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WO2006128497A1 - Formule pharmaceutique de facteur xi - Google Patents

Formule pharmaceutique de facteur xi Download PDF

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Publication number
WO2006128497A1
WO2006128497A1 PCT/EP2005/052511 EP2005052511W WO2006128497A1 WO 2006128497 A1 WO2006128497 A1 WO 2006128497A1 EP 2005052511 W EP2005052511 W EP 2005052511W WO 2006128497 A1 WO2006128497 A1 WO 2006128497A1
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WIPO (PCT)
Prior art keywords
fxi
polypeptide
pharmaceutical formulation
buffer
formulation according
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PCT/EP2005/052511
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English (en)
Inventor
Simon Bjerregaard Jensen
Dorthe Viuff
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Novo Nordisk A/S
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Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to PCT/EP2005/052511 priority Critical patent/WO2006128497A1/fr
Publication of WO2006128497A1 publication Critical patent/WO2006128497A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21027—Coagulation factor XIa (3.4.21.27)

Definitions

  • the present invention relates to the therapeutic use of human Factor Xl for the prevention and/or treatment of bleeding episodes, methods for the purification of factor Xl and factor Xl polypeptides from biological fluids, as well as pharmaceutical formulations.
  • Human Factor Xl is a serine protease consisting of two identical subunits, each having a molecular mass of about 80 kDa.
  • FXI circulates in plasma as a disulfide-linked homodimer having a molecular mass of ⁇ 160KDa.
  • FXI is activated by cleavage of each monomer between Arg 369 and He 370 to form an amino-terminal heavy chain of 50 kDa and a carboxy-terminal light chain of 35 kDa, which are disulfide-linked.
  • the protein is encoded by a 23 kb gene located on chromosome 4 (4q35) 15 exons and 14 introns coding for a mRNA consisting of 2,097 nucleotides, which in turn encodes an amino-terminal signal (leader) peptide of 18 amino acids and the 607 amino acids present in each monomer of the mature protein.
  • Exons Hl-X encode four tandem repeats sequences (Apple domains) homologous to similar domains found in human plasma PK (58% identity).
  • Exons XI-XV encode the typical trypsin-like catalytic domain, which is activated by proteolytic cleavage of the zymogen at an internal Arg 369-lle 370 bond to yield a heavy chain containing four Apple domains (369 amino acids) and the light chain or catalytic domain (238 amino acids).
  • TF tissue factor
  • FVII plasma Factor VII
  • FVIIa plasma Factor VII
  • FVIIa binding of Factor X to the TF-FVIIa complex and its proteolytic conversion to activated Factor X
  • FXa proteolytic conversion by FXa of prothrombin to thrombin
  • TFPI tissue factor pathway inhibitor
  • thrombin burst The relatively small amount of thrombin produced during this phase results in the activation of FXI to FXIa (which activates Factor IX to FIXa) and the activation of Factor V on the surface of platelets and the further activation of Factor X. These events further promote the formation of sufficient amounts of thrombin (the so-called "thrombin burst") to convert fibrinogen into fibrin, thereby stabilizing an initial platelet plug and resulting in appropriate hemostasis.
  • Dimeric FXI circulates in plasma as a zymogen in a non-covalent complex with the cofactor high molecular weight kininogen (HK) that promotes the binding of FXI to negatively charged surfaces and its activation by its cognate proteases, FXIIa, FXIa, and thrombin.
  • HK cofactor high molecular weight kininogen
  • the HK binding site to FXI involves multiple Apple domains (A1 , A2, A4), with the A2 domain being the most important.
  • Complex formation with HK in the presence of Zn 2+ ions has been shown to promote the binding of FXI to activated platelets.
  • FXI The interaction of FXI with the surface of activated platelets has been shown to be mediated via residues Ser 2 48-Val 2 7i within the A3 domain of FXI; residues Ser 248 , Arg 25 o, LyS 255 , Phe 26 o and Gln 26 3 have also been implicated in this interaction.
  • the A3 domain of FXI also contains a heparin binding site within residues Thr 249 -Phe 2 6o and residues LyS 252 and LyS 253 have been implicated in the binding to platelets.
  • FXI and HK circulate in plasma in a non-covalent complex, and HK has been shown to bind to the surface of activated platelets, the interaction of FXI with the platelet surface apparently does not require binding of HK-FXI complex. Instead, it appears that the FXI dimer binds directly to a high-affinity, specific site on activated platelets (approx. 1500 sites/platelet; K d at approx 10 nM). The isolated recombinant A3 domain of FXI binds to the same number of sites on activated platelets and with the same affinity as the FXI dimer.
  • the activated enzyme, FXIa has also been shown to bind to high-affinity, saturable sites on activated platelets (Kd at approx 800 pM; 500 sites/platelet) and can activate FIX at rates similar to those observed in solution.
  • the substrate FIX binding site in FXI involves both a subdomain (Alai 34 -Leui 72 ) in the A2 domain and two subdomains ( I lew VaIi 92 and Ser 259 -Ser 265 ) within the A3 domain. Binding to the platelet surface is mediated by the glycoprotein I b-V-IX complex utilizing one polypeptide chain of the FXI dimer, thereby presenting the other monomer as a substrate binding site for FIX. It is likely that FIXa generation serves to localize FIXa-catalyzed FX activation to the platelet surface which also promotes prothrombin activation by FXa.
  • FXIa is also subject to regulation by a variety of plasma and platelet protease inhibitors whose functional activity appears to depend on whether FXIa is bound to the platelet surface or whether it is free in solution.
  • PNII is a potent inhibitor of FXIa with a Ki of 300-500 pM that is significantly enhanced in the presence of heparin. Binding of FXI to the platelet surface in the presence of HK and Zn 2+ ions or in the presence of prothrombin and Ca 2+ is protected from inactivation by both PNII and ⁇ -1 -protease inhibitor showing that FXIa activity generated on the surface of platelet is localized to the haemostatic thrombus whereas the site of regulation of FXIa by PNII and other protease inhibitors occurs in solution.
  • endothelial cells which contain heparan sulphate glycosaminoglycans, might promote the assembly of FXIa/PNII complexes thereby potentiating the inhibition of FXIa on the endothelium.
  • FXI thrombin-activatable fibrinolysis inhibitor
  • platelets and megakaryocytes apparently synthesize a second form of FXI, designated platelet-derived FXI (pd-FXI), which differs from the circulating form in lacking Exon V, which is the first exon of the two exons encoding the second Apple domain, and in vitro studies have shown that the preferred substrate for platelet factor XIa may be plasma FXI and not FIX.
  • Platelet FXI (Mr 220 KDa) has been found to be associated with the platelet plasma membrane. Platelets contain about 300 molecules of pd-FXI/cell.
  • FXI deficiency is an autosomal recessive syndrome characterized by a variable tendency to bleed. Even if severe, the deficiency may be clinically asymptomatic until the patient is challenged by surgical trauma; however, in some cases bleeding can occur regardless of the severity of the deficiency.
  • Optimal management of patients with FXI deficiency requires attention to a number of features in addition to the FXI level. First, it is important to evaluate the bleeding tendency in an individual with partial deficiency and whether additional factors are making a significant contribution. Such assessment should include measurement of FVIIIC and von Willebrand factor levels, the bleeding time and platelet aggregation.
  • Fresh frozen plasma has been used to treat the first known cases of FXI deficiency and was the main treatment until the development of FXI concentrate.
  • the main disadvantages of plasma are the large volumes required, allergic reactions and the potential for transmission of infectious agents.
  • Two FXI concentrates are currently available.
  • the FXI concentrate from Bio Products Laboratory (BPL) (England) is formulated with a high concentration of antithrombin (mean 102 iu/ml) and heparin (10u/ml) which is thought to protect against any residual FXIa.
  • a second FXI concentrate is produced by Hemoleven (France) and the product is formulated with 3-5 u/ml heparin, 2-3 iu/ml of antithrombin and C1 inhibitor. Furthermore, it has been reported that it is possible to pasteurize pooled fresh frozen plasma with preservation of 75-95% activity of FXI. Patients with mild FXI deficiency are generally treated with fresh frozen plasma, though patients with severe FXI deficiency may be treated with a FXI concentrate.
  • WO2003007983 discloses the use of a combination of factor Vila and FXI for treatment of bleeding episodes.
  • the present invention provides stable pharmaceutical formulations comprising recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form.
  • the present invention provides a pharmaceutical formulation comprising (i) isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, and (ii) a pharmaceutically acceptable carrier or excipient, wherein said formulation is stable against significant activation of said uncleaved zymogen FXI polypeptide for more than 3 months at 5 0 C.
  • a pharmaceutical formulation comprising: (i) isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, and (ii) a pharmaceutically acceptable carrier or excipient, wherein said formulation exhibits less than about 1% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 1 month at 5 Q C.
  • the present invention further provides methods and compositions for treating bleeding episodes.
  • the methods are carried out by administering to a patient in need thereof a preparation comprising a factor Xl (FXI) polypeptide, in an amount effective for such treatment.
  • the methods of the invention result in one or more of: reduced clotting time; enhancement of hemostasis; increase in clot lysis time; increase in clot strength; and/or increase in overall clot quality (OCQ) in said patient.
  • the patient following administration of a FXI polypeptide, the patient exhibits an effective FXI plasma concentration of at least about 5 nM, 10 nM, 30 nM, 60 nM, or 120 nM.
