WO2005115442A1 - Use of coagulation factor xiii for treatment of post surgical bleedings - Google Patents

Abstract

The present invention concerns the post surgical administration of factor XIII alone or in combination with other haemostatic agents e.g. factor VIIa or factor XI for the treatment of post surgical bleeding episodes. These haemostatic agent(s) are effective when administrated post surgically and therefore only need to be administrated to the patients showing uncontrollable bleedings after surgery.

Description

USE OF COAGULATION FACTOR XIII FOR TREATMENT OF POST SURGICAL BLEEDINGS

FIELD OF THE INVENTION The present invention relates to a method for treating post surgical bleeding episodes by the post surgical administration of factor XIII to a patient in need thereof.

BACKGROUND OF THE INVENTION Post surgical bleedings can be a serious complication after various surgical procedures. In the case of cardiac surgery 3-5 % of the patients need reoperation due to uncontrollable post surgical bleeding. It is possible to reduce blood loss by administrating aprotinin or ε-amino capronic acid; however these agents are only effective if administered prior to surgery. Due to the cost of aprotinin, this drug is in many hospitals only administered to high risk patients. Factor XIII (FXIII), the fibrin stabilising factor, is a transglutaminase that binds to and cross-links fibrin monomers in the haemostatic plug thereby providing a fibrin structure with increased mechanical strength and resistance against f ibrinolysis (see Ariens et al, Blood 100(3), 743-754 (2002)). Factor XIII is also known as "fibrinoligase" and "fibrin stabilizing factor". When activated, factor Xllla is able to form intermolecular gamma-glutamyl-ε-lysine cross-links between side chains of fibrin molecules and other substrates. Factor XIII is found in plasma and in platelets. The enzyme exists in plasma as a tetrameric zymogen consisting of two A-subunits (also referred to as "a") and two B subunits (also referred to as "b") (this tetrameric zymogen is designated A₂B₂ or a₂b₂) and in platelets as a zymogen consisting of two A-subunits (this dimeric zymogen is designated A₂- or a₂-dimer). Both zymogens are activated by thro bin and Ca²⁺. Ca²⁺ is released from the platelets upon aggregation at the site of injury. Thrombin cleaves off the N-terminal 37 amino acid residues of the A subunit of factor XIII. In case of the A₂B₂-zymogen, the B-subunits are dissociated from the activated A-subunits. Ca²⁺ binds equally well to the zymogen and to the thrombin modified molecule. Following thrombin and Ca²⁺ activation the active centre cysteine on the A-subunit is exposed and the fully activated enzyme is formed. Subjects with severe thrombocytopenia have been found to have low plasma levels of factor XIII. WO 93/12813 (ZymoGenetics) concerns the use of factor XIII for reducing perioperative blood loss in a subject undergoing surgery. The factor XIII is administered to the subject prior to or during surgery. Not all patients undergoing surgery experiences post surgical bleedings. Therefore it will be an advantage to have haemostatic agents effective in controlling post surgical bleedings when administrated post surgery in case excessive bleeding is observed.

SUMMARY OF THE INVENTION The present invention concerns methods of reducing post surgical bleeding and methods of reducing post surgical drainage in a patient after surgery, wherein the method comprises post surgical administration to said patient of an effective amount of factor XIII.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the overall clot formation after surgery using factor XIII alone and factor XIII in combination with factor Vila. Figure 2 shows the post surgical blood loss from six pigs undergoing cardiac surgery with three hours cardio pulmonary bypass. Data from individual animals as well as median value (line) are shown.

