AU2002354846B2 - Pharmaceutical composition comprising factor VII polypeptides and factor XI polypeptides - Google Patents

Description

PHARMACEUTICAL COMPOSITION COMPRISING FACTOR VII POLYPEPTIDES AND FACTOR XI POLYPEPTIDES FIELD OF THIS INVENTION The present invention relates to a pharmaceutical composition comprising Sfactor VII or a factor VII-related polypeptide and factor XI or a factor XI-related 00 polypeptide. The invention also relates to the use of a combination of a factor VII or a V) factor VII-related polypeptide and a factor XI or a factor XI-related polypeptide for the manufacture of a medicament for treatment of subjects suffering from bleeding episodes, S 10 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 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 stabilizes the initial platelet plug. This process is compartmentalized, localised to the site of TF expression or exposure, thereby minimizing the risk of a systemic activation 2 of the coagulation system. The insoluble fibrin forming the plug is furthermore stabilised by FXII-catalysed cross-linking of the fibrin fibre SFVllIa 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 O offers a rapid and highly effective pro-haemostatic response in haemophilic subjects 00 with bleedings that cannot be treated with coagulation factor products due to antibody t formation. Also bleeding subjects with a factor VII deficiency or subjects having a C 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) has been encountered.

Extra exogenously administered FVIIa 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 FvIIa increases the thrombin formation.

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

FXI is a component of the intrinsic pathway of coagulation. A deficiency of FXI is associated with a mild to moderate bleeding disorder especially from tissues with a high local fibrinolytic activity. In contrast, it is believed that high levels of FXI are a risk factor for venous thrombosis. FXI is the zymogen of a trypsin-like serine protease that is activated by FXIIa, thrombin and FXIa. Activated FXI (FXIa) participates in the activation of FIX, which in turn (in combination with FVIII) further activates FX and thus gives rise to generation of thrombin.

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 Sassociated with the risk of transferring human viruses (hepatitis, HIV, parvovirus, and other 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 making any IND treatment extremely difficult and unfortunately often unsuccessful. Therefore a major 00 goal in surgery as well as in the treatment of major tissue damage is to avoid or minit mise the bleeding. To avoid or minimise such bleeding it is of importance to ensure the (N 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 FvIIa since the short half-life of FvIIa (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.

Japanese patent application JP59116213 concerns an aerosol composition for use as a tissue glue containing a blood coagulant as an active component. The blood coagulant may be selected from blood coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, and XIII, prekallikrein, high polymer kininogen and thrombin. A combination of FXIII and thrombin is preferred.

European Patent No. 225.160 (Novo Nordisk) concerns compositions of FVIIa 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 VIIa 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 FVIIa.

US Patent No. 5,252,217 concerns a process for preparing a human factor XI concentrate intended for therapeutic use.

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, Strauma, or other forms of tissue damage; induced coagulopathy, including coagulopathy in multi-transfused subjects; congenital or acquired coagulation or bleeding disorders, including diminished liver function ("liver disease"); defective platelet function or IDdecreased platelet number; lacking or abnormal essential clotting "compounds" 00 platelets or von Willebrand factor protein); increased fibrinolysis; anticoagulant therapy t or thrombolytic therapy; or stem cell transplantation.

C€3 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 amount of FVIIa needed for achieving full haemostasis is lowered and methods wherein the time to bleeding arrest is shortened.

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.

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".

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.

In a first aspect, the invention provides a pharmaceutical composition comprising factor VII or a factor VII-related polypeptide, and factor XI or a factor XI-related polypeptide.

SIn a second aspect, the present invention provides a kit of parts when used in the Streatment of bleeding episodes comprising; a) an effective amount of a preparation of a factor VII or factor VIIrelated polypeptide and a pharmaceutically acceptable carrier in a first unit dosage form; ND b) an effective amount of a preparation of a factor XI or factor XI- OO related polypeptide and a pharmaceutically acceptable carrier in a sec- Sond unit dosage form; and Sc) container means for containing said first and second dosage forms.

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 a factor VII or factor VIIrelated polypeptide and a pharaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a preparation of a factor XI or factor XI-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.

In different embodiments thereof, the kit further contains an effective amount of a TFPI-inhibitor and/or a factor VIII; the TFPI-inhibitor or the factor VIII (or the combination of the two) may be present in seperate unit dosage forms or may be present in one of the unit dosage forms containing either factor VII or factor VII-related polypeptide, or the factor XI or factor XI-related polypeptide.

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

In a fourth aspect, the invention provides the use of a composition according to the first aspect, for the manufacture of a medicament for treating bleeding episodes in a subject.

In different embodiments thereof, the medicaments are for reducing clotting time, prolonging the clot lysis time, and increasing clot strength.

In another embodiment, the medicament is formulated for intravenous administration, preferably injection or infusion, in particular injection.

6 i In one embodiment, the medicament is formulated in single-unit dosage form; in another it is formulated in the form of a first unit dosage form comprising a preparation of a factor VII of factor VII-related polypeptide and a second unit dosage form comprising a preparation of a factor XI or factor XI-related polypeptide.

In different embodiments, the medicaments are for treatment of subjects I experiencing bleeding episodes due to surgery, trauma, or other forms of tissue damage; 00 coagulopathy, including coagulopathy in multi-transfused subjects; congenital or t acquired coagulation or bleeding disorders, including decreased liver function ("liver disease"); defective platelet function or decreased platelet number; lacking or abnormal S 10 essential clotting "compounds" platelets or von Willebrand factor protein); increased fibrinolysis, anticoagulant 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 (bleeding in joints), chronic haemophilic arthropathy, haematomas, muscular, retroperitioneal, sublingual and retropharyngeal), bleedings in other tissue, haematuria (bleeding from 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 a factor VII or factor VII-related polypeptide and a second amount of preparation of a factor XI or a factor XI-related polypeptide, wherein the first and second amount together are effective to treat bleedings.