  • the FXI polypeptide comprises the sequence of SEQ ID NO:1 , or a fragment thereof that retains at least one FXI-associated biological activity. In some embodiments, the FXI polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity. In some embodiments, the FXI polypeptide comprises a chemically modified derivative of SEQ ID NO:
  • the FXI polypeptide has the sequence of SEQ ID NO:1. In some embodiments, the FXI polypeptide has the sequence of SEQ ID NO:2. In some embodiments, the patient does not suffer from a congenital FXI deficiency.
  • the bleeding episodes are secondary to surgery, a dental procedure, trauma, or hemodilution.
  • the patient suffers from aquired FXI deficiency.
  • the patient suffers from aquired FXI deficiency, wherein the patient has a FXI plasma concentration lower than 90 % of normal human plasma FXI concentration, such as lower than 80 % of normal human plasma FXI concentration, such as lower than 70 % of normal human plasma FXI concentration, such as lower than 60 % of normal human plasma FXI concentration, such as lower than 50 % of normal human plasma FXI concentration, such as lower than 40 % of normal human plasma FXI concentration, such as lower than 30 % of normal human plasma FXI concentration, such as lower than 20 % of normal human plasma FXI concentration, such as lower than 10 % of normal human plasma FXI concentration.
  • the invention also provides methods and compositions for preventing bleeding episodes.
  • the methods are carried out by administering to a patient in need thereof a preparation comprising a FXI polypeptide, in an amount effective to prevent bleeding.
  • the methods of the invention further comprise, prior to administration of a FXI polypeptide: (a) obtaining a sample of blood from said patient; (b) determining at least one of: FXI concentration, ratio of FXIa:FXI, or amount of exogenous FXI necessary to restore coagulation; and (c) based on the results of step (b), determining said amount of FXI effective for treatment.
  • the methods of the invention does not comprise administration of a Factor Vll/Factor Vila coagulation agent.
  • a Factor Vll/Factor Vila coagulation agent is a Factor VII polypeptide or a Factor Vll-related polypeptide as described in WO2003007983.
  • the invention also provides methods and compositions for treating bleeding episodes in which a patient is administered (i) a first amount of a preparation comprising a
  • FXI polypeptide and (ii) a second amount of a preparation comprising a non-Factor Vll/Factor
  • Non-limiting examples of non-Factor Vll/Factor Vila coagulation agents include: Factor XII, phospholipids, Factor XIlI; tissue factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis inhibitor (TAFI); plasminogen activator inhibitor-1 (PAI-1); Factor V; protein C inhibitor; protein S inhibitor; tissue plasminogen activator (tPA) inhibitor; prothrombin, Factor VIII, fibrinogen, and Factor
  • the invention also provides pharmaceutical formulations comprising (i) isolated recombinant FXI polypeptide and (ii) a pharmaceutically acceptable carrier or excipient.
  • the invention further provides methods for purifying a factor Xl polypeptide from a biological material, the method comprising subjecting the material to sequential chromatography on an cation-exchange chromatographic material, a hydrophobic interaction chromatographic material, and a Hydroxyapatite chromatographic material.
  • Figure 1 is a graphic representation of the effect of increasing amounts of FXI on overall clot quality in blood obtained from patients before and after cardiac surgery.
  • Figure 2 is a graphic representation of the effect of increasing amounts of FXI on overall clot quality in blood obtained from normal subjects.
  • Figure 3 is a graphic representation of the biological activity of different FXI formulations after storage at 5 0 C for 96 days.
  • Figure 4 is the preparative chromatogram of factor Xl polypeptide-containing fractions from first cation-exchange chromatography using Obelix ST CIEX (cat no 11 -0010) as described in example 7.
  • Figure 5 is the preparative chromatogram of factor Xl polypeptide-containing fractions from Hydrophobic interaction chromatography using Butyl Sepharose High Performance High Substitution (cat no 17-3100) as described in example 8.
  • Figure 6 is the preparative chromatogram of factor Xl polypeptide-containing fractions from Hydroxyapatite chromatography using CHT Hydroxyapatite Type I BioRad cat no 157-0020) as described in example 9.
  • the present invention is based on the finding of stable pharmaceutical formulations comprising recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form.
  • One broad aspect of the present invention provides a pharmaceutical formulation comprising (i) isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, and (ii) a pharmaceutically acceptable carrier or excipient, wherein said formulation is stable against significant activation of said uncleaved zymogen FXI polypeptide for more than 3 months at 5 0 C.
  • a pharmaceutical formulation comprising: (i) isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, and (ii) a pharmaceutically acceptable carrier or excipient, wherein said formulation exhibits less than about 1% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 1 month at 5 9 C.
  • the present invention provides methods for determining the dose for administration to a patient of isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, to replace the aquired FXI deficiency.
  • assays are described herein, which provides a unique opportunity for measuring FXI i human plasma and for the determination of dose to replace the aquired FXI deficiency.
  • FXI replacement may be calculated from the deficiency data:
  • concentration of FXI in plasma is measured in a patient with aquired FXI deficiency using the assays described herein or any other suitable assay for measuring FXI plasma concentrations.
  • the percentage of the deficiency compared to the concentration in normal human plasma is determined. Normal plasma FXI concentration is 5 ⁇ g/ml.
  • the present invention is also based on the surprising finding that exogenously administered Factor Xl (FXI) can be effective as a general hemostatic agent in human blood without the administration of Factor Vll/Factor Vila coagulation agents.
  • the therapeutic use of FXI according to the invention may provide one or more of: a shortened clotting time, a firmer clot, and an increased resistance of the formed clots to fibrinolysis and a reduction of bledding-associated complications.
  • the present invention provides methods and compositions useful in the therapeutic use of FXI in human patients for treating or preventing bleeding episodes, for enhancing hemostasis, for increasing clot lysis time, and/or for increasing clot strength.
  • the methods are carried out by administering to the patient an effective amount of Factor Xl for achieving one or more of these desired therapeutic goals.
  • the compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise FXI.
  • the compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise isolated FXI.
  • the compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise recombinant FXI.
  • compositions include pharmaceutical formulations for the therapeutic use of FXI that comprise isolated recombinant FXI.
  • the present invention relates to administration of FXI to normal human patients.
  • a "normal" human is one who does not suffer from a congenital deficiency in Factor Xl (i.e., Hemophilia C, see, Seligsohn (1993), Thromb. Haemost.
  • normal humans include, without limitation, patients exhibiting thrombocytopenia (lowered count or activity of platelets), patients contemplating or undergoing a surgical or dental procedure, and patients who have been subjected to trauma or organ damage and who, as a consequence, may exhibit lowered platelet counts and/or lowered levels of fibrinogen, FVIII, and/or other coagulation proteins.
  • Normal human patients encompass patients experiencing a transient decrease in the plasma levels of FXI (or any other coagulation-related protein or factor) due to bleeding, trauma, chemotherapy, liver disease, hemodilution (such as, e.g., may result from the infusion of plasma expanders or salt solutions to maintain blood volume or prevent shock), or any other circumstances not directly related to a congenital defect in a FXI gene.
  • FXI coagulation-related protein or factor
  • the present invention relates to administration of isolated and/or recombinant FXI to human patients suffering from a congenital FXI deficiency.
  • the present invention relates to administration of isolated and/or recombinant FXI to human patients suffering from aquired FXI deficiency.
  • any FXI polypeptide may be used that is effective in preventing or treating bleeding.
  • FXI polypeptides derived from blood or plasma or from platelets or those produced by recombinant means in any suitable host organism or cell.
  • FXI polypeptides in their uncleaved (zymogen) form as well as those that have been proteolytically processed to yield their respective bioactive forms (designated FXIa).
  • FXI polypeptides encompass, without limitation, FXI as well as FXI- related polypeptides.
  • FXI is intended to encompass, without limitation, polypeptides having the amino acid sequence of wild-type human plasma FXI, as described, e.g., in Fujikawa et al., Biochem. 25:2417 (1986), as well as wild-type FXI derived from other species, such as, e.g., bovine, porcine, canine, murine, rabbit, and salmon FXI.
  • FXI proteins syngeneic with the subject in order to reduce the risk of inducing an immune response.
  • Preparation and characterization of non-human FXI has been described, e.g., by Gailani (1997), Blood 90:1055.
  • the present invention also encompasses the use of such factor Xl proteins within veterinary procedures.
  • the FXI polypeptide is wild-type human plasma FXI (SEQ ID NO:1 ).
  • the FXI is platelet-derived FXI (pd-FXI) (SEQ ID NO:2), as described, e.g., in Hsu et al. (1998), J. Biol. Chem. 273:13787-93.
  • FXI polypeptides further encompass natural allelic variations of FXI that may exist and differ from one individual to another. Also, the degree and location of glycosylation or other post-translational modifications may vary in some circumstances, depending on the source of FXI-encoding nucleic acid, the host cells in which the FXI is produced, and the conditions in which the FXI-producing cells are maintained.
  • FXI-related polypeptides include, without limitation, FXI polypeptides that have either been chemically modified relative to human FXI (i.e., FXI derivatives) and/or contain one or more amino acid sequence alterations relative to human FXI (i.e., FXI variants). Such FXI-related polypeptides may exhibit an alteration in one or more aspects of biological activity relative to human FXI, including, without limitation, altered stability, altered phospholipid binding, altered specific enzymatic activity, altered immunogenicity, altered bioavailability, altered binding to one or more FXI binding partners, altered binding to FXI inhibitors, and the like.