DESCRIPTION OF THE INVENTION The present invention concerns the post surgical administration of factor XIII alone or in combination with other haemostatic agents e.g. factor Vila or factor XI for the treatment of post surgical bleeding episodes. These haemostatic agent(s) are effective when administrated post surgically and therefore only need to be administrated to the patients showing uncontrollable bleedings after surgery. The present invention provides improved methods for reducing post surgical blood loss in patients having undergone surgery, in particular, in patients coming out of major thoracic or abdominal surgery or other surgeries having the potential for loss of large volumes of blood. These methods and compositions reduce or eliminate the need for whole donor blood or blood products, thereby reducing the risk of infection and other adverse side effects, as well as the cost of surgery. The methods are thus useful in reducing post surgical blood loss in normal patients, i.e. those not suffering from inborn or other pre-operative bleeding disorders such as defects or deficiencies in coagulation factors, such as congenital factor XIII deficiency. The reduction in blood loss is typically seen as reduced post surgical drainage. According to the present invention, an effective amount of factor XIII is combined with a biologically compatible vehicle and administered to a patient. The present invention provides a method of reducing post surgical blood loss in a patient after surgery, wherein the method comprises post surgical administration to said patient of an effective amount of factor XIII. In one embodiment, the present invention provides a method of reducing post surgical drainage in a patient after surgery, wherein the method comprises post surgical administration to said patient of an effective amount of factor XIII. By factor XIII (FXIII) means any protein with transglutaminase activity similar to plasma derived coagulation factor XIII, i.e. plasma derived factor XIII, platelet derived factor XIII, recombinant factor XIII A₂B₂, A₂ subunits, or A subunit, as well as genetically modified variants or chemically modified derivatives hereof, wherein these genetically modified variants or chemically modified derivatives have retained at least part of the activity of factor XIII. Non-limiting examples of factor XIII variants include: factor XIII in which one or more N-linked or O-linked glycosylation consensus sites have been modified, and cysteine variants in which one or more cysteine residues are eliminated or relocated, including, but not limited to, alterations that change the disulfide bonding pattern of the monomer or dimer. In one embodiment, the factor XIII variant is selected among factor XIII variants, which are amino acid sequence variants having no more than 20 amino acids replaced, deleted or inserted compared to wild-type factor XIII (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950), In one embodiment, the factor XIII is selected among factor XIII variants, which have no more than 15 amino acids replaced, deleted or inserted; in one embodiment, the factor XIII is selected among factor XIII variants, which have no more than 10 amino acids replaced, deleted or inserted; in one embodiment, the factor XIII is selected among factor XIII variants, which have no more than 8 amino acids replaced, deleted or inserted; in one embodiment, the factor XIII is selected among factor XIII variants, which have no more than 6 amino acids replaced, deleted or inserted; in one embodiment, the factor XIII is selected among factor XIII variants, which have no more than 5 amino acids replaced, deleted or inserted; in one embodiment, the factor XIII is selected among factor XIII variants, which have no more than 3 amino acids replaced, deleted or inserted compared to wild-type factor XIII. Non-limiting examples of factor XIII derivatives include: wild-type factor XIII or factor XIII variants that have been modified by phosphorylation, sulfation, PEGylation, or by the action of one or more glycosyltransferases and/or glycosidases, whether in vivo or in vitro. Also encompassed by the invention are chimeric or fusion polypeptides between all or part of the factor XIII sequence and other heterologous peptide sequences. In one embodiment, a binding site for LDL Receptor-associated protein (LRP) (such as, e.g., a peptide comprising residues Phe342-Asn346 of Factor IXa, which has been shown to contribute to the interaction with LRP, Rohlena et al., J. Biol. Chem. 278, 9394 (2003)) is attached to the sequence of factor XIII to modify its pharmacokinetic properties. In one embodiment of the present invention, the factor XIII protein used is a factor XIII protein syngeneic with the patient in order to reduce the potential risk of inducing an immune response. Preparation and characterization of non-human factor XIII has been disclosed by Nakamura et al. (J. Biochem. 78: 1247-1266, 1975). The present invention encompasses the use of such factor XIII proteins within veterinary procedures. Within the present invention an "effective amount" of a factor XIII, and other compounds for use in the present invention, is defined as the amount of the compound, which is sufficient to reduce bleeding or blood loss after surgery. It is understood that the effective amount of these compounds may be higher, when the factors are administered without the other compounds being administered than when these compounds are administered to the same patients, whether or not the administration takes place at the same time. An effective amount may for instance be an amount sufficient to reduce blood loss after surgery by at least 15%. The amount of factor XIII administered will be sufficient to provide a supranormal plasma level of factor XIII. An effective amount of factor XIII will generally be in the range of about 0.1 to 1.0 mg per kg of patient weight (i.e. in this example a dose of about 10 mg to about 70 mg for a 70 kg patient). Doses may also be in the range of about 0.15 mg to 0.4 mg per kg of patient weight, for instance about 0.25 mg per kg of patient weight. The actual amount of factor XIII administered will depend in part on such factors as the nature of the surgery and overall patient condition, including pre-existing factor XIII levels. Factor XIII for use within the present invention may be prepared from plasma according to known methods, such as those disclosed by Cooke and Holbrook (Biochem. J. 141, 79-84, 1974) and Curtis and Lorand (Methods Enzymol. 45: 177-191 , 1976), incorporated herein by reference. The A₂ dimer form of factor XIII may be prepared from placenta as disclosed in U.S. Patents 3,904,751 ; 3,931 ,399; 4,597,899 and 4,285,933, incorporated herein by reference. Factor XIII for use within the present invention may also be recombinant factor XIII so as to avoid the use of blood- or tissue-derived products that carry a risk of disease transmission. Methods for preparing recombinant factor XIII are known in the art. See, for example, Davie et al., EP 268,772 and Grundmann et al., AU-A-69896/87, which are incorporated herein by reference in their entirety. In one embodiment, the factor XIII is prepared cytoplasmically in the yeast Pichica pastoris as described in Park et al., Biotechnology Letters 24(2), 97-101 (2002). In one embodiment, the factor XIII is prepared in Escherichia coli as described in Lai