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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or a factor XI-related polypeptide wherein 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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-related polypeptide wherein the first and second amount together are effective to enhance haemostasis.

IND In an eighth aspect, the invention provides a method for prolonging the clot lysis 00 time in a subject, the method comprising administering to a subject in need thereof a first t amount of a preparation of a factor VII or factor VII-related polypeptide and a second C amount of a preparation of a factor XI or factor XI-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 a factor VII or factor VII-related polypeptide and a second amount of preparation of a factor XI or factor XI-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 XI or factor XI-related polypeptide are administered in a single-unit dosage form.

In another series of embodiments the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide are administered in the form of a firstunit dosage form comprising a preparation of a factor VII or factor VII-related polypeptide and a second-unit dosage form comprising a preparation of a factor XI or factor XIrelated 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 minutes.

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

SIn an eleventh aspect, the present invention provides a method of treating bleeding episodes in a subject, the method comprising administering a composition according to Sthe invention.

0 In a twelfth aspect, the present invention provides use of a factor VII or factor VIIrelated polypeptide in combination with a factor XI or a factor XI-related polypeptide j for the manufacture of a medicament for reducing clotting time.

00 In a thirteenth aspect, the present invention provides use of a factor VII or factor r VII-related polypeptide in combination with a factor XI or a factor XI-related polypeptide for the manufacture of a medicament for enhancing haemostasis.

In a fourteenth aspect, the present invention provides use of a factor VII or factor VII-related polypeptide in combination with a factor XI or a factor XI-related polypeptide for the manufacture of a medicament for prolonging clot lysis time.

In a fifthteenth aspect, the present invention provides use of a factor VII or factor VII-related polypeptide in combination with a factor XI or a factor XI-related polypeptide for the manufacture of a medicament for increasing clot strength.

In one series of embodiments of the invention, the factor VII or factor VIIrelated 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 Vila (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 the factor VIIrelated 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 it's activated form. In one preferred embodiment of the invention the factor VII is recombinant human factor Vila.

In one series of embodiments the factor XI or factor XI-related polypeptide is a factor XI-related polypeptide. In one embodiment the factor XI-related polypeptide is a Sfactor XI amino acid sequence variant. In one embodiment the ratio between the activity of said factor XI-related polypeptide and the activity of native human plasma factor XI (wild-type FXI) is at least about 1.25 when tested in the "FXI chromogenic assay" as described in the present description. In one embodiment the factor XI or factor XIrelated polypeptide is a factor XI polypeptide. In one embodiment the factor XI is hu- IND man factor XI. In one embodiment the factor XI is bovine, porcine, canine, equine, mur- 00 ine, or salmon factor XI. In a preferred embodiment the factor XI is recombinantly t made. In another embodiment the factor XI is derived from plasma. In another em-

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bodiment the factor XI is platelet-derived factor XI. In a preferred embodiment the factor XI is recombinant human plasma factor XI. In one series of embodiments of the invention the factor XI or factor XI-related polypeptide is in its activated form. In one embodiment the factor XI-related polypeptide is a fragment of factor XI. In one embodiment the factor XI-related polypeptide is a hybrid factor XI polypeptide, a procine/human hybrid. In one embodiment, the factor XI is human plasma activated factor XI (FXIa).

WO 03/007983 PCT/DK02/00505 7 In one embodiment the factor VII or factor VII-related polypeptide and the factor XI or factor-XI related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII:factor XI).

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-FVIIa, L305T-FVIIa, F374P-FVIIa, V158T/M298Q-FVIla, V158D/E296V/M298Q-FVIla, K337A-FVlla, M298Q-FVlla, V158D/M298Q-FVIla, L305V/K337A-FVIla, V158D/E296V/M298Q/L305V-FVIIa, V158D/E296V/M298Q/K337A-FVIIa, V158D/E296V/M298Q/L305V/K337A-FVIla, 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 Vila. 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 XI or factor XI-related polypeptide are recombinant human factor Vila and recombinant human plasma factor XI or recombinant human factor Vila and recombinant human plasma factor Xla.

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 having a deficiency of one or more coagulation factors; in another 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 XI-deficient human blood.

In one series of embodiments, the blood is plasma.

In one embodiment, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide prolong the in vitro clot lysis time in normal human plasma. In WO 03/007983 PCT/DK02/00505 8 another embodiment, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide increase the maximal clot strength clot lysis time in normal human plasma in vitro. In another embodiment, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide shorten the clotting time in normal human plasma in vitro.

In one embodiment of the invention, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-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 XI or factor XI-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 XI or factor XI-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 XI or factor XI-related polypeptide are the sole active agents administered to the patient. In one embodiment, the composition is substantially free of 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 a factor VII or factor VII-related polypeptide as a first-unit dosage form and a preparation of a factor XI or factor XI-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 XI or factor XI-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 XI or factor XI-related polypeptide are administered in the form of a first unit dosage form comprising a preparation of a factor VII or factor VII-related polypeptide and a second unit dosage form comprising a preparation of a factor XI or factor XI-related polypeptide.

In one embodiment of the invention, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide are administered simultaneously. In another embodiment, the factor VII or factor VII-related polypeptide and the factor XI or factor XIrelated polypeptide are administered sequentially. In one embodiment, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide are administered with a time separation of no more than 15 minutes, preferably 10, more preferred 5, more preferred WO 03/007983 PCT/DK02/00505 9 2 minutes. In one embodiment, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-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 a factor XI or factor XI-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 XI or factor XI-related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII:factor XI) In one embodiment of the present invention, the pharmaceutical composition is in single-dosage form and consists essentially of a preparation of a factor VII or factor VII-related polypeptide and a preparation of a factor XI or factor XI-related polypeptide, and one or more of the components selected from the list of pharmaceutical acceptable excipients or 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 hasa lowered plasma concentration of factor VIII.