  • FXI-related polypeptides 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 "FXIa-related polypeptides" or "activated FXI-related polypeptides".
  • FXI derivatives include: wild-type FXI or FXI 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. (1999), Thromb. Haemost. 82:1283-8).
  • FXI variants include: FXI in which one or more N-linked or O-linked glycosylation consensus sites have been modified, single-chain FXI (i.e., FXI 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.
  • Cysn which is not believed to participate in inter- or intramolecular disulfide bonding is eliminated or substituted.
  • the FXI variant has decreased half-life in plasma relative to wild-type human FXI. In one embodiment the FXI variant has a half-life lower than 50 hours. In one embodiment the FXI variant has a half-life lower than 24 hours. In one embodiment the FXI variant has a half-life lower than 12 hours. In one embodiment the FXI variant has a half-life lower than 6 hours. In one embodiment the FXI variant has a half-life lower than 3 hours.
  • FXI variants are polypeptides in which N-linked glycosylation at one or more sites has been disrupted by modified of the cognate N-linked glycosylation consensus sites, such as, e.g., by independent substitution with any amino acid of N72, N108, N335, N432, N473, or combinations of any of the foregoing.
  • Non-limiting examples of such variants include FXI-N72Q; FXI-N108Q; FXI-N335Q, FXI-N432Q, FXI- N473Q; FXI-N72Q/N108Q; FXI-N72Q/N108Q/N335Q; FXI-N72Q/N108Q/N335Q/N432Q; FXI-N72Q/N108Q/N335Q/N432Q/N473Q; FXI-N72Q/N432Q; FXI-N72Q/N473Q; FXI- N108Q/N432Q; FXI-N108Q/N473Q; and FXI-N432Q/N473Q.
  • Disruption of N-linked glycosylation at one or more of the sites may also be achieved, e.g., by: (i) independent deletion of any of residues 72-74, 108-110, 335-337, 432-434, and 473-475 (i.e., one or more residues at each site may be deleted and not substituted with any another amino acid) (ii) independent substitution of the N+2 residue (such as, e.g., substituting T74 to any residue other than S, substitution of S110 to any residue other than T, substitution of S337 to any residue other than S, substitution of S434 to any residue other than T, substitution of T475 to any residue other than T; (iii) substitution of the N+1 residues with a glycosylation-disrupting amino acid (exemplified by, but not limited to, proline (P). It will be understood that any combination of the above means may be used to independently disrupt glycosylation at different sites within the FXl polypeptide.
  • chimeric or fusion polypeptides between all or part of the FXI sequence and other heterologous peptide sequences.
  • one or more of the four Apple domains may be substituted by similar apple domains from other polypeptides (see, e.g., Gailani et al.(1999) Blood 94:621 a) or one or more of the Apple domains may be deleted in its entirety.
  • a binding site for LDL Receptor-associated protein (LRP) (such as, e.g., a peptide comprising residues Phe 342 - Asn 346 of Factor IXa, which has been shown to contribute to the interaction with LRP, Rohlena et al. (2003), J. Biol. Chem. 278:9394) is attached to the sequence of a FXI polypeptide to modify its pharmacokinetic properties.
  • FXI in its active form also enables preparations for use in the present invention that comprise FXI heterodimers, i.e., combinations of two non-identical FXI (or FXI-related) monomer polypeptides.
  • FXI heterodimers i.e., combinations of two non-identical FXI (or FXI-related) monomer polypeptides.
  • the only requirement is that the heterodimer exhibit one or more beneficial aspects of FXI bioactivity.
  • FXI polypeptides for use in the present invention include, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild- type human FXI, as well as polypeptides in which the FXI biological activity has been substantially modified or reduced relative to the activity of wild-type human FXI.
  • compositions comprising FXIa or FXIa- related polypeptides 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%, or at least about 130%, of the specific activity of compositions comprising solely wild-type FXI, when the wild-type FXI equivalent is one that has been obtained from the same source or produced in the same cell type, and when the activity comparison is made by parallel testing in an identical FXI activity assay.
  • the terms “activity” and “specific activity” apply, individually or in aggregate, to any aspect or aspects of FXI bioactivity.
  • the ratio between the specific proteolytic activity of a FXI- related polypeptide and the proteolytic activity of wild-type human FXI is at least about 1.25 when tested a FXI amidolytic assay; in other embodiments, the ratio is at least about 2.0; in further embodiments, the ratio is at least about 4.0.
  • one or more different aspects of FXI bioactivity may be quantified and used, e.g., in (i) selection of: appropriate FXI compositions for therapeutic administration, formulations, methods for FXI production or purification, and the like; and/or (ii) assessment of the efficacy of different therapeutic modalities.
  • “specific activity" of FXIa for any of these aspects of bioactivity is expressed as units of activity per unit mass of FXIa polypeptides.
  • Amidolytic activity may be quantified in vitro using a suitable chromogenic substrate, such as, e.g., S2355 (Chromogenix), as described in Ekdahl et al. (1999), Thromb. Haemost. 82:1283-8.
  • a suitable chromogenic substrate such as, e.g., S2355 (Chromogenix), as described in Ekdahl et al. (1999), Thromb. Haemost. 82:1283-8.
  • the measured activity is compared with a standard FXIa preparation having a defined specific activity (Enzyme Research Laboratories) and values are expressed as AU of FXIa activity.
  • FXI activation activity may be quantified directly in vitro by measuring the proteolytic conversion of factor IX to IXa as described for example, in Gailani et al. (2001 ), Blood 97: 3117-3122.
  • Wild-type human FXI has a number of binding partners, including prekallikrein (PK), high-molecular weight kininogen (HK), thrombin/prothrombin, Factor IX (FIX), and the platelet-associated FXI receptor designated GPI b-V-IX.
  • PK prekallikrein
  • HK high-molecular weight kininogen
  • FIX Factor IX
  • GPI b-V-IX the platelet-associated FXI receptor designated GPI b-V-IX.
  • any conventional binding assay may be used to quantify the affinity of FXI polypeptides for any of these (or other) binding partners.
  • binding assays include, but are not limited to, competition binding assays in which either binding partner is labelled.
  • FXl polypeptide binding partners are FXI active site inhibitors, including, without limitation, antithrombin III, C1 inhibitor, ⁇ 2 antitrypsin, PAM , protein C inhibitor, and protease nexin Il (PNII).
  • FXI active site inhibitors including, without limitation, antithrombin III, C1 inhibitor, ⁇ 2 antitrypsin, PAM , protein C inhibitor, and protease nexin Il (PNII).
  • the affinity of these compounds for FXI polypeptides may be quantified by use of conventional binding assays; alternatively, the inhibitory activity of such compounds for the proteolytic activity of particular FXI polypeptide preparations may be measuring using an amidolytic or FIX-activation assay.
  • Clotting time, clot lysis time, and clot strength are clinical parameters used for assaying the status of patient's haemostatic system. Blood samples are drawn from the patient at suitable intervals after administration of a FXI polypeptide and one or more of these parameters are assayed. Alternatively, a FXI polypeptide or preparation may be used for in vitro/ex vivo treatment of blood that has been drawn from a human subject. Clotting time may be assayed by means of standard PT or aPTT assays. Clot lysis time and clot strength may be measured by thromboelastograpy as described by, e.g., Vig et al. (2001) Blood coagulation & fibrinolysis, Vol.
  • clot strength may be assayed as described by Carr et al, (1991 ), Am. J. Med. ScL 302: 13-8.
  • OCQ all clot quality
  • Wild-type human FXI is believed to have a half-life in plasma of approximately 50 hours, which is mediated at least in part to its interaction with HK.
  • FXI polypeptides which exhibit pharmacokinetic properties that differ from native FXI, may be used.
  • Non-limiting examples include FXI polypeptides that have been treated with sialidase to remove one or more terminal sialic acid residues from FXI-associated oligosaccharides, FXI polypeptides that have been modified by PEGylation, and FXI polypeptides exhibiting an altered interaction with HK.
  • pharmacokinetic properties may be calculated using, e.g., WinNonlin Professional Version 3.1 (Pharsight Inc., Mountain View, CA, USA). Calculations are performed using mean concentration values at each time point, if more than one value was present.
  • the following pharmacokinetic parameters may be calculated: AUC, AUCo /oE ⁇ t r a P , Cmax, t m ax, ⁇ z , t 1/2 , CL, and V z using the following formulas:
  • the linear trapezoidal rule is used from time 0 to t ma i ⁇ j
  • the log trapezoidal rule is used from time t max to the last time point t:
  • FXI polypeptides for use in the present invention may be prepared from plasma or from recombinant sources using any suitable method known in the art.
  • isolated refers to FXI polypeptides that have been separated from the cell in which they were synthesized or the medium in which they are found in nature (e.g., 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 nonadherent cells; and the like.
  • FXI polypeptides may be further purified.