TS et al., Protein Expression and Purification 5, (2) 125-132 (1994). In one embodiment, the factor XIII A₂ dimer is prepared cytoplasmically in the yeast Saccharomyces cerevisiae as disclosed in United States Patent Application Publication No. US 5,612,456 and PCT application WO93/03147, incorporated herein by reference in their entirety. The cells are harvested and lysed, and a cleared lysate is prepared. The lysate is fractionated by anion exchange chromatography at neutral to slightly alkaline pH using a column of derivatized agarose, such as DEAE Fast-FlowSepharoseTM (Pharmacia) or the like. Factor XIII is then precipitated from the column eluate by concentrating the eluate and adjusting the pH to 5.2-6.5, such as by diafiltration against ammonium succinate buffer. The precipitate is then dissolved and further purified using conventional chromatographic techniques, such as gel filtration and hydrophobic interaction chromatography. A "post surgical" time period encompasses the time period starting immediately after surgery and ending about 48 hours later. In one embodiment, the "post surgical" time period encompasses the time period starting immediately after surgery and ending about 36 hours later. In one embodiment, the post surgical time period encompasses the time period starting immediately after surgery and ending about 24 hours later. In one embodiment, the post surgical time period encompasses the time period starting immediately after surgery and ending about 18 hours later. A post surgical bleeding episode is a bleeding episode which starts within the post surgical time period. The post surgical bleeding episode - whether treated according to the present invention or not - may end after the expiry of the post surgical time period. A post surgical bleeding episode may for instaiace encompass post surgical drainage. A post surgical administration is an administration regime, which is initiated within the post surgical time period. A post surgical administration regime may comprise a single administration, which is then performed within the post surgical time period. However, a post surgical administration regime may also comprise two or more administrations of which only the first one needs to be performed within the post surgical time period. The. purpose of a post surgical administration regime according to the present invention is to treat one or more post surgical bleeding episodes. In one embodiment, the patient does not suffer from congenital factor XIII deficiency. In one embodiment, the factor XIII is a recombinant factor XIII. In one embodiment, the factor XIII is a factor XIII A₂ dimer. In one embodiment, the factor XIII is a factor XIII A monomer. In one embodiment, the factor XIII is administered at a dose of from about 0.1 to about 1.0 mg per kg of patient weight. In one embodiment, the factor XIII is administered at a dose of from about 0.15 to about 0.4 mg per kg of patient weight. In one embodiment, the factor XIII is administered at a dose of about 0.25 mg per kg of patient weight. In one embodiment, no pre-operative administration of factor XIII takes place. In one embodiment, no pre-operative administration of factor XIII takes place within 1 hour prior to surgery. In one embodiment, no pre-operative administration of factor XIII takes place within 2 hours prior to surgery. In one embodiment, no pre-operative administration of factor XIII takes place within 4 hours prior to surgery. In one embodiment, no pre-operative administration of factor XIII takes place within

