated form.

9. A composition according to claim 8, wherein said factor VII is recombinant human factor Vila. A composition according to any one of claims 1-9, wherein said factor V or factor V-related polypeptide is a factor V-related polypeptide.

11. A composition according to claim 10, wherein said factor V-related polypeptide is a factor V amino acid sequence variant.

12. A composition according to claim 10 or claim 11, wherein the ratio between the activity of said factor V-related polypeptide and the activity of native human factor V (wild-type factor V) is at least about 1.25 when tested in the "factor Vassay" as described in the present description. -39-

13. A composition according to any one of claims I to 9, wherein said factor V or factor V-related polypeptide is a factor V polypeptide.

14. A composition according to claim 13, wherein said factor V is human factor V. A composition according to claim 14, wherein said factor V is platelet- or plasma- derived factor V.

16. A composition according to claim 14 or claim 15, wherein said factor V is recombinant human factor V.

17. A composition according to claim 16, wherein said factor V is recombinant human activated factor V.

18. A composition according to any one of claims 1 to 17, wherein said factor VII or factor VII-related polypeptide and said factor V or factor V-related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII:factor V).

19. A composition according to any one of claims 1 to 18, wherein the composition further comprises pharmaceutically acceptable excipients suitable for injection or infusion, in particular injection. A kit of parts when used in the treatment of bleeding episodes comprising; a) an effective amount of a preparation of factor VII or a factor VII-related polypeptide and a pharmaceutically acceptable carrier in a first-unit dosage form; b) an effective amount of a preparation of factor V or a factor V-related polypeptide and a pharmaceutically acceptable carrier in a second-unit dosage form; and c) container means for containing said first and second dosage forms. O O 21. A kit according to claim 20, wherein said factor VII or factor VII-related polypeptide is a factor VII-related polypeptide.

22. A kit according to claim 21, wherein said factor VII-related polypeptides are factor VII amino acid sequence variants.

23. A kit according to claim 21 or claim 22, wherein the ratio between the activity of Ssaid factor VII-related polypeptide and the activity of native human factor Vlla (wild- N type FVIIa) is at least about 1.25 when tested in the in Vitro Hydrolysis Assay" as described in the present description. (Nl

24. A kit according to claim 20, wherein said factor VII or factor VII-related polypeptide is factor VII.

25. A kit according to claim 24, wherein said factor VII is human factor VII.

26. A kit according to claim 25, wherein said factor VII polypeptide is recombinant human factor VII.

27. A kit according to any one of claims 20 to 26, wherein said factor VII or factor VII- related polypeptide is in its activated form.

28. A kit according to claim 27, wherein said factor VII is recombinant human factor VIIa.

29. A kit according to any one of claims 20-28, wherein said factor V or factor V-related polypeptide is a factor V-related polypeptide. A kit according to claim 29, wherein said factor V-related polypeptide is a factor V amino acid sequenced variant.

31. A kit according to claim 29 or claim 30, wherein the ratio between the activity of said factor V-related polypeptide and the activity of native human factor V (wild-type -41 factor V) is at least about 1.25 when tested in the "factor V assay" as described in the present description.

32. A kit according to any one of claims 20 to 28, wherein said factor V or factor V- related polypeptide is factor V.

33. A kit according to claim 32, wherein said factor V is human factor V.

34. A kit according to claim 33, wherein said factor V is platelet- or plasma-derived factor V. A kit according to claim 33 or claim 34, wherein said factor V is recombinant human factor V.

36. A kit according to claim 35, wherein said factor V is recombinant human activated factor V.

37. A kit according to any one of claims 20 to 36, wherein said factor VII or factor VII- related polypeptide and factor V or factor V-related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII: factor V).

38. Use of factor VII or a factor VII-related polypeptide in combination with a factor V or a factor V-related polypeptide for the manufacture of a medicament for treating bleeding episodes.

39. Use of a composition according to any one of claims 1 to 19, for the manufacture of a medicament for treating bleeding episodes. Use according to claim 38 or claim 39, wherein the medicament is for reducing clotting time.

41. Use according to claim 38 or claim 39, wherein the medicament is for prolonging the clot lysis time. -42-

42. Use according to claim 38 or claim 39, wherein the medicament is for increasing clot strength.

43. Use according to any one of claims 38 to 39, wherein the medicament is formulated for injection or infusion, in particular injection.

44. Use according to any one of claims 38 to 43, wherein the bleeding episodes are due to trauma, or surgery, or lowered count or activity of platelets.

45. Use according to any one of claims 38 to 43, wherein the bleeding episodes are due to lowered amount or activity of coagulation factors VIII or IX or von Willebrand's factor.

46. Use according to any one of claims 38 to 45, wherein the medicament is in single- dosage form.

47. Use according to any one of claims 38 to 45, wherein the medicament is prepared in the form of a first unit dosage form comprising a preparation of factor VII or a factor VII-related polypeptide and a second unit dosage form comprising a preparation of factor V or a factor V-related polypeptide.

48. A method for treating bleeding episodes in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of factor V or a factor V-related polypeptide, wherein the first and second amount together are effective to treat bleedings.

49. A method for reducing clotting time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of factor V or a factor V-related polypeptide wherein the first and second amount together are effective to reduce clotting time. -43- A method to enhance haemostasis in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII- related polypeptide and a second amount of a preparation of factor V or a factor V- related polypeptide wherein the first and second amount together are effective to enhance haemostasis.

51. A method for prolonging the clot lysis time in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of a preparation of factor V or a factor V-related polypeptide wherein the first and second amount together are effective to prolong the clot lysis time.

52. A method for increasing clot strength in a subject, the method comprising administering to a subject in need thereof a first amount of a preparation of factor VII or a factor VII-related polypeptide and a second amount of preparation of factor V or a factor V-related polypeptide wherein the first and second amount together are effective to increase clot strength.

53. Method according to any one of claims 48 to 52, wherein the factor VII or factor VII-related polypeptide, and the factor V or factor V-related polypeptide are administered in single-dosage form.

54. Method according to any one of claims 48 to 52, wherein the factor VII or factor VII-related polypeptide and the factor V or factor V-related polypeptide are administered in the form of a first dosage form comprising a preparation of factor VII or a factor VII-related polypeptide and a second dosage form comprising a preparation of factor V or a factor V-related polypeptide. Method according to claim 54, wherein the first dosage form and the second dosage form are administered with a time separation of no more than 15 minutes.

56. A kit containing a treatment for bleeding episodes comprising; -44- a) an effective amount of factor VII or a factor VII-related polypeptide and an effective amount of factor V or a factor V-related polypeptide and a pharmaceutically acceptable carrier in a single-unit dosage form; and b) container means for containing said single-unit dosage form.

57. Method of treating bleeding episodes comprising the step of administering to a subject in need of a composition according to any one of claims 1 to 19.

58. Use according to claim 38 or 39 wherein the medicament is for treating bleeding episodes.

59. A pharmaceutical composition comprising factor VII or a factor VII-related polypeptide, and factor V or a factor V-related polypeptide; a kit of parts when used in the treatment of bleeding episodes; use of factor VII or a factor VII-related polypeptide in combination with factor V or a factor V-related polypeptide for the manufacture of a medicament; use of composition comprising factor VII or a factor VII-related polypeptide, and factor V or a factor V-related polypeptide; for the manufacture of a medicament for treating bleeding episodes; a method for treating bleeding episodes or reducing clotting or enhancing haemostasis or prolonging the clot lysis time or increasing clot strength in a subject; or a kit containing a treatment for bleeding episodes, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.