  • Purification may be achieved using any method known in the art, including, without limitation, affinity chromatography, such as, e.g., on an anti-FXI antibody column or a peptide affinity column (non-limiting examples of which include Heparin, Blue, Red, L-arginine, Benzamidine peptide, other dyes, or RP-chromatography); hydrophobic interaction chromatography; ion- exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., any precipitation or crystallization using, e.g., salt, pH, ammonium sulphate, or other additives), or extraction and the like, as described in more detail above.
  • 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-FXI polypeptides derived from the host cell.
  • FXI from plasma may also be achieved by known methods, including, without limitation, those disclosed by Koide et al. (1977), Biochem. 16: 2279 and Bouma et al. (1977), J.Biol.Chem. 252:6432, incorporated herein by reference.
  • Methods for preparing recombinant FXI are known in the art. See, for example, Kemball-Cook et al. (1994), Gene 139:275, Fujikawa et al. (1986), Biochem. 25:2417, and Meijers et al.(1992), Blood 79:1435, which are incorporated herein by reference in their entirety.
  • FXIa is also commercially available from Enzyme Research Laboratories, South Bend, IN.
  • the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to chromatography on a cation-exchange chromatographic material.
  • the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to chromatography on a a hydrophobic interaction chromatographic material.
  • the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to chromatography on a hydroxyapatite chromatographic material.
  • the present invention further concerns a method for purifying a FXI polypeptide, such as recombinant FXI, from other biological material, the method comprising subjecting the material to sequential chromatography on a cation-exchange chromatographic material, a hydrophobic interaction chromatographic material and Hydroxyapatite chromatographic material. It is to be understood that a sequential chromatography is performed in the order as described.
  • the term "Hydroxyapatite chromatographic material" as used herein means any combination of chromatographic material.
  • Hydroxyapatite chromatographic material known in the art which is capable of binding a FXI polypeptides, such as a Hydroxyapatite matrix.
  • cation-exchange chromatographic material means any cation-exchange chromatographic material known in the art which is capable of binding a FXI polypeptides, such as a cation-exchange matrix.
  • hydrophobic interaction chromatographic material means any hydrophobic interaction chromatographic material known in the art which is capable of binding a FXI polypeptides, such as a hydrophobic interaction matrix.
  • the present invention concerns a method for purifying a FXI polypeptide from a biological material, the method comprising the step of: subjecting a biological material comprising a FXI polypeptide to chromatography on a first cation-exchange chromatographic material, said chromatography comprising:
  • the present invention concerns a method for purifying a FXI polypeptide from a biological material, the method comprising the steps of: subjecting the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) to chromatography using a hydrophobic interaction chromatographic material, said chromatography comprising: (v) applying the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) to said hydrophobic interaction chromatographic material; (vi) eluting unbound material from said hydrophobic interaction chromatographic material with buffer B, which buffer B is suitable for eluting material not bound to the hydrophobic interaction chromatographic material; and
  • the present invention concerns a method for purifying a FXI polypeptide from a biological material, the method comprising the steps of: subjecting the eluate from step (vii) or a fluid prepared by use of the eluate from step (vii) to chromatography using a Hydroxyapatite chromatographic material, said chromatography comprising: (viii) applying the eluate from step (vii) or a fluid prepared by use of the eluate from step (vii) to said hydroxyapatite chromatographic material;
  • the present invention concerns a method for purifying a FXI polypeptide from a biological material, the method comprising the steps of:
  • step (v) applying the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) to said hydrophobic interaction chromatographic material;
  • step (vii) eluting said FXI polypeptide from said hydrophobic interaction chromatographic material by gradient-elution with buffer B 1 , which buffer B 1 is suitable for eluting FXI polypeptide from said hydrophobic interaction chromatographic material; (c) subjecting the eluate from step (vii) or a fluid prepared by use of the eluate from step (vii) to chromatography using a Hydroxyapatite chromatographic material, said chromatography comprising:
  • step (viii) applying the eluate from step (vii) or a fluid prepared by use of the eluate from step (vii) to said hydroxyapatite chromatographic material; (ix) eluting unbound material from the hydroxyapatite chromatographic material with buffer C, which buffer C is suitable for eluting material not bound to the hydroxyapatite chromatographic material; and
  • Purification of a FXI polypeptide is the process of increasing the concentration of the FXI polypeptide in a sample in relation to other components of said sample, resulting in an increase of the purity of the FXI polypeptide. It should be understood that the concentration of a FXI polypeptide in a sample in relation to other components of said sample is not equivalent to the concentration of FXI polypeptide in the sample.
  • the increase in the purity of the FXI polypeptide may be followed measured by use of methods known in the art, such as for instance by use of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), HPLC (High Performance Liquid Chromatography) or Berichrome assays (Dade Behring Diagnostics), or Clot activity assay.
  • Biological material may be any material derived from or containing cells, cell components or cell products.
  • a biological material may be a biological fluid.
  • a biological fluid may be any fluid derived from or containing cells, cell components or cell products.
  • Biological fluids include, but are not limited to cell cultures, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood, plasma, serum, all of which may also be homogenizates and filtrates, and fractions thereof, for instance collected by chromatography of unfractionated biological fluids.
  • the FXI polypeptides may be purified from a wide variety of biological materials, including cell culture supematants, which naturally produce a FXI polypeptide, but also of cells which have been genetically modified to produce a FXI polypeptide, such as mammalian cells (for instance CHO cells) transformed with DNA coding for a FXI polypeptide.
  • cell culture supematants which naturally produce a FXI polypeptide
  • cells which have been genetically modified to produce a FXI polypeptide such as mammalian cells (for instance CHO cells) transformed with DNA coding for a FXI polypeptide.
  • the biological material may be treated by use of a number of methods prior to application on the first cation-exchange chromatographic material. Such methods include, but a not limited to, centrifugation, filtration.
  • the biological material is a biological fluid.
  • the biological fluid is the supernatant of a cell lysate.
  • the biological fluid is the supernatant of a yeast cell lysate.
  • the FXI polypeptide is purified from a cell culture, such as a mammalian cell culture, as described above.
  • a cell culture such as a mammalian cell culture
  • the mammalian cells Prior to the chromatography in step (a), the mammalian cells may be separated from cell culture supernatant by centrifugation and / or filtration.
  • Inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCI may be included before being subjected to chromatographic step (a).
  • a buffer is a solution comprising a substance, which substance is capable of preventing significant changes in the pH of solutions to which small amounts of acids or bases are added and thereby of maintaining largely the original acidity or basicity of the solution.
  • a buffer usually comprises a weak acid or weak base together with a salt thereof.
  • the pH of the biological fluid may be adjusted to the pH of buffer A prior the chromatography in step (a), for instance by using 1 M HCI or 1 M NaOH or by other means known in the art.
  • the first cation-exchange chromatographic material may be any cation-exchange chromatographic material known in the art which is capable of binding a FXI polypeptide under one set of conditions and releasing it under a different set of conditions, such as an cation-exchange chromatographic material comprising a sulphopropyl group.
  • cation-exchange chromatographic materials include derivatised dextrans, agarose, cellulose, polyacrylamide, and specialty silicas, such as carboxymetyl.
  • Suitable cation-exchange chromatographic material may be identified by subjecting a biological fluid comprising FXI polypeptide to chromatography on the cation-exchange chromatographic material of choice, collecting fractions and determining the purity and content of the fractions, for instance by use of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), HPLC (High Performance Liquid Chromatography), clotactivity or Berichrome assays (Dade Behring Diagnostics), monitoring the absorbance of the eluate at 280 nm and by use of other methods known in the art.
  • SDS-PAGE sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
  • HPLC High Performance Liquid Chromatography
  • Berichrome assays Dade Behring Diagnostics
  • Suitable cation-exchange chromatographic materials include, but are not limited to Streamline SP XL (Amersham Biosciences cat no 17-5073), Obelix ST CIEX (Amersham Biosciences cat no 11-0010), Streamline Direct CST (Amersham Biosciences 17-5266), S-Support Unosphere, BioRad cat no 156-0113 or Toyopearl SP-550C Toso Haas cat no 14028. In one embodiement Obelix ST CIEX is used.
  • the first cation-exchange chromatographic material may be pre-equilibrated with buffer A prior to application of the biological material.
  • Buffer A may comprise protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCI, but other commercially available protease inhibitors may also be used.
  • protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCI, but other commercially available protease inhibitors may also be used.
  • the pH of buffer A is between 6.5 and 9. In a further embodiment, the pH of buffer A is between 7 and 9. In a further embodiment, the pH of buffer A is about 8.
  • the conductivity of buffer A is less than about 40 mS/cm.
  • Buffer A" is used for the elution of the FXI .
  • concentration of one or more of the components of the buffer used for washing in step (ii), in this case buffer A and A' is increased or decreased during the course of elution or a new component is added to the buffer, and the concentration of this component . This increase or decrease may take place continuously or in discrete steps as it is known in the art.
  • a salt for instance NaCI
  • This specific cation exchanger can also be used as a hydrophobic interaction chromatographic resin, for this kind of resin it is customary to add a propandiol / glycerol to buffer A creating buffer A 1 . If both NaCI and Propandiol / glycerol is added to buffer A then FXI can be eluted.
  • the determination of which fractions containing FXI polypeptide to pool for further processing, for instance to exclude undesired impurities eluting at the beginning or the end of the FXI polypeptide elution, is within the knowledge of a person skilled in the art.
  • the general art of performing an cation-exchange chromatography with regard to for instance pre-equilibration, elution time, washing, reconstitution of the cation-exchange chromatographic material etc is well-known.