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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-related polypeptide, wherein the first and second amounts together are effective to enhance formation of thrombin.

In 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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-related polypeptide, wherein the first and second amounts together are effective to enhance formation of thrombin.

WO 03/007983 PCT/DK02/00505 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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-related polypeptide, wherein the first and second amounts together are effective in treating bleedings.

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

In one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the composition further comprises an inhibitor of the fibrinolytic system, including, without limitation, aprotinin, s -aminocaproic acid or tranexamic acid. In one embodiment, the composition further contains a TFPI inhibitor and/or FVIII.

In one embodiment, the composition further contains a factor VIII. In one embodiment, the factor VIII is an activated factor VIII (factor Villa). In a further embodiment the factor VIII is a recombinant factor Villa. In a further embodiment the factor VIII is recombinant human factor Villa.

In another aspect, the invention relates to the use of a factor Vila in combination with a factor XI 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 thereof a first amount of a preparation of a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-related polypeptide, wherein the first and second amounts together are effective in treating bleedings.

In one embodiment of the present invention, the pharmaceutical composition (when in single-preparation form) consists essentially of a factor Vila and a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor.

WO 03/007983 PCT/DK02/00505 11 In another embodiment of the present invention, the pharmaceutical composition (when in single-preparation form) consists essentially of a factor Vila and a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor, and a TFPI-inhibitor.

In another embodiment of the present invention, the pharmaceutical composition (when in single-preparation form) consists essentially of a factor Vila and a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor, and a factor VIII, and, optionally, a TFPI-inhibitor.

In another embodiment, the pharmaceutical composition (when in form of a kit) consists of a first unit dosage form consisting essentially of a factor Vila and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor; and a second unit dosage form consisting essentially of a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor.

In another embodiment, the pharmaceutical composition (when in form of a kit) consists of a first unit dosage form consisting essentially of a factor Vila and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor; and a second unit dosage form consisting essentially of a factor XI, and, optionally, a pharmaceutical acceptable excipient or carrier, and, optionally, a stabiliser, and, optionally, a detergent, and, optionally, a neutral salt, and, optionally, an antioxidant, and, optionally, a preservative, and, optionally, a protease inhibitor; wherein either the first unit dosage form or the second unit dosage form or both dosage forms further contain a factor VIII and/or a TFPI-inhibitor.

>0 LIST OF FIGURES Figure 1: Addition of FVIIa results in a dose-dependent prolongation of the clot lysis time. This effect was optimal at 10 nM FVIIa.

Figure 2: In the presence of 10 nM FVIIa, addition of FXI resulted in a further prolongation of the clot lysis time. The effect was dose-dependent and optimal at 30 nM FXI.

Figure 3: Thromboelastography (roTEG) measurements were utilized to analyze the effect of FVlla and FXI on the Maximal Clot Firmness (MCF), as well as the clots resistance to t-PA mediated lysis.

WO 03/007983 PCT/DK02/00505 12 Figure 4: These results demonstrate that FVlla and FXI when added to plasma in a synergistic fashion shorten the clotting time in NHP.

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.

WO 03/007983 PCT/DK02/00505 13 Thus, major objectives in treatment of bleedings are to obtain haemostasis in a minimum of time, thus keeping the blood loss at a minimum.

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 a factor VII or factor VII-related polypeptide, factor Vila, in combination with a preparation of a factor XI or factor XI-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 XI is administered alone.

The administration of a preparation of a factor VII or factor VII-related polypeptide, factor Vila, in combination with a preparation of a factor XI or factor XI-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 XI is administered alone. The present invention provides a beneficial effect of simultaneous or sequential dosing of a preparation of a factor XI or factor X-related polypeptide and a preparation of a factor VII or 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 a factor VII or factor VII-related polypeptide and a preparation of a factor XI or factor XI-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 a factor VII or factor VIIrelated polypeptide and a factor XI or a factor XI-related polypeptide is an advantageous product ensuring the formation of solid, stable and quickly formed haemostatic plugs.

WO 03/007983 PCT/DK02/00505 14 The present inventors have shown that a combination of a factor Vila and a factor XI can reduce the clotting time of normal human plasma more effectively than either factor Vila or factor XI alone. It has also been shown that a combination of a factor Vila and a factor XI can increase the firmness of the clot more effectively than either factor Vila or factor Xl alone. By combining a factor Vila at a concentration where no further increase in clot firmness was observed with a factor XI, also at a concentration where no further increase in clot firmness was observed, it was unexpectedly shown that a further increase in clot firmness was obtained. It has also been shown that combination of a factor Vila and a factor XI can prolong the in vitro clot lysis time in normal human plasma more effectively than either factor Vila or factor XI alone. It has also been shown that combination of a factor Vila and a factor XI can prolong the half-clot lysis time in normal human plasma more effectively than either factor Vila or factor XI alone. It has also been shown that combination of a factor Vila and a factor XI can protect the clot from fibrinolysis, in particular tPA-mediated fibrinolysis, in normal human plasma more effectively than either factor Vila or factor XI 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 a factor VII and a factor XI.

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 conse- WO 03/007983 PCT/DK02/00505 quence, 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 level 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 prematurely 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.

In the presence of a factor XI it is believed that lower concentrations of factor Vila may be sufficient to ensure a sufficient haemostasis.

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 WO 03/007983 PCT/DK02/00505 16 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.

"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 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 WO 03/007983 PCT/DK02/00505 17 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%, 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 FVlla) 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 FVIlla) 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 WO 03/007983 PCT/DK02/00505 18 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 0 2 2 2776).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/D3095-FVII, L3051-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V1 5 8D/E296V/M298Q/K337A-FVII, V1 5 8D/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 WO 03/007983 PCT/DK02/00505 19 (iv) Measuring hydrolysis of a synthetic substrate by factor Vlla and/or a factor Vlla -related polypeptide ("In Vitro Hydrolysis Assay", see below); and Measuring generation of thrombin in a TF-independent in vitro system.