6 hours prior to surgery. In one embodiment, no pre-operative administration of factor XIII takes place within 12 hours prior to surgery. In one embodiment, no pre-operative administration of factor XIII takes place within 24 hours prior to surgery. In one embodiment, no pre-operative administration of factor XIII takes place within 48 hours prior to surgery. In one embodiment, no pre-operative administration of factor XIII takes place within seven days prior to surgery. In one embodiment, the patient is treated with an effective amount of factor Vila prior to, during or after surgery. The term factor VII (FVII) includes zymogen factor VII (single-chain factor VII), activated factor VII (FVIIa) as well as genetically modified variants or chemically modified derivatives hereof, wherein these genetically modified variants or chemically modified derivatives have retained at least part of the activity of factor VII or Vila. Non-limiting examples of factor VII variants having substantially the same or better biological activity compared to wild-type factor Vila include, but are not limited to, those described in Danish Patent Applications Nos. PA 2000 00734, PA 2000 01360, PA 2000 01361 , and PA 2001 00477. In one embodiment, the factor Vila variant is selected among factor Vila variants, which are amino acid sequence variants having no more than 20 amino acids replaced, deleted or inserted compared to wild-type factor Vila (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950), In one embodiment, the factor Vila is selected among factor Vila variants, which have no more than 15 amino acids replaced, deleted or inserted; in one embodiment, the factor Vila is selected among factor Vila variants, which have no more than 10 amino acids replaced, deleted or inserted; in one embodiment, the factor Vila is selected among factor Vila variants, which have no more than 8 amino acids replaced, deleted or inserted; in one embodiment, the factor Vila is selected among factor Vila variants, which have no more than 6 amino acids replaced, deleted or inserted; in one embodiment, the factor Vila is selected among factor Vila variants, which have no more than 5 amino acids replaced, deleted or inserted; in one embodiment, the factor Vila is selected among factor Vila variants, which have no more than 3 amino acids replaced, deleted or inserted compared to wild-type factor Vila. In one embodiment, the factor Vila variants are selected from the list of [L305V]-FVIIa, [L305V/M306D/D309S]-FVIIa, [L305l]-FVIIa, [L305T]-FVIIa, [F374P]-FVIIa, [V158T/M298Q]- FVIIa, [V158D/E296V/M298Q]-FVIIa and [K337A]-FVIIa. Non-limiting examples of factor VII derivatives include: wild-type factor VII or factor

VII variants that have been modified by phosphorylation, sulfation, PEGylation, or by the action of one or more glycosyltransferases and/or glycosidases, whether in vivo or in vitro. Also encompassed by the invention are chimeric or fusion polypeptides between all or part of the factor VII sequence and other heterologous peptide sequences. In one embodiment, a binding site for LDL Receptor-associated protein (LRP) (such as, e.g., a peptide comprising residues Phe342-Asn346 of Factor IXa, which has been shown to contribute to the interaction with LRP, Rohlena et al., J. Biol. Chem. 278, 9394 (2003)) is attached to the sequence of factor VII to modify its pharmacokinetic properties. Factor VII polypeptides for use in the present invention include, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild-type human factor VII, as well as polypeptides in which the factor VII biological activity has been substantially modified or reduced relative to the activity of wild-type human factor VII. In one embodiment, the patient is treated with an effective amount of factor VIII prior to, during or after surgery. The term factor VIII (FVIII) includes factor VIII polypeptides derived from blood or plasma or from platelets or those produced by recombinant means in any suitable host organism or cell. Also encompassed are factor VIII polypeptides in their uncleaved (zymogen) form, factor VII polypeptides that have been proteolytically processed to yield their respective bioactive forms (designated factor Villa) as well as genetically modified variants or chemically modified derivatives hereof, wherein these genetically modified variants or chemically modified derivatives have retained at least part of the activity of factor VIII. Non-limiting examples of factor VIII variants include: factor VIII in which one or more N-linked or O-linked glycosylation consensus sites have been modified, and cysteine variants in which one or more cysteine residues are eliminated or relocated, including, but not limited to, alterations that change the disulfide bonding pattern of the monomer or dimer. In one embodiment, the factor VIII variant is selected among factor VIII variants, which are amino acid sequence variants having no more than 20 amino acids replaced, deleted or inserted compared to wild-type factor VIII (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950), In one embodiment, the factor VIII is selected among factor VIII variants, which have no more than 15 amino acids replaced, deleted or inserted; in one embodiment, the factor VIII is selected among factor VIII variants, which have no more than 10 amino acids replaced, deleted or inserted; in one embodiment, the factor VIII is selected among factor VIII variants, which have no more than 8 amino acids replaced, deleted or inserted; in one embodiment, the factor VIII is selected among factor VIII variants, which have no more than 6 amino acids replaced, deleted or inserted; in one embodiment, the factor VIII is selected among factor VIII variants, which have no more than 5 amino acids replaced, deleted or inserted; in one embodiment, the factor VIII is selected among factor VIII variants, which have no more than 3 amino acids replaced, deleted or inserted compared to wild-type factor VIII. Non-limiting examples of factor VIII derivatives include: wild-type factor VIII or factor