  • the eluate containing the FXI polypeptides protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and Benzamidine is added and then taken to step (v) .
  • the eluate may also be kept at, for instance, 4 0 C for 24 hours or longer, or at, for instance, -80 0 C.
  • the hydrophobic interaction chromatographic material for use in step (b) may be any hydrophobic interaction chromatographic material known in the art, which is capable of binding a FXI polypeptide under one set of conditions and releasing it under a different set of conditions, such as a hydrophobic interaction chromatographic material derivatised with phenyl, butyl or octyl groups, or polyacrylic resins.
  • Non-limiting examples of suitable hydrophobic interaction chromatographic material are AmberchromTM CG 71 (Tosoh Bioscience), Phenyl SepharoseTM High Performance (Amersham, cat no 17-1082), Phenyl SepharoseTM 6 Fast Flow High Substitution (Amersham, cat no 17-0973), Toyopearl® Butyl 650 (Tosoh Bioscience), Toyopearl® Phenyl (Tosoh Bioscience), SourceTM 15Phe (Amersham, cat no 17-0147), Butyl SepharoseTM High Performance High Substitution
  • the hydrophobic interaction chromatographic material uses butyl as a ligand.
  • Buffer B and NaCI may be added to the eluate from stage (iv) or a fluid prepared by use of the eluate from stage (iv) prior to the chromatography in step (b) in an amount of about one to two volumes or more, or a concentrated version of buffer B, comprising the same ingredients as buffer B, but in, e.g., twice the concentration, is added to the eluate from stage (iv) or a fluid prepared by use of the eluate from stage (iv) in an amount corresponding to the strength of the concentrated buffer (a twice-concentrated buffer is added in the amount of 1 ,5 volumes).
  • Buffer B may have a pH from about 5 to about 9, for instance about 8.
  • buffer B has a conductivity of more than 25 mS/cm, for instance more than 70 mS/cm. This may be achieved, for example, by use of a phosphate buffer or by other means known in the art, e.g. NaCI.
  • the conductivity of the eluate from step (iv) or a fluid prepared by use of the eluate from step (iv) is adjusted to a conductivity of at least about 60 mS/cm.
  • Buffer B' is used for the elution of the FXI polypeptide by gradient elution.
  • the composition of buffer B 1 is changed during the course of elution.
  • the concentration of one or more of the components of the buffer used for washing in step (vi), in this case buffer B is increased or decreased during the course of elution, or a new component is added to the buffer and the concentration of this component is then increased during the course of elution. This increase or decrease may take place continuously or in discrete steps, as is well known in the art.
  • the eluate from stage (vii) or a fluid prepared by use of the eluate from stage (vii) is treated by use of a method comprising a step of
  • This step, and optionally other steps of post-processing known in the art, may be carried out alone or in combination, and the order in which the steps are performed is not critical. The person skilled in the art will be able to determine how and when to perform these steps.
  • a stabilizing agents which are capable of increasing the physical and/or chemical stability of the FXI polypeptide is added to the fractions containing FXI.
  • the term "physical stability" of the FXI polypeptide as used herein refers to the potential tendency of the protein to form biologically inactive and/or insoluble aggregates or multimers of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces.
  • Physical stability of the FXI polypeptide when present in buffer A may be evaluated by means of visual inspection and/or turbidity measurements after exposing the formulation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods. Visual inspection of the FXI polypeptide when present in buffer may be performed in a sharp focused light with a dark background.
  • the turbidity of the composition may be characterized by a visual score ranking the degree of turbidity, for instance on a scale from 0 to 3 (a composition showing no turbidity then corresponding to a visual score 0, and a composition showing visual turbidity in daylight corresponding to visual score 3).
  • a composition is classified as physically unstable with respect to protein aggregation when it shows visual turbidity in daylight.
  • the turbidity of the composition may be evaluated by simple turbidity measurements well-known to the skilled person, for instance by measuring the optical density of the solution at a wavelength of 405 nm (OD 405 ).
  • Physical stability of the aqueous protein compositions may also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein.
  • the probe is preferably a small molecule that preferentially binds to a non-native conformer of the protein.
  • a small-molecule spectroscopic probe of protein structure is Thioflavin T.
  • Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
  • hydrophobic patch probes that bind preferentially to exposed hydrophobic patches of a protein.
  • the hydrophobic patches are generally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature.
  • these small molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like.
  • spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.
  • chemical stability of the FXI polypeptide when used herein refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potentially lower biological potency and/or potentially increased immunogenic properties compared to the native protein structure.
  • chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoided, and an increase in amounts of chemical degradation products is often seen during storage and use of the protein composition, as well-known to a person skilled in the art.
  • Most proteins are prone to deamidation, a process in which the side-chain amide group in glutaminyl or asparaginyl residues is hydrolysed to form a free carboxylic acid.
  • Any agent which is capable of significantly improving the physical and/or chemical stability of FXI polypeptide when present in buffer B' may be used as a stabilizing agent.
  • An agent suitable for use as stabilizing agent for instance, be a salt (e.g. sodium chloride), a sugar, an alcohol (such as an C 4 -C 8 alcohol), an alditol, an amino acid (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), a polyethyleneglycol (e.g. PEG400), or a mixture of one or more thereof.
  • Any sugar such as a mono-, di-, or polysaccharide, or a water-soluble glucan, may be used.
  • An alditol is a polyalcohol of structure HOCH 2 -[CH(OH)] n -CH 2 OH, where n is 1, 2, 3....etc.
  • substances which are sugars, alcohols or alditols are fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, glycerol (glycerine), propan-1 ,2-diol (propylene glycol), propan-1 ,3-diol, and butan-1 ,3-diol.
  • sugars, alcohols and alditols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the substance is soluble in the liquid preparation and improves the physical stability of a FXI polypeptide in solution. In this respect, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
  • one or more stabilizing agents of the polyalcohol type is added.
  • one or more stabilizing agents selected from the group consisting of glycerol (propan-1 ,2,3-triol), propylene glycol (propan-1 ,2-diol), propan-1 ,3-diol, propyl alcohol (1 -propanol) and isopropyl alcohol (2-propanol) is added.
  • one or more stabilizing agents selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol is added.
  • the stabilizing when the stabilizing is a liquid alcohol or liquid polyalcohol [such as, e.g., glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol or isopropyl alcohol], the stabilizing agent is present in a concentration of from about 5% by volume (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 20% (v/v).
  • a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 20% (v/v).
  • a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of about 10% (v/v). In a still further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of about 20% (v/v).
  • the stabilizing agent mentioned should be capable of increasing the physical and/or chemical stability, as described above, of the FXI polypeptide. Any agent which is capable of significantly improving the physical and/or chemical stability of FXI polypeptide (e.g. as determined by measuring turbidity at OD 405 over a period of time) may be used as a stabilizing agent.
  • the eluate from step (vii) may be used for the preparation of a pharmaceutical composition. This may involve a change of buffer, and/or adjustment of the conductivity and/or pH to physiological values, and/or other actions to render the eluate acceptable for use in mammals, such as humans; means of rendering such an eluate acceptable for use in this manner are well known in the art.
  • the eluate may also be kept at, e.g., 4 0 C for 24 hours or longer, or at, e.g., -8O 0 C.
  • the method further comprises a step of subjecting the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to chromatography on a hydroxyapatite chromatographic material, said chromatography comprising: (viii) applying the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to said hydroxyapatite chromatographic material;
  • step (x) eluting said FXI polypeptide from the hydroxyapatite chromatographic material with buffer C, which buffer C is suitable for eluting FXI polypeptides which bind to the hydroxyapatite chromatographic material in step (ix).
  • a fluid prepared by use of the eluate from stage (vii) may, for instance, be prepared before application.
  • the conductivity of the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vi), is adjusted to less than about 20 mS/cm by adding water. pH is adjusted to 5,8 to 9. In one embodiment pH is adjusted to 6,0.
  • the components of buffer C and buffer C may be chosen with a view to the desired final pharmaceutical composition of the FXI polypeptide. Such considerations are within the knowledge of a person skilled in the art.
  • buffer C comprises one or more stabilizing agents, which stabilizing agents are capable of increasing the physical and/or chemical stability, as described above, of the FXI polypeptide.
  • Any agent which is capable of significantly improving the physical and/or chemical stability of FXI polypeptide when present in buffer C e.g. as determined by measuring turbidity at OD 405 over a period of time
  • An agent suitable for use as stabilizing agent in buffer C may, for instance, be a salt (e.g. sodium chloride), a sugar, an alcohol (such as an C 4 -C 8 alcohol), an alditol, an amino acid (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), a polyethyleneglycol (e.g. PEG400), or a mixture of one or more thereof.
  • Any sugar such as a mono-, di-, or polysaccharide, or a water-soluble glucan, may be used.
  • Non-limiting examples of substances which are sugars, alcohols or alditols are fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, glycerol (glycerine), propan-1 ,2- diol (propylene glycol), propan-1 ,3-diol, and butan-1 ,3-diol.
  • sugars, alcohols and alditols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the substance is soluble in the liquid preparation and improves the physical stability of a FXI polypeptide in solution. In this respect, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
  • buffer C comprises one or more stabilizing agents of the polyalcohol type. In one embodiment of the present invention, buffer C comprises NaCI.