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 XI polypeptides: In practicing the present invention, any factor XI polypeptide may be used that is effective in preventing or treating bleeding. This includes factor XI polypeptides derived from blood or plasma, or produced by recombinant means. Furthermore, platelets may contain a structurally different form of FXI (possible due to alternative splicing of the FXI gene). Platelet factor XI is described in Lipscomb, M.S. Walsh, P.N. (1979), Journal of Clinical Investigation, 63, 1006-1014.

As used herein, "factor XI polypeptide" encompasses, without limitation, factor XI, as well as factor XI-related polypeptides. The term "factor XI" is intended to encompass, without limitation, polypeptides having the amino acid sequence as described in Sun, Y. Gailani, D.

(1996), J. Biol. Chem. 271: 29023-29028 (wild-type human factor XI, plasma), as well as wild-type factor XI derived from other species, such as, bovine, porcine, canine, murine, and salmon factor XI. In some embodiments, the factor XI polypeptide is wild-type human factor XI, as disclosed, in Sun, Y. Gailani, D. (1996), J. Biol. Chem. 271: 29023-29028.

It further encompasses natural allelic variations of factor XI that may exist and occur from one individual to another. Also, degree and location of glycosylation or other posttranslation modifications may vary depending on the chosen host cells and the nature of the host cellular environment. The term "factor XI" is also intended to encompass factor XI 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 Xla.

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

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

WO 03/007983 PCT/DK02/00505 The term "factor XI-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 Xla-related polypeptides" or "activated factor XI-related polypeptides".

As used herein, "factor XI-related polypeptides" encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wildtype human factor XI, as well as polypeptides, in which the factor XI biological activity has been substantially modified or reduced relative to the activity of wild-type human factor XI. These polypeptides include, without limitation, factor XI or factor Xla that has been chemically modified and factor XI 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 XI.

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

Factor XI-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 XI that has been produced in the same cell type, when tested in the factor XI activity assay as described in the present specification.

Factor XI-related polypeptides, including variants, having substantially the same or improved biological activity relative to wild-type factor XI 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 XI that has been produced in the same cell type when tested in one or more of the specific factor XI activity assay as described. For purposes of the invention, factor XI biological activity may be quantified as described later in the present description ("assay part").

Factor XI-related polypeptides, including variants, having substantially reduced biological activity relative to wild-type factor XI are those that exhibit less than about preferably less than about 10%, more preferably less than about 5% and most preferably less WO 03/007983 PCT/DK02/00505 21 than about 1% of the specific activity of wild-type factor XI that has been produced in the same cell type when tested in one or more of the specific factor XI activity assays as described above.

Non-limiting examples of factor XI polypeptides include plasma-derived human factor XI as described, in Gailani Broze (1993), Blood Coagul. Fibrinolysis, 4:15-20, or Kerbiriou Griffin (1979), J. Biol. Chem., 254:12020-12207, or, In some embodiments the factor XI are factor XI-related polypeptides wherein the ratio between the activity of said factor XI polypeptide and the activity of native human factor XI (wild-type factor XI) is at least about 1.25 when tested in the FXI chromogenic assay" (see below); in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about Factor XI-related polypeptides also include fragments of factor XI or factor XI-related polypeptides retaining their characteristic haemostasis-related activity. The haemostasis-related activity of a factor XI polypeptide may, for example, be measured using the factor XI-activity assay described in the present specification.

In preferred embodiments, the factor XI is human plasma factor XI or activated human plasma factor Xla. In one embodiment, the FXI is platelet factor XI. In another embodiment, the FXI is recombinantly made.

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 WO 03/007983 PCT/DK02/00505 Glutamic Acid Glu E Aspartic Acid Asp D The term "factor Vila" or "FVIla" may be used interchangeably. The term factor Vila includes zymogen factor VII (single-chain factor VII). The term "factor XI" or "FXI" may be used interchangeably. The term "factor VIII" or "FVIII" may be used interchangeably. The term "factor VIII" or "FVIII" included activated factor VIII (FVIIla), variants and truncated forms retaining the characteristic FVIII-related haemostatic activity; the term includes recombinantly made FVIII and plasma-derived FVIII. Human FVIII and human recombinant FVIII are preferred 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 with 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 a factor VII or factor VII-related polypeptide and a preparation of a factor XI or factor XI-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 XI or factor XI-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 XI or factor XI-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 WO 03/007983 PCT/DK02/00505 23 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 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 a factor VII or factor VIIrelated polypeptide and a preparation of a factor XI or factor XI-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 XI or factor XI-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 a factor VII or factor VII-related polypeptide and a preparation of a factor XI or factor XI-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 XI or factor XI-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 a factor VII or factor VII-related polypeptide and a preparation of a preparation of a factor XI or factor XI-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 XI or factor XI-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 a factor VII or factor VII-related polypeptide and a preparation of a preparation of a factor XI or factor XI-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 XI or factor XI-related WO 03/007983 PCT/DK02/00505 24 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 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 WO 03/007983 PCT/DK02/00505 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 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 lIb/llla 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 XI; 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, WO 03/007983 PCT/DK02/00505 26 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 tissue or organ, or it may be an unspecified bleeding. Prophylactic administration of a preparation of a factor VII or factor VII-related polypeptide and a preparation of a factor Xl or factor Xl-related 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 factor VII or factor VII-related polypeptides and factor XI or factor Xl-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 a factor VII or factor VII-related polypeptide as a first unit dosage form and a preparation of a factor XI or factor XI-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 a factor VII or factor VII-related polypeptide and a preparation of a factor XI or factor XI-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 separatiori of no more than 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 30 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 XI" or "factor XI level" is meant the level of the patient's clotting factor XI 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 XI" or "reduced factor XI level" is meant a decrease in the presence or activity of factor XI in the blood stream compared to the mean factor XI level in a population of subjects having no coagulation factor XI deficiency or inhibitors to coagulation factor Xl. The level of circulating factor XI can be measured by either a coagulant or an immunologic assay, factor XI procoagulant activity is determined by the ability of the patient's plasma to correct the clotting time of factor XI-deficient plasma an APTT assay, see below; see also "assay part" of the present description).