VIII variants that have been modified by phosphorylation, sulfation, PEGylation, or by the action of one or more glycosyltransferases and/or glycosidases, whether in vivo or in vitro. Also encompassed by the invention are chimeric or fusion polypeptides between all or part of the factor VIII sequence and other heterologous peptide sequences. In one embodiment, a binding site for LDL Receptor-associated protein (LRP) (such as, e.g., a peptide comprising residues Phe342-Asn346 of Factor IXa, which has been shown to contribute to the interaction with LRP, Rohlena et al., J. Biol. Chem. 278. 9394 (2003)) is attached to the sequence of factor VIII to modify its pharmacokinetic properties. Factor VIII polypeptides for use in the present invention include, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild-type human factor VIII, as well as polypeptides in which the factor VIII biological activity has been substantially modified or reduced relative to the activity of wild-type human factor VIII. In one embodiment, the patient is treated with an effective amount of factor XI prior to, during or after surgery. The term factor XI (FXI) includes factor XI polypeptides derived from blood or plasma or from platelets or those produced by recombinant means in any suitable host organism or cell. Also encompassed are factor XI polypeptides in their uncleaved (zymogen) form, factor XI polypeptides, that have been proteolytically processed to yield their respective bioactive forms (designated factor Xla)as well as genetically modified variants or chemically modified derivatives hereof, wherein these genetically modified variants or chemically modified derivatives have retained at least part of the activity of factor XI. Non-limiting examples of factor XI variants include: factor XI in which one or more N- linked or O-linked glycosylation consensus sites have been modified, single-chain factor XI (i.e., factor XI in which the monomer polypeptides are not subject to intrachain proteolytic cleavage as in the wild-type), and cysteine variants in which one or more cysteine residues are eliminated or relocated, including, but not limited to, alterations that change the disulfide bonding pattern of the monomer or dimer. In one embodiment, Cys11 (which is not believed to participate in inter- or intramolecular disulfide bonding) is eliminated or substituted. In one embodiment, the factor XI variant is selected among factor XI variants, which are amino acid sequence variants having no more than 20 amino acids replaced, deleted or inserted compared to wild-type factor XI (i.e., a polypeptide having the amino acid sequence disclosed in U.S. Patent No. 4,784,950), In one embodiment, the factor XI is selected among factor XI variants, which have no more than 15 amino acids replaced, deleted or inserted; in one embodiment, the factor XI is selected among factor XI variants, which have no more than 10 amino acids replaced, deleted or inserted; in one embodiment, the factor XI is selected among factor XI variants, which have no more than 8 amino acids replaced, deleted or inserted; in one embodiment, the factor XI is selected among factor XI variants, which have no more than 6 amino acids replaced, deleted or inserted; in one embodiment, the factor XI is selected among factor XI variants, which have no more than 5 amino acids replaced, deleted or inserted; in one embodiment, the factor XI is selected among factor XI variants, which have no more than 3 amino acids replaced, deleted or inserted compared to wild-type factor XI. « Non-limiting examples of factor XI derivatives include: wild-type factor XI or factor XI variants that have been modified by phosphorylation, sulfation, PEGylation, or by the action of one or more glycosyltransferases and/or glycosidases, whether in vivo or in vitro (see, e.g., Ekdahl et al., Thromb. Haemost. 82, 1283-8 (1999)). Also encompassed by the invention are chimeric or fusion polypeptides between all or part of the factor XI sequence and other heterologous peptide sequences. For example, one or more of the four Apple domains may be substituted by similar apple domains from other polypeptides (see, e.g., Gailani et al., Blood 94, 621a (1999)) or one or more of the Apple domains may be deleted in its entirety. In one embodiment, a binding site for LDL Receptor-associated protein (LRP) (such as, e.g., a peptide comprising residues Phe342- Asn346 of Factor IXa, which has been shown to contribute to the interaction with LRP, Rohlena et al., J. Biol. Chem. 278, 9394 (2003)) is attached to the sequence of factor XI to modify its pharmacokinetic properties. Due to the dimeric nature of factor XI in its active form (and the asymmetric function of the two monomers in, e.g., platelet binding and FIX activation), factor XI also encompasses factor XI heterodimers, i.e., combinations of two non- identical factor XI (or factor Xl-related) monomer polypeptides. The only requirement is that the heterodimer exhibit one or more beneficial aspects of factor XI bioactivity. Factor XI polypeptides for use in the present invention include, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild-type 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. In one embodiment, the patient is treated with an effective amount of a TFPI inhibitor prior to, during or after surgery. 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. The present invention also provides the use of factor XIII for the preparation of a pharmaceutical composition for reducing post surgical blood loss in a patient after surgery. In one embodiment, the pharmaceutical composition is for administration after surgery. In one embodiment, the patient does not suffer from congenital factor XIII deficiency. In one embodiment, the factor XIII is recombinant factor XIII. In one embodiment, the factor XIII is a factor XIII A₂ dimer. In one embodiment, the factor XIII is a factor XIII A monomer. In one embodiment, the pharmaceutical composition is to be administered at a dose of from about 0.1 to about 1.0 mg per kg of patient weight. .