  • One or more stabilizing agents selected from the group consisting of glycerol (propan-1 ,2,3-triol), propylene glycol (propan-1 ,2-diol), propan-1 ,3-diol, propyl alcohol (1 -propanol) and isopropyl alcohol (2-propanol) is added to the fluid from (x).
  • one or more stabilizing agents selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol is added.
  • propylene glycol is added.
  • a stabilizing agent of the liquid alcohol or liquid polyalcohol type when it is present in a concentration of from about 5% (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of from about 10% (v/v) to about 50% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type used is present in a concentration of from about 10% (v/v) to about 20% (v/v). In a further embodiment, a stabilizing agent of the liquid alcohol or liquid polyalcohol type is present in a concentration of about 10% (v/v).
  • Buffer C is used for the elution of the FXI polypeptide by gradient elution, wherein the composition of buffer C is changed during the course of elution.
  • concentration of one or more of the components of the buffer used for washing in step (ix), in this case buffer C is increased or decreased during the course of elution, or a new component is added to the buffer and the concentration of this component is then increased during the course of elution. This increase or decrease may take place continuously or in discrete steps, as is well known in the art.
  • a salt e.g.
  • the use of one or more stabilizing agents in any or all of the solutions used in purification of FXI results in an increase in the physical and/or chemical stability of FXI by at least 10%, 25%, 50%, or 100% over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
  • the use of one or more stabilizing agents in any or all of the solutions used in purification of FXI results in an increase in the physical and/or chemical stability of FXI by at least 2-fold, 5-fold, 10-fold, or 20-fold over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
  • Wild-type human FXI is normally activated by proteolytic cleavage between Arg 360 and He 370 , which may be catalyzed by FXIa, FXIIa, or thrombin. If desired, activation of FXI for use in the present invention may be achieved using FXIa or FXIIa (both from Enzyme Research Laboratories, South Bend, IN) or thrombin (Sigma). See, e.g., Sun et al. (1999) J. Biol Chem 51 :36373-36373 and Baglia (2003) J. Biol Chem 24:21744-21750. It is also within the scope of the invention to utilize other proteases to activate FXI polypeptides and in particular, FXI-related polypeptides.
  • the present invention encompasses methods and compositions for the therapeutic administration of FXI that utilize preparations having different FXI activation levels.
  • the methods and compositions employ FXI polypeptides that have not been subjected to any activation procedure.
  • the preparation of FXI or FXI- related polypeptide exhibits a ratio (by mass) of activated :zymogen FXI or FXI-related polypeptide of between about 1 :99 to about 99:1 , such as, e.g., between about 5:95 to about 95:5; about 10:90 to about 90:10; about 20:80 to about 80:20; about 30:70 to about 70:30; about 40:60 to about 60:40; and about 50:50.
  • the preparation contains not more than about 5% FXIa relative to the total FXI on a molar basis; more preferably, not more than about 2.5%, even more preferably, not more than about 1%, most preferably not more than about 0.5% or 0.1%. In some embodiments, the preparation contains not more than about 0.01-0.05% FXIa on a molar basis. In some embodiments, the preparation contains not more than about 0.01 -0.04% FXIa on a molar basis. In some embodiments, the preparation contains not more than about 0.01 -0.03% FXIa on a molar basis.
  • the invention also relates to FXI-related polypeptides that exhibit a differential capacity to be activated relative to wild-type FXI, such as, e.g., FXI-related polypeptides that are more easily activated by FXIIa than by thrombin, and vice versa; polypeptides that are constitutively activated, even in the absence of proteolytic cleavage; heterodimers in which one monomer (by virtue of mutation or chemical modification) cannot be proteolytically activated; and the like. Furthermore, the invention also relates to FXI-related polypeptides that are resistant to autoactivation, i.e. variants where the ratio between rate of activation by thrombin (and/or FXIIa) versus rate of activation by FXIa is higher than for wild-type FXI.
  • the methods and compositions of the invention may also employ treatment, pre- treatment, storage, or co-administration of a FXI polypeptide with additional agents that inhibit and/or promote activation.
  • agents that inhibit activation include C1 esterase inhibitor (C1 Inb), ⁇ -2 antiplasmin, ( ⁇ 2AP), ⁇ 1 -antitrypsin ( ⁇ 1AT), protease Nexin II, benzamidine, heparin, and antithrombin III;
  • agents that promote activation include FXIa, FXIIa, and thrombin.
  • compositions comprising FXI encompasses pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide for prophylactic and/or therapeutic treatment.
  • compositions or formulations according to the invention comprise a a FXI polypeptide, such as, e.g., at concentrations between 0.001 -100 mg/ml, that is preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier or diluent.
  • pharmaceutical compositions suitable for use according to the present invention are made by mixing a preparation comprising FXI and/or a FXI-related polypeptide, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent.
  • aqueous carriers such as water, buffered water, 0.4% saline, 0.3% glycine, sugars, detergents, salts, buffers, glycerols, preservatives, protease inhibitors, glycols, and the like.
  • the preparations of the 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.
  • compositions may contain pharmaceutically acceptable auxiliary substances or adjuvants, including, without limitation, pH adjusting and buffering agents, tonicity adjusting agents, preservatives, stabilizers, surfactants, chelating agents, and the like.
  • auxiliary substances or adjuvants including, without limitation, pH adjusting and buffering agents, tonicity adjusting agents, preservatives, stabilizers, surfactants, chelating agents, and the like.
  • 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.
  • the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a pH adjusting and buffering agent. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a tonicity adjusting agent. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a preservative. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a stabilizer. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a surfactant. In one embodiment of the invention, the pharmaceutical compositions comprising a preparation of FXI or FXI-related polypeptide further comprises a chelating agent.
  • Non-limiting examples of suitable buffers include acetate buffers, carbonate buffers, citrate buffers, glycylglycine buffers, histidine buffers, glycine buffers, lysine buffers, arginine buffers, phosphate buffers (containing, e.g.,sodium dihydrogen phosphate, disodium hydrogen phosphate or trisodium phosphate), TRIS [tris(hydroxymethyl)aminomethane] buffers, bicine buffers, tricine buffers, malate buffers, succinate buffers, maleate buffers, fumarate buffers, tartrate buffers, aspartate buffers, and mixtures thereof.
  • phosphate buffers containing, e.g.,sodium dihydrogen phosphate, disodium hydrogen phosphate or trisodium phosphate
  • TRIS [tris(hydroxymethyl)aminomethane] buffers bicine buffers, tricine buffers, malate buffers, succinate buffers, maleate buffer
  • Non-limiting examples of pharmaceutically acceptible preservatives include phenol, o-cresol, m-cresol, p-cresol, chlorocresol, methyl p-hydroxybenzoate, ethyl p- hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, 2- phenylethanol, benzyl alcohol, chlorobutanol, thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, benzethonium chloride, chlorphenesine (3-p- chlorphenoxypropane-1 ,2-diol), benzamidine and mixtures thereof.
  • the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In one further embodiment of the present invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In another further embodiment of the present invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In another further embodiment of the present invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml.
  • the use of a preservative in pharmaceutical compositions is well-known to the skilled person (see, e.g., Remington: The Science and Practice of Pharmacy, 19th edition, 1995).
  • Non-limiting examples of tonicity-adjusting agents (which are normally incorporated for the purpose of rendering the formulation substantially isotonic include salts (e.g. sodium chloride), sugars, alcohols (such as C 4 -C 8 alcohols), alditols, amino acids (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), polyethyleneglycols (e.g. PEG400), and mixtures thereof. Any sugar, such as a mono-, di-, or polysaccharide, or a water-soluble glucan, may be used.
  • salts e.g. sodium chloride
  • sugars e.g. sodium chloride
  • alcohols such as C 4 -C 8 alcohols
  • alditols e.glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threon
  • Non-limiting examples of substances which are sugars, alcohols or alditols are fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, glycerol (glycerine), propan-1 ,2-diol (propylene glycol), propan-1 ,3- diol, and butan-1 ,3-diol.
  • the sugars, alcohols and alditols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the substance is soluble in the liquid preparation.
  • the tonicity-adjusting agent is present in a concentration of from about 1 mg/ml to about 150 mg/ml. In a further embodiment of the present invention, the tonicity-adjusting agent is present in a concentration of from about 1 mg/ml to about 50 mg/ml. In one embodiment, the tonicity-adjusting agent is NaCI. In one embodiment, the tonicity-adjusting agent is NaCI present in a concentration of from about 1 mg/ml to about 150 mg/ml. In a further embodiment of the present invention, the tonicity-adjusting agent is NaCI present in a concentration of from about 1 mg/ml to about 50 mg/ml.
  • Non-limiting examples of chelating agents include salts of EDTA, citric acid and aspartic acid, and mixtures thereof.
  • a chelating agent is present in a concentration from 0.1 mg/ml to 5 mg/ml; from 0.1 mg/ml to 2 mg/ml; or from 2 mg/ml to 5 mg/ml.
  • compositions of the present invention may include as a therapeutically active component a polypeptide that possibly may exhibit aggregate formation during storage in liquid pharmaceutical compositions.
  • aggregate formation is intended to indicate a physical interaction between the polypeptide molecules that results in formation of oligomers which may remain soluble, or of large visible aggregates that precipitate from the solution.