WO 03/007983 PCT/DK02/00505 27 One unit of factor XI has been defined as the amount of factor XI present in one millilitre of normal (pooled) human plasma (corresponding to a factor XI 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 gg protein. After activation 50 units correspond to about 1 lag protein.

By "deficiency" is meant a decrease in the presence or activity of, factor XI in plasma compared to that of normal healthy individuals. The term may, where appropriate, be used interchangeably with "reduced factor XI 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 XI-responsive syndrome" is meant a syndrome where exogenous factor XI 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 XI, bleeding disorders caused by inhibitors to factor XI. A factor XI-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 Vlla, 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 a factor VII or factor VII-related polypeptide or a factor XI or factor XI-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 Vlla, 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 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.

WO 03/007983 PCT/DK02/00505 28 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 XI coagulation 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 XI protein- containing preparation with an anti-FXI antibody immobilised onto the elisa plate, subsequently contacting the immobilised antibody-factor XI complex with a second anti FXI 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 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 XI or factor XI-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, e.g., on an anti-factor VII or anti-factor XI 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.

The term "TFPI inhibitor" means compounds inhibiting the anti-coagulative activity of TFPI (tissue factor pathway inhibitor). The term includes compounds such as those disclosed in European Patent No. 558 529, WO 96/28153 and US 5,622,988. "TFPI" and "EPI" (extrinsic pathway inhibitor) may be used interchangeably.

Within the present invention an "effective amount" of a factor VII polypeptide and a factor XI polypeptide is defined as the amount of a factor VII polypeptide, FVlla, and a factor XI 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 03/007983 PCT/DK02/00505 29 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.

The composition according to the invention may further comprise a TFPI-inhibitor. The composition according to the invention may further comprise a factor VIII. Such a composition should preferably be administered to subjects who do not have inhibitors to factor VIII.

Abbreviations TF tissue factor FVII factor VII in its single-chain, unactivated form FVIla factor VII in its activated form rFVlla recombinant factor VII in its activated form FXI factor XI in its zymogenic, unactivated form FXIa factor XI in its activated form rFXI recombinant FXI rFXIa recombinant FXIa FVIII factor VIII in its zymogenic, unactivated form rFVIII recombinant FVIII FVIIIa factor VIII in its activated form rFVIlla recombinant FVIIla TFPI tissue factor pathway inhibitor 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.Invest. 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 Xlla, 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 WO 03/007983 PCT/DK02/00505 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 XI-molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the factor VII or factor VII-related polypeptide or factor XI or factor XI-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 et al., 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 et al., Biochem. 27:7785, 1988); hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures preparative isoelectric focusing (IEF), differential solubility WO 03/007983 PCT/DK02/00505 31 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 less than about 1 of non-factor VII or factor VII-related polypeptides derived from the host cell.

Factor VII or factor VII-related polypeptides may be activated by proteolytic cleavage, using factor Xlla 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 XI for use within the present invention may be prepared from plasma according to known methods, such as those disclosed by Koide et al. (Biochemistry 16: 2279-2286, 1977) and Bouma et al. (J.Biol.Chem. 252: 6432-6437, 1977), incorporated herein by reference. It is preferred, however, to use recombinant factor XI so as to avoid to the use of blood- or tissuederived products that carry a risk of disease transmission. Methods for preparing recombinant factor XI are known in the art. See, for example, Kemball-Cook et al. (Gene 139(2): 275-279, 1994), Fujikawa et al. (Biochemistry 25: 2417-2424, 1986), Meijers et al. (Blood 79(6): 1435-1440, 1992), which are incorporated herein by reference in their entirety.

Factor XI -related polypeptides may produced by modification of wild-type factor XI or by recombinant technology, factor XI -related polypeptides with altered amino acid sequence when compared to wild-type factor XI may be produced by modifying the nucleic acid sequence encoding wild-type factor XI 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 XI 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 XI or factor XI-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 XI 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 XI or factor XI-related polypeptides derived from the host cell. The re- WO 03/007983 PCT/DK02/00505 32 suiting activated factor XI or factor XI-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 XI and factor Vila proteins syngeneic with the subject in order to reduce the risk of inducing an immune response. Preparation and characterization of non-human factor XI has been disclosed by, for example, Gailani (Blood 90(3): 1055-1064, 1997). The present invention also encompasses the use of such factor XI 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 a factor VII or factor VII-related polypeptide and a preparation of a factor XI or factor XI-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 XI or a factor XI-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 a factor Vila, or a factor XI, or a factor Vila in combination with a factor XI, 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 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 WO 03/007983 PCT/DK02/00505 33 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 XI or factor XIrelated polypeptide, or factor VII or factor VII-related polypeptide in combination with factor XI or factor XI-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 XI or factor Xlrelated 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 XI or factor XI-related polypeptide 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 XI or factor XI-related polypeptide in another dosage form.