„, In one embodiment, the pharmaceutical composition is to be administered at a dose of from about 0.15 to about 0.4 mg per kg of patient weight. In one embodiment, the pharmaceutical composition is to be administered at a dose of about 0.25 mg per kg of patient weight. In one embodiment, the patient is treated with an effective amount of factor Vila prior to, during or after surgery. In one embodiment, the patient is treated with an effective amount of factor VIII prior to, during or after surgery. In one embodiment, the patient is treated with an effective amount of factor XI prior to, during or after surgery. In one embodiment, the patient is treated with an effective amount of a TFPI inhibitor prior to, during or after surgery. In one embodiment, the present invention provides the use of factor XIII for the preparation of a pharmaceutical composition for the stated purposes, wherein the pharmaceutical composition also comprises factor Vila. In one embodiment, the present invention provides the use of factor XIII for the preparation of a pharmaceutical composition for the stated purposes, wherein the pharmaceutical composition also comprises factor VIII. In one embodiment, the present invention provides the use of factor XIII for the preparation of a pharmaceutical composition for the stated purposes, wherein the pharmaceutical composition also comprises factor XI. In one embodiment, the present invention provides the use of factor XIII for the preparation of a pharmaceutical composition for the stated purposes, wherein the pharmaceutical composition also comprises factor Vila and factor VIII. In one embodiment, the present invention provides the use of factor XIII for the preparation of a pharmaceutical composition for the stated purposes, wherein the pharmaceutical composition also comprises factor Vila and factor XI. In one embodiment, the present invention provides the use of factor XIII for the preparation of a pharmaceutical composition for the stated purposes, wherein the pharmaceutical composition also comprises factor Vila, factor VIII and factor XI. In one embodiment, the present invention provides the use of factor XIII for the preparation of a pharmaceutical composition for the stated purposes, wherein the pharmaceutical composition also comprises factor VIII and factor XI. The present invention encompasses pharmaceutical compositions comprising a preparation of factor XIII for post surgical administration. In one embodiment, the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly. They may also be administered by continuous or pulsatile infusion. It will be understood that any effective method for administering factor XIII may be used, including, e.g., using mucosal or inhalation methods of administration. Pharmaceutical compositions according to the invention comprise a factor XIII, preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent. Briefly, pharmaceutical compositions suitable for use according to the present invention are made by mixing a preparation comprising factor XIII, 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, saline (for instance 0.4%), glycine (for instance 0.3%), sugars, detergents, salts, buffers, glycerols, conservating agents, protease inhibitors, glycols, and the like. The pharmaceutical compostions of the present invention can also be formulated using non-aqueous carriers, such as, e.g., 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, e.g., 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 adjusting agents, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The compositions 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 according to the present invention in 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. Local delivery of the preparations of the present invention, such as, for example, topical application, may be carried out, e.g., by means of a spray, perfusion, double balloon catheters, stent, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, incorporation into gauze or other bandage materials, or other well established methods. In one embodiment, the invention provides a pharmaceutical product comprising (a) a composition comprising factor XIII alone or combined with one or more other haemostatic agents, such as for instance factor Vila, factor VIII or factor XI, (b) a pharmaceutically acceptable carrier, vehicle, excipient, diluent, preservative, stabilizer, binder, flavouring agent, an antioxidant, a colorant, adjuvant, disintegrating agent, solvent, a solubilizer, a suspending agent, a isotonizing/isotonic agent, a buffer, a soothing agent, or combination of any thereof as exemplified above, and, optionally, (c) a notice associated with said container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by said agency of said pharmaceutical product for human or veterinary administration to reduce post surgical blood loss. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a unit dosage form; and b) container means for containing said dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a factor Vila and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) container means for containing said dosage form. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a TFPI inhibitor and, optionally, a i, pharmaceutically acceptable carrier in a unit dosage form; and • b) container means for containing said dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; c) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing said first, second and third dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor Vila and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a factor Vila and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a third dosage form; and d) an effective amount of a TFPI-inhibitor and, optionally, a pharmaceutically acceptable carrier in a fourth dosage form; and e) container means for containing said first, second, third and fourth dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a factor Vila and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; c) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing said first, second and third dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a third unit dosage form; and d) container means for containing said first, second and third dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a factor VIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) an effective amount of a TFPI-inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and d) container means for containing said first, second and third dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a factor Vila and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor VIII and a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a TFPI-inhibitor and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor VIII and a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor XIII and a factor VIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a TFPI-inhibitor and a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) container means for containing said first and second dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor Vila and a factor VIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) an effective amount of a TFPI inhibitor and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and d) container means for containing said first, second and third dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a factor Vila and a TFPI-inhibitor and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form;