  • the term “during storage” refers to a liquid pharmaceutical composition or formulation which, once prepared, is not immediately administered to a subject. Rather, following preparation, it is packaged for storage in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject.
  • dried form refers to a liquid pharmaceutical composition or formulation dried by freeze-drying [i.e. lyophilization; see, for example, Williams and PoIIi (1984), J. Parenteral Sci. Technol. 38:48-59], by spray-drying [see Masters (1991 ) in Sprav-Dryin ⁇ Handbook (5th ed; Longman Scientific and Technical, Essex, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; and
  • the pharmaceutical composition comprises an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition.
  • amino acid base indicates an amino acid or a combination of amino acids where any given amino acid is present either in its free base form or in its salt form.
  • amino acids for use in preparing compositions of the present invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid or glutamic acid. Any stereoisomer of a particular amino acid (e.g.
  • compositions of the present invention may be present in pharmaceutical compositions of the present invention so long as the particular amino acid is present either in its free base form or its salt form.
  • the L-stereoisomer is used.
  • Compositions of the present invention may also be formulated with analogues of these amino acids.
  • amino acid analogue is intended a derivative of the naturally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the present invention.
  • Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogues include ethionine and buthionine and suitable cysteine analogues include S-methyl-L cysteine.
  • the amino acid analogues are incorporated into the compositions in either their free base form or their salt form.
  • the compound imidazole is also to be regarded as an amino acid analogue in the context of the present invention.
  • the amino acids or amino acid analogues are used in a concentration which is sufficient to prevent or delay aggregation of the protein.
  • the pharmaceutical formulation comprises methionine (or another sulfur-containing amino acid or amino acid analogue) to inhibit oxidation of methionine residues to their sulfoxide form when the factor Xl polypeptide is a polypeptide comprising at least one methionine residue susceptible to such oxidation.
  • the term "inhibit oxidation” is intended to indicate minimization of accumulation of oxidized species (of methionine) with time. Inhibition of methionine oxidation results in greater retention of the polypeptide in its proper molecular form. Any stereoisomer of methionine (L, D or DL isomer) or combinations thereof can be used.
  • the amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of sulfoxide form of methionine is acceptable to regulatory agencies. Typically, this means that the composition contains no more than from about 10% to about 30% methionine sulfoxide form. This can in general be achieved by adding methionine in an amount such that the ratio of added methionine to methionine residues ranges from about 1 :1 to about 1000:1 , such as 10:1 to about 100:1.
  • Non-limiting examples of stabilizers include high-molecular-weight polymers or low- molecular-weight compounds, such as, e.g., polyethylene-glycols (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose and derivatives thereof (including HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulfur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, various salts (e.g. sodium chloride), glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol (1 -propanol) and isopropyl alcohol (2-propanol).
  • PEG 3350 polyethylene-glycols
  • PVA polyvinyl alcohol
  • PVC polyvinylpyrrolidone
  • carboxy-/hydroxycellulose and derivatives thereof including HPC, HPC-
  • Non-limiting examples of surfactants include detergents, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (e.g. poloxamers such as Pluronic ® F68, poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives ("Tweens", e.g.
  • Tween-20, Tween-40, Tween-80 and Brij-35 monoglycerides and ethoxylated derivatives thereof, diglycerides and polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (eg. phosphatidyl-serine, phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidyl-inositol, diphosphatidyl-glycerol and sphingomyelin), derivatives of phospholipids (e.g. dipalmitoyl-phosphatidic acid) and lysophospholipids (e.g.
  • phospholipids e.g. dipalmitoyl-phosphatidic acid
  • lysophospholipids e.g.
  • cholines ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DGP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (e.g. cephalins), glyceroglycolipids (e.g. galactopyranoside), sphingoglycolipids (e.g. ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives (e.g.
  • long-chain fatty acids e.g. C 6 -Ci 2 fatty acids (such as oleic acid or caprylic acid)] and salts thereof, acylcarnitines and derivatives thereof, NP-acylated derivatives of lysine, arginine and histidine, side-chain acylated derivatives of lysine and arginine, N ⁇ -acylated derivatives of dipeptides comprising any combination of lysine, arginine and histidine and a neutral or acidic amino acid, N ⁇ -acylated derivatives of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no [577-11-7]), docusate calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl s
  • the pharmaceutical formulation comprises a surfactant in a concentration of about 0.01 mg/ml to about 50 mg/ml.
  • the pharmaceutical formulation comprises Tween-80.
  • the pharmaceutical formulation comprises poloxamer 188.
  • the pharmaceutical formulation comprises an electrolyte. In one embodiment, the pharmaceutical formulation comprises an electrolyte, such as NaCI. In one embodiment, the pharmaceutical formulation comprises an electrolyte, such as KCI. In one embodiment an electrolyte, such as NaCI, such as in a concentration of 150 mM, is employed when Tween 80 is employed as stabiliser. In one embodiment aggregation following storage is avoided.
  • the use of one or more stabilizing agents used in a pharmaceutical formulation comprising a preparation of FXI or FXI-related polypeptide results in an increase in the physical and/or chemical stability of FXI by at least 10%, 25%, 50%, or 100% over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
  • the use of one or more stabilizing agents in a pharmaceutical formulation comprising a preparation of FXI or FXI-related polypeptide results in an increase in the physical and/or chemical stability of FXI by at least 2-fold, 5-fold, 10-fold, or 20-fold over the physical and/or chemical stability of a control (i.e., FXI subjected to the same treatment but in the absence of the stabilizing agent).
  • a suitable formulation that allows recovery of active FXI after freeze-drying contains:
  • Buffer 20 mM buffer (Histidine or TRIS) (pH 5.5, 6.5 of 7.4), 25 mg/ml Mannitol (bulking agent), 2.5 mg/ml NaCI (bulking agent), with 0.01 % Tween 80
  • the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly; intravenously being most preferred. They may also be administered by continuous or pulsatile infusion. It will be understood that any effective method for administering a FXI polypeptide may be used, including, e.g., using mucosal or inhalation methods of administration.
  • Local delivery of the preparations of the present invention 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.
  • compositions of the present invention may be administered in various dosage forms, e.g. as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules (e.g. hard gelatin capsules or soft gelatin capsules), suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solutions, in situ transforming solutions (e.g. in situ gelling, in situ setting, in situ precipitating or in situ crystallizing), infusion solution, or as implants.
  • in situ transforming solutions e.g. in situ gelling, in situ setting, in situ precipitating or in situ crystallizing
  • compositions of the present invention may further be compounded in, or bound or conjugated to (e.g. via covalent, hydrophobic or electrostatic interactions), a drug carrier, drug delivery system or advanced drug delivery system in order to further enhance stability of the factor Xl polypeptide, to increase bioavailability, to increase solubility, to decrease adverse effects, to achieve chronotherapy well known to those skilled in the art, and/or to increase patient compliance.
  • a drug carrier e.g. cellulose and derivatives thereof, other polysaccharides (e.g.
  • thermogelling systems such as block co-polymeric systems well known to those skilled in the art
  • micelles such as block co-polymeric systems well known to those skilled in the art
  • liposomes such as polyethylene glycol
  • microspheres such as polypropylene glycol
  • nanoparticulates such as polypropylene glycol
  • liquid crystals and dispersions thereof L2 phase and dispersions thereof well known to those skilled in the art of phase behaviour in lipid-water systems
  • polymeric micelles such as block co-polymeric systems well known to those skilled in the art
  • multiple emulsions self -emulsifying and self-microemulsifying
  • cyclodextrins and derivatives thereof such as dendrimers.
  • compositions comprising a factor Xl polypeptide prepared by use of a method according to the present invention are suitable for use in the formulation of solids, semisolids, powders and solutions for pulmonary administration using, for example, a metered dose inhaler, dry powder inhaler or a nebulizer, all of which are devices well known to those skilled in the art.
  • Pharmaceutical compositions comprising a factor Xl polypeptide prepared by use of a method according to the present invention are suitable for use in the formulation of controlled-release, sustained-release, protracted-release, retarded-release or slow-release drug delivery systems.
  • compositions comprising a factor Xl polypeptide prepared by use of a method according to the present invention are, for instance, useful in formulation of parenteral controlled-release and sustained-release systems (both systems leading to a many-fold reduction in number of administrations) of types well known to those skilled in the art, such as controlled-release and sustained-release systems for subcutaneous administration.
  • parenteral controlled-release and sustained-release systems both systems leading to a many-fold reduction in number of administrations
  • examples of useful controlled-release systems and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres and nanoparticles,
  • Methods for producing controlled release systems useful for pharmaceutical compositions comprising a factor Xl polypeptide prepared by use of a method according to the present invention include, but are not limited to, crystallization, condensation, co- crystallization, precipitation, co-precipitation, emulsification, dispersion, high-pressure homogenisation, encapsulation, spray-drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes.
  • Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, for example a syringe in a device of the pen type.
  • parenteral administration can be performed by means of an infusion pump.
  • a further option for administration of a composition in the form of a solution or suspension containing a factor Xl polypeptide prepared by use of a method according to the present invention is administration as a nasal or pulmonary spray.
  • pharmaceutical compositions containing a factor Xl polypeptide prepared by use of a method according to the present invention may be adapted to transdermal administration, e.g. by needleless injection, by application of a patch (such as an iontophoretic patch) or by transmucosal (e.g. buccal) administration.