WO 03/007983 PCT/DK02/00505 34 It is to be understood that the amount of factor VII or factor VII-related polypeptide and the amount of factor XI or factor XI-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 XI 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 a factor VII or factor VII-related polypeptide and a preparation of a factor XI or factor XI-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 Vll-related polypeptide and the amount of factor XI or factor XI-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 XI or factor XI-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 a factor VII or factor VII-related polypeptide and a preparation of a preparation of a factor XI or factor XI-related polypeptide in suitable concentrations. The composition may also be in the form of a kit-of-parts consisting of a first unit dosage form comprising a preparation of a preparation of a factor VII or factor VII-related polypeptide and a second unit dosage form comprising a preparation of a preparation of a factor XI or factor XI-related polypeptide. In this case, the factor VII or factor VII-related polypeptide and the factor XI or factor XI-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, WO 03/007983 PCT/DK02/00505 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 XI or factor XI-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 XI 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 XI or factor XI-related polypeptide ranges from what corresponds to about 0.05 mg to about 500 mg/day of wild-type factor XI, 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 a factor Vila and a factor XI shows a synergistic effect in an in vitro clot firmness- and fibrinolysis time-assay. Moreover, the combination of a factor Vila and a factor XI shows a synergistic effect in forming stable fibrin clots, increasing the half-clot lysis time, increasing clot strength and increasing resistance to fibrinolysis.

The composition may be in the form of a single preparation comprising both a factor Vila and a factor XI in suitable concentrations. The composition may also be in the form of a kit consisting of a first unit dosage form comprising a factor Vila and a second unit dosage form comprising a factor XI and, optionally, one or more further unit dosage forms comprising a factor VIII and/or an TFPI inhibitor. In this case, the factor Vila and the factor XI should be administered sequentially, preferably within about 1-2 hours of each other, for example within 30 minutes of WO 03/007983 PCT/DK02/00505 36 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 (5-2288, 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 SpectraMaxT M 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 405 nm factor Vila variant)/(A 4 05nm 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 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.

WO 03/007983 PCT/DK02/00505 37 In Vitro Proteolysis Assay Native (wild-type) factor Vila and factor Vila variant (both hereafter referred to as "factor Vlla") 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 FVIla, 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 XI or factor XI-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 XI activity: A suitable assay for testing for factor XI amidolytic activity and thereby selecting suitable factor XI variants can be performed as a simple in vitro test using a chromogenic substrate as described, for example, in Gailani et al. (Blood 97(10): 3117-3122, 2001) ("the FXI chromogenic assay").

Factor XI biological activity may also be performed as a simple in vitro test measuring the activation of factor IX to IXa as described for example, in Gailani et al. (Blood 97(10): 3117- 3122, 2001).

Test for factor VIII activity: Suitable assays for testing for factor VIII activity, and thereby providing means for selecting suitable factor VIII variants for use in the present invention, can be performed as simple in vitro tests as described, for example, in Kirkwood TBL, Rizza CR, Snape TJ, Rhymes IL, Austen WO 03/007983 PCT/DK02/00505 38 DEG. Identification of sources of interlaboratory variation in factor VIII assay. B J Haematol 1981; 37; 559-68.; or Kessels et al., British Journal of Haematology, Vol. 76 (Suppl.1) pp. 16 (1990)).

factor VIII activity may also be measured by a two-step chromogenic assay based on the amidolytic activity of generated FXa (Wagenvoord et al, 1989, Haemostasis, 19(4):196-204).

factor VIII biological activity may also be quantified by measuring the ability of a preparation to correct the clotting time of factor VII-deficient plasma, as described in Nilsson et al., 1959.(Nilsson IM, Blombaeck M, Thilen A, von Francken Carriers of haemophilia A A laboratory study, Acta Med Scan 1959; 165:357). In this assay, biological activity is expressed as units/ml plasma (1 unit corresponds to the amount of FVIII present in normal pooled plasma.

Aspects of the invention: Aspect 1: A pharmaceutical composition comprising a factor VII or a factor VII-related polypeptide, and a factor XI or factor XI-related polypeptide.

Embodiment 2: The composition as in aspect 1, wherein said factor VII or factor VIIrelated polypeptide is a factor VII-related polypeptide.

Embodiment 3: The composition as in aspect 1, wherein the factor Vila is human factor Vila Embodiment 4: The composition as in aspect 1 or aspect 3, wherein the factor Vila and the factor XI is recombinant human factor Vila and recombinant human factor XI.

Embodiment 5: The composition as in any one of aspects 1-4, wherein the factor XI is platelet factor XI.

Embodiment 6: The composition as in any one of aspects 1-5, wherein the factor XI is activated factor XI.

Embodiment 7: The composition as in any one of aspects 1-6, wherein the composition further contains a TFPI inhibitor.

Embodiment 8: The composition as in any one of aspects 1-7, wherein the composition further contains a factor VIII.

Aspect 2: A kit containing a treatment for bleeding episodes comprising a) an effective amount of a factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms.

Embodiment 10: The composition as in aspect 2, comprising a) an effective amount of a factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form; WO 03/007983 PCT/DK02/00505 39 b) an effective amount of a factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; c) an effective amount of a TFPI inhibitor and a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing said first, second and third dosage forms.

Aspect 3: A kit containing a treatment for bleeding episodes comprising a) an effective amount of a factor Vila and a TFPI inhibitor and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XI and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms.

Aspect 4: A kit containing a treatment for bleeding episodes comprising a) an effective amount of a factor Vila and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XI and a TFPI inhibitor and a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms.

Embodiment 9: A kit as in any one of embodiments 2-4 further containing a factor VIII, either formulated in a separate unit dosage form, or contained within a unit dosage form also containing one or more of the compounds selected from the list of a factor Vila, a factor Xl or a TFPI inhibitor.

Aspect 6: Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for treating bleeding episodes in a subject.

Aspect 7: Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for reducing clotting time in a subject.

Aspect 8: Use of a factor Vila in combination with a factor Xl for the manufacture of a medicament for prolonging the clot lysis time in normal mammalian plasma.

Aspect 9: Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for increasing clot strength in normal mammalian plasma.

Aspect 10: Use of a factor Vila in combination with a factor XI for the manufacture of a medicament for enhancing fibrin clot formation in normal human plasma.

Aspect 11: A method of enhancing fibrin clot formation in a subject, which method comprises administering to a subject an effective amount of a factor Vila in combination with an effective amount of a factor XI.