_* b) an effective amount of a factor XIII and, optionally j,.a pharmaceutically acceptable carrier in a second unit dosage form; and c) an effective amount of a factor VIII and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and d) container means for containing said first, second and third dosage forms. In one embodiment the present invention provides a kit containing a treatment for post surgical bleeding episodes, said kit comprising a) an effective amount of a TFPI-inhibitor and a factor VIII and, optionally, a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a factor XIII and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and c) an effective amount of a factor Vila and, optionally, a pharmaceutically acceptable carrier in a second unit dosage form; and d) container means for containing said first, second and third dosage forms. Recombinant factor XIII A or A₂ subunits has been produced and tested for effect on mechanical strength of clots formed ex vivo using blood from patients undergoing cardiac surgery as will be shown in the following non-limiting examples. None of the patients suffered from congenital factor XIII deficiency. Factor XIII alone or in combination with factor Vila improves the mechanical strength of the clots formed.

EXAMPLES

Example 1 Recombinant factor XIII A or A₂ subunits has been produced and tested for effect on mechanical strength and resistance against fibrinolysis of clots formed ex vivo using blood from patients undergoing cardiac surgery. Factor XIII alone or in combination with factor Vila improves the mechanical strength of the clots formed. Blood was obtained before and after surgery from 3 patients undergoing cardiac surgery with cardiopulmonary bypass. The effect of factor XIII on clot formation and stability was evaluated using rotational thromboelastography (roTEG), using the method of Vig et al. Blood Coag Fibrinol. 12, 555 (2001 ). Briefly, coagulation in whole citrated blood was initiated by adding lipidated tissue factor (innovin (Dade Behring) at a final dilution of 1 :50,000) and CaCI₂ (final concentration 15 nM), in the presence or absence of factor XIII (120 nM A subunit) or factor XIII (120nM A subunit) in combination with factor Vila (25nM). Fibrinolysis was initiated by addition of 4 nM tPA (American Diagnostica). Measurements were made using a ROTEG-04 Whole Blood Haemostasis System Rotation Thrombelastography apparatus (Pentapharm GmBH). Overall Clot Quality (OCQ) is calculated as: „ Max Vel X (t _min vel - t max vel) / t max vel where Max vel is the maximal velocity, t max vel is the time until maximal velocity is obtained, and t_mi_n ei is the time until minimal velocity (corresponding to maximal fibrinolysis) is obtained. OCQ is then normalized to the control sample (the values obtained in the absence of any haemostatic agents). The results are shown in Figure 1. Factor XIII as well as factor XIII in combination with factor Vila considerably improved the overall clot quality and showed an increased resistance to fibrinolysis.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law). All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. A description herein of an aspect or embodiment of the invention using terms such as "comprising", "encompassing", "having," "including," or "containing" a particular element is intended to provide support for an aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element, unless otherwise stated or clearly contradicted by context. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

Claims

1. A method of reducing post surgical blood loss in a patient after surgery, wherein the method comprises post surgical administration to said patient of an effective amount of factor XIII.