  • a pharmaceutical composition comprising a factor Xl polypeptide prepared by use of a method according to the present invention is stable for more than 6 weeks of usage and for more than 3 years of storage.
  • a pharmaceutical composition comprising a factor Xl polypeptide prepared by use of a method according to the present invention is stable for more than 4 weeks of usage and for more than 3 years of storage.
  • a pharmaceutical composition comprising a factor Xl polypeptide prepared by use of a method according to the present invention is stable for more than 4 weeks of usage and for more than 2 years of storage.
  • a pharmaceutical composition comprising a factor Xl polypeptide prepared by use of a method according to the present invention is stable for more than 2 weeks of usage and for more than 2 years of storage.
  • FXI polypeptide formulations have a pH from about 4.0 to about 10.0. In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to about 8.0. In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to about 7.0. In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to about 6.5. In some embodiments, FXI polypeptide formulations have a pH from about 4.0 to about 6.0. In some embodiments, FXI polypeptide formulations have a pH of about 6.5 or below, such as, e.g., between about pH 5.0 and about 6.5; such as between about 5.5 and 6.5.
  • the present invention provides stable pharmaceutical formulations comprising recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form.
  • the present invention provides a pharmaceutical formulation comprising (i) isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, and (ii) a pharmaceutically acceptable carrier or excipient, wherein said formulation is stable against significant activation of said uncleaved zymogen FXI polypeptide for more than 3 months at 5 0 C.
  • a pharmaceutical formulation comprising: (i) isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, and (ii) a pharmaceutically acceptable carrier or excipient, wherein said formulation exhibits less than about 1 % conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 1 month at 5 Q C.
  • the pharmaceutical formulation exhibits less than about 1%, such as less than about 0.5%, such as less than about 0.1 %, such as less than about 0.05%, such as less than about 0.01% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 2 month at 5 Q C. In one series of embodiments the pharmaceutical formulation exhibits less than about 1%, such as less than about 0.5%, such as less than about 0.1%, such as less than about 0.05%, such as less than about 0.01% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 6 month at 5-C.
  • the pharmaceutical formulation exhibits less than about 1 %, such as less than about 0.5%, such as less than about 0.1%, such as less than about 0.05%, such as less than about 0.01% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 12 month at 5 Q C. In one series of embodiments the pharmaceutical formulation exhibits less than about 1%, such as less than about 0.5%, such as less than about 0.1%, such as less than about 0.05%, such as less than about 0.01% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 18 month at 5 Q C.
  • the pharmaceutical formulation is a liquid formulation.
  • the pharmaceutical formulation comprises an activated FXI polypeptide in a ratio by mass of activated :zymogen FXI or FXI-related polypeptide of below 1 :99, such as between about 1 :9999 to about 1 :99, such as below 1 :999, such as between about 1 :9999 to about 1 :999.
  • the pharmaceutical formulation comprises an activated FXI polypeptide in a concentration of less than about 0.05 mg/ml, such as less than about 0.025 mg/ml, such as less than about 0.02 mg/ml, such as less than about 0.01 mg/ml, such as less than about 0.005 mg/ml, such as less than about 0.0001 mg/ml.
  • the pharmaceutical formulation comprises an additional agent that inhibit activation of said FXI or FXI-related polypeptide in its uncleaved zymogen form.
  • the pharmaceutical formulation comprises a protease inhibitor selected from the list consisting of: serine protease inhibitors such as ⁇ 1 -antitrypsin ( ⁇ 1AT), ⁇ 1 -antichymotrypsin, ⁇ -2 antiplasmin ( ⁇ 2AP), Complement 1 -inhibitor, Neuroserpin, Plasminogen activator inhibitor-1 and 2, Protein Z-related protease inhibitor; C1 esterase inhibitor (C1 Inb), Protease Nexin-I, Protease Nexin-ll, amidine compounds, benzamidine and analogs of benzamidine, heparin, and Antithrombins I-VI, such as Antithrombin III; storage proteins such as ovalbumin, carriage proteins such as thyroxine-binding globulin and steroid- binding globulin, hormone precursors, such as angiotensinogen, and aprotinin.
  • serine protease inhibitors such as ⁇ 1 -antitrypsin ( ⁇ 1AT),
  • the pharmaceutical formulation comprises a protease inhibitor as described in WO 05/016365 (herein also called stabilizing agent), the content of which is hereby incorporated by reference in its entirety.
  • Z 1 and Z 2 independently are selected from the group consisting of -O-, -S- , -NR H - and a single bond, where R H is selected from the group consisting of hydrogen, Ci -4 - alkyl, aryl and arylmethyl, and R 1 and R 2 independently are selected from the group consisting of hydrogen, optionally substituted Ci -6 -alkyl, optionally substituted C 2-6 -alkenyl, optionally substituted aryl, optionally substituted heterocyclyl, or
  • Ci- 6 -alkyP' is intended to encompass acyclic and cyclic saturated hydrocarbon residues which have 1 -6 carbon atoms and which can be linear or branched. Particular examples are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl, cyclopropylmethyl, n-pentyl, isopentyl, n-hexyl, etc. Similarly, the term “Ci- 6 -alkyP' is intended to encompass acyclic and cyclic saturated hydrocarbon residues which have 1 -6 carbon atoms and which can be linear or branched. Particular examples are methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl, cyclopropyl
  • Ci- 4 -alkyl encompasses acyclic and cyclic saturated hydrocarbon residues which have 1-4 carbon atoms and which can be linear or branched.
  • C 2 -6-alkenyl is intended to encompass acyclic and cyclic hydrocarbon residues which have 2-6 carbon atoms and comprise one unsaturated bond, which can be linear or branched.
  • Examples of C 2 . 6 -alkenyl groups are vinyl, allyl, but-1 -en-1 - yl, but-2-en-1 -yl, pent-1 -en-1 -yl, and hex-1-en-1 -yl.
  • Ci -6 -alkyl and C 2 - 6 -alkenyl groups is intended to denote that the group in question may be substituted one or several times, preferably 1-3 times, with group(s) selected from the group consisting of hydroxy, C 1-6 - alkoxy (i.e.
  • Ci -6 -alkyl-oxy C 2-6 -alkenyloxy, oxo (forming a keto or aldehyde functionality), aryl, aryloxy, arylcarbonyl, heterocyclyl, heterocyclyloxy, heterocyclylcarbonyl, amino, mono- and di(Ci -6 -alkyl)amino, halogen, where any aryl and heterocyclyl may be substituted as specifically described below for optionally substituted aryl and heterocyclyl.
  • "Halogen” includes fluoro, chloro, bromo, and iodo.
  • aryl is intended to denote a fully or partially aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, 1 ,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferred example.
  • heterocyclyl is intended to denote a saturated, partially unsaturated, partially aromatic or fully aromatic carbocyclic ring or ring system where one or more of the carbon atoms have been replaced with heteroatoms, e.g.
  • heterocyclyl groups are oxazolyl, oxazolinyl, oxazolidinyl, isoxazolyl, isoxazolinyl, isoxazolidinyl, oxadiazolyl, oxadiazolinyl, oxadiazolidinyl, thiazolyl, isothiazolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imida ⁇ olinyl, imidazolidinyl, pyra ⁇ olyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzoxozolyl, diazolyl,
  • heterocyclyl groups are 5-, 6- or 7- membered monocyclic groups such as isoxazolyl, isoxazolinyl, oxadiazolyl, oxadiazolinyl, pyrrolyl, pyrrolinyl, diazolyl, diazolinyl, triazolyl, triazolinyl, imidazolyl, imidazolinyl, etc.
  • heterocyclic ring is intended to mean a ring corresponding to those defined under “heterocyclyl”.
  • aryl In connection with the terms “aryl”, “heterocyclyl” and “heterocyclic ring”, the term “optionally substituted” is intended to denote that the group in question may be substituted one or several times, preferably 1 -3 times, with group(s) selected from hydroxy (which when present in an enol system may be represented in the tautomeric keto form), Ci -6 -alkyl, C 2 .
  • -C-NH-Z 2 -R 2 may form part of a heterocyclic ring selected from the group consisting of a 1 ,2-diazoline ring, an isoxazoline ring, a 1 ,2,4-triazoline ring, and a 1 ,2,4- oxadiazoline ring.
  • Such heterocyclic rings may be substituted as described above.
  • At least one of R 1 and R 2 is hydrogen, e.g. both are hydrogen.
  • at least one of Z 1 and Z 2 is a single bond, e.g. both are a single bond.
  • R 1 and R 2 are both hydrogen, and Z 1 and Z 2 are both a single bond.
  • C 6 H 4 denotes an optionally substituted benzene ring, i.e. an o-amino-benzamidine, a m-amino-benzamidine or a p-amino-benzamidine, of which a p-amino-benzamidine is the currently most preferred.
  • EP 1 162 194 A1 and EP 1 270 551 A1 are hereby incorporated by reference in its entirety.
  • guanidine compounds are those selected from the group consisting of arginine, arginine derivatives and peptides of 2-5 amino acid residues comprising at least one arginine residue.
  • Arginine constitutes a particular embodiment (see the Experimental section).
  • the term "arginine derivatives" is intended to encompass arginine homologues, N- terminal functionalised arginines (e.g. N-methylated and N-