Aspect 12: A method for treating bleeding episodes in a subject comprising administering to a subject an effective amount of a factor Vila in combination with an effective amount of a factor XI.

WO 03/007983 PCT/DK02/00505 Embodiment 10: Method as in aspect 19 or 20, wherein the factor Vila and the factor XI are administered in one-dosage form.

Embodiment 11: Method as in aspect 19 or 20, wherein the factor Vila and the factor XI is administered sequentially.

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 Improving Haemostatic Clot Stability by Combining Coagulation factors Vila and XI

METHODS:

Clot lysis assay: Normal human plasma diluted 10-fold with buffer (20 mM HEPES, 150 mM NaCI, mM CaCI, pH 7.4) containing Innovin (Dade Behring, 2000-fold dilution), rFVlla (Novo Nordisk A/S, Bagsvaerd, Denmark; various concentrations) and t-PA (American Diagnostics, 8 nM) was added to 96-well ELISA plates and turbidity at 650 nm was measured over time at room temperature. Where indicated, purified human FXI (Haematologic Technologies, various concentrations) was included.

Rotatonal thromboelastography (roTEG): Measurements was conducted on citrated normal human plasma added 5 nM t-PA and the effect of addition of 1 nM FVIIa alone or in combination with 30 nM FXI (Haematologic Technologies) was analyzed. Clotting was initiated by addition of Innovin (final concentration 2000-fold diluted, Dade Behring 526945) and calcium (final concentration 15 mM) in a 20 mM HEPES, 150 mM NaCI, pH 7.4 buffer.

RESULTS:

Clot lysis assay: Addition of FVIIa results in a dose-dependent prolongation of the clot lysis time (Fig. This effect was optimal at 10 nM FVIlla. In the presence of 10 nM FVlla, addition of FXI resulted in a further prolongation of the clot lysis time (Fig. The effect was dose-dependent and optimal at 30 nM FXI.

Thromboelastography: roTEG measurements were utilized to analyze the effect of FVIla and FXI on the Maximal Clot Firmness (MCF), as well as the clots resistance to t-PA mediated lysis. Prior to addition of FVIIa/FXI, the MCF was 25 mm and the time required for half clot lysis was 12.3 WO 03/007983 PCT/DK02/00505 41 minutes (Fig. Addition of FXI (30 nM) did not alter MCF but prolonged the half-clot lysis time to 16.1 min (Fig. Similarly, addition of FVlla (1 nM) resulted in clot protection from t-PAmediated fibrinolysis (half-clot lysis time; 16.7 min) without any effect on MCF (Fig. However, addition of FVIlla (1 nM) together with FXI (30 nM) increases the MCF (29 mm), as well as the half-clot lysis time (24.2 min, Fig. 3).

CONCLUSION:

These results demonstrate that FVIlla and FXI addition to plasma in a synergistic fashion improve clot mechanical strength and resistance to t-PA mediated fibrinolysis.

Example 2 Shortening the clotting time in normal human plasma by Combining Coagulation factors Vila and XI

METHODS:

Clot assay: Aliquots (55 pl) of rFVIla (1 pg/ml) alone, FXI (25 nM) alone, or rFVlla and FXI in mM Pipes, 100 mM NaCI, 2 mM EDTA, 1% BSA, pH 7.2, were incubated for 5 min with a 55 pl aliquot containing 100 pM PC/PS vesicles and 50 mM CaCl2 in the same buffer. A 55 pI aliquot of normal human plasma (NHP) was then added and clotting followed for 400 seconds in an ACL clotting machine using the standard APTT program.

RESULTS:

Clot assay: Prior to addition of rFVlla or FXI, NHP did not clot within the 400 seconds monitoring time. Following addition of FXI (25 nM) the clotting time was still longer than 400 s. Addition of FVlla (1 pg/ml) reduced the clot time to 159.4 1.4 seconds (Fig Addition of both FVIIa (1 pg/ml) and FXI (25 nM) reduced the clot time to 95.0 1.4 seconds (Fig 4).

CONCLUSION:

These results demonstrate that FVIlla and FXI addition to plasma in a synergistic fashion shorten the clotting time in NHP.

Claims (45)

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

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

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

12. A composition according to claim 10 or claim 11, wherein the ratio between the activity of said factor XI-related polypeptide and the activity of native human plasma factor XI (wild-type FXI) is at least about 1.25 when tested in the "FXI chromogenic assay" as described in the present description. 43 O 1 13. A composition according to any one of claims 1 to 9, wherein said factor XI or c factor XI-related polypeptide is a factor XI polypeptide.

14. A composition according to claim 13, wherein said factor XI is human factor XI. A composition according to claim 14, wherein said factor XI is recombinant 0 5 plasma human factor XI. 00

16. A composition according to claim 14, wherein said factor XI is recombinant platelet human factor XI.

17. A composition according to any one of claims 1 to 16, wherein said factor VII or factor VIl-related polypeptide and said factor XI or factor XI-related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII: factor XI).

18. A composition according to any one of claims 1 to 17, wherein the composition further comprises pharmaceutically acceptable excipients suitable for injection or infusion, in particular injection.

19. A kit of parts when used in the treatment of bleeding episodes comprising; a) an effective amount of a preparation of a factor VII or factor VII-related polypeptide and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a preparation of a factor XI or factor XI-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. A kit according to claim 19, wherein said factor VII or factor VII-related polypep- tide is a factor VII-related polypeptide.

21. A kit according to claim 20, wherein said factor VII-related polypeptides are factor VII amino acid sequence variants.

22. A kit according to claim 20 or claim 21, 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 description. WO 03/007983 PCT/DK02/00505 44

23. A kit according to claim 19, wherein said factor VII or factor VII-related polypeptide is factor VII.

24. A kit according to claim 23, wherein said factor VII is human factor VII A kit according to claim 24, wherein said factor VII polypeptide is recombinant human factor VII.