2. A method of reducing post surgical drainage in a patient after surgery, wherein the method comprises post surgical administration to said patient of an effective amount of factor XIII.

3. A method according to claim 1 or claim 2, wherein the patient does not suffer from congenital factor XIII deficiency.

4. A method according to any of claims 1 to 3, wherein the factor XIII is a recombinant factor XIII.

5. A method according to any of claims 1 to 4, wherein the factor XIII is a factor XIII A₂ dimer.

6. A method according to any of claims 1 to 4, wherein the factor XIII is a factor XIII A monomer.

7. A method according to any of claims 1 to 6, wherein the factor XIII is administered at a dose of from about 0.1 to about 1.0 mg per kg of patient weight.

8. A method according to claim 7, wherein the factor XIII is administered at a dose of from about 0.15 to about 0.4 mg per kg of patient weight.

9. A method according to claim 8, wherein the factor XIII is administered at a dose of about 0.25 mg per kg of patient weight.

10. A method according to any of claims 1 to 9, wherein no pre-operative administration of factor XIII takes place.

11. A method according to any of claims 1 to 9, wherein no pre-operative administration of factor XIII takes place within 1 hour prior to surgery.

12. A method according to claim 11 , wherein no pre-operative administration of factor XIII takes place within 2 hours prior to surgery.

13. A method according to claim 12, wherein no pre-operative administration of factor XIII takes place within 4 hours prior to surgery.

14. A method according to claim 13, wherein no pre-operative administration of factor XIII takes place within 6 hours prior to surgery.

15. A method according to claim 14, wherein no pre-operative administration of factor XIII takes place within 12 hours prior to surgery.

16. A method according to claim 15, wherein no pre-operative administration of factor XIII takes place within 24 hours prior to surgery.

17. A method according to claim 16, wherein no pre-operative administration of factor XIII takes place within 48 hours prior to surgery.

18. A method according to claim 17, wherein no pre-operative administration of factor XIII takes place within seven days prior to surgery.

19. A method according to any of claims 1 to 18, wherein the patient is treated with factor Vila prior to, during or after surgery.

20. A method according to any of claims 1 to 19, wherein the patient is treated with factor VIII prior to, during or after surgery.

21. A method according to any of claims 1 to 20, wherein the patient is treated with factor XI prior to, during or after surgery.

22. A method according to any of claims 1 to 21 , wherein the patient is treated with a TFPI inhibitor prior to, during or after surgery.

23. Use of factor XIII for the preparation of a pharmaceutical composition for reducing post surgical blood loss in a patient after surgery.

24. Use according to claim 23, wherein the pharmaceutical composition is for administration after surgery.

25. Use according to claim 23 or claim 24, wherein the patient does not suffer from congenital factor XIII deficiency.

26. Use according to any of claims 23 to 25, wherein the factor XIII is recombinant factor XIII.

27. Use according to any of claims 23 to 26, wherein the factor XIII is a factor XIII A₂ dimer.

28. Use according to any of claims 23 to 26, wherein the factor XIII is a factor XIII A monomer.

29. Use according to any of claims 23 to 28, wherein pharmaceutical composition is to be administered at a dose of from about 0.1 to about 1.0 mg per kg of patient weight.

30. Use according to claim 29, wherein pharmaceutical composition is to be administered at a dose of from about 0.15 to about 0.4 mg per kg of patient weight.

31. Use according to claim 30, wherein pharmaceutical composition is to be administered at a dose of about 0.25 mg per kg of patient weight.

32. Use according to any of claims 23 to 31 , wherein the patient is treated with factor Vila prior to, during or after surgery.

33. Use according to any of claims 23 to 32, wherein the patient is treated with factor VIII prior to, during or after surgery.

34. Use according to any of claims 23 to 33, wherein the patient is treated with factor XI prior to, during or after surgery.

35. Use according to any of claims 23 to 34, wherein the patient is treated with a TFPI inhibitor prior to, during or after surgery.