26. A kit according to any one of claims 19 to 25, wherein said factor VII or factor VII-related polypeptide is in its activated form.

27. A kit according to claim 26, wherein said factor VII is recombinant human factor Vila.

28. A kit according to any one of claims 19-27, wherein said factor XI or factor XI-related polypeptide is a factor XI-related polypeptide.

29. A kit according to claim 28, wherein said factor XI-related polypeptide is a factor XI amino acid sequence variant.

30. A kit according to claim 28 or claim 29, wherein the ratio between the activity of said factor XI-related polypeptide and the activity of native human plasma factor XI (wild-type FXI) is at least about 1.25 when tested in the "FXI chromogenic assay" as described in the present descrip- tion.

31. A kit according to any one of claims 19 to 27, wherein said factor XI or factor XI-related poly- peptide is factor XI.

32. A kit according to claim 31, wherein said factor XI is human factor XI

33. A kit according to claim 32, wherein said factor XI is recombinant plasma human factor XI.

34. A kit according to claim 32, wherein said factor XI is recombinant platelet human factor XI. A kit according to any one of claims 19 to 34, wherein said factor VII or factor VII-related polypeptide and factor XI or factor XI-related polypeptide are present in a ratio by mass of between about 100:1 and about 1:100 (w/w factor VII:factor XI) WO 03/007983 PCT/DK02/00505

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

37. Use of a composition according to any one of claims 1 to18, for the manufacture of a me- dicament for treating bleeding episodes in a subject.

38. Use according to claim 36 or claim 37, wherein the medicament is for reducing clotting time.

39. Use according to claim 36 or claim 37, wherein the medicament is for prolonging the clot lysis time. Use according to claim 36 or claim 37, wherein the medicament is for increasing clot strength.

41. Use according to any one of claims 36 to 40, wherein the medicament is formulated for injec- tion or infusion, in particular injection.

42. Use according to any one of claims 36 to 41, wherein the bleeding episodes are due to trauma, or surgery, or lowered count or activity of platelets.

43. Use according to any one of claims 36 to 42, wherein the medicament is in single-dosage form.

44. Use according to any one of claims 36 to 42, wherein the medicament is prepared in the form of a first unit dosage form comprising a preparation of a factor VII or factor VII-related polypeptide and a second unit dosage form comprising a preparation of a factor XI or factor XI- related polypeptide. 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 a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI-related polypep- tide, wherein the first and second amount together are effective to treat bleedings.

46. 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 a factor VII or factor VII-related poly- peptide and a second amount of a preparation of a factor XI or factor XI-related-polypeptide wherein the first and second amount together are effective to reduce clotting time. C

47. A method to enhance haemostasis in a subject, the method comprising administer- t ing to a subject in need thereof a first amount of a preparation of a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI- O related polypeptide wherein the first and second amount together are effective to enhance haemostasis. 0 48. A method for prolonging the clot lysis time in a subject, the method comprising t administering to a subject in need thereof a first amount of a preparation of a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or 0factor XI-related polypeptide wherein the first and second amount together are effective to prolong the clot lysis time.

49. A method for increasing clot strength in a subject, the method comprising adminis- tering to a subject in need thereof a first amount of a preparation of a factor VII or factor VII-related polypeptide and a second amount of a preparation of a factor XI or factor XI- related polypeptide wherein the first and second amount together are effective to in- crease clot strength. Method according to any one of claims 45 to 49, wherein the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide are administered in single-dosage form.

51. Method according to any one of claims 45 to 49, wherein the factor VII or factor VII-related polypeptide and the factor XI or factor XI-related polypeptide are administered in the form of a first dosage form comprising a preparation of a factor VII or factor VII-related polypeptide and a second dosage form comprising a preparation of a factor XI or factor XI-related polypeptide.

52. Method according to claim 51, wherein the first dosage form and the second dosage form are administered with a time separation of no more than 15 minutes.

53. A kit containing a treatment for bleeding episodes comprising; a) an effective amount of a factor VII or factor VII-related polypeptide and an effective amount of a factor XI or factor XI-related polypeptide and a pharmaceutically acceptable carrier in a single-unit dosage form; and b) container means for containing said single-unit dosage form. -47- C 54. Method of treating bleeding episodes in a subject, the method comprising Sadministering a composition according to any one of claims 1 to 18. Use of a factor VII or factor VII-related polypeptide in combination with a factor O XI or a factor XI-related polypeptide for the manufacture of a medicament for reducing clotting time. 00 56. Use of a factor VII or factor VII-related polypeptide in combination with a factor t XI or a factor XI-related polypeptide for the manufacture of a medicament for enhancing C haemostasis.

57. Use of a factor VII or factor VII-related polypeptide in combination with a factor XI or a factor XI-related polypeptide for the manufacture of a medicament for prolong- ing clot lysis time.

58. Use of a factor VII or factor VII-related polypeptide in combination with a factor XI or a factor XI-related polypeptide for the manufacture of a medicament for increasing clot strength.

59. A pharmaceutical composition according to claim 1, a kit of parts when used in the treatment of bleeding episodes according to claim 19, use of a factor VII or factor VII- related polypeptide in combination with factor XI and a factor XI-related polypeptide for the manufacture of a medicament for treating bleeding episodes in a subject according to claim 36, use of a composition according to any one of claims 1 to 18 for the manufac- ture of a medicament for treating bleeding episodes in a subject, a method for treating bleeding episodes in a subject according to claim 45, a method for reducing clotting time in a subject according to claim 46, a method to enhance haemostasis in a subject accord- ing to claim 47, a method for prolonging the clot lysis time in a subject according to claim 48, a method for increasing clot strength in a subject according to claim 49, a kit containing a treatment for bleeding episodes according to claim 53, or use of a factor VII or factor VII-related polypeptide in combination with a factor XI or a factor XI-related polypeptide for the manufacture of a medicament according to any one of claims 55 to 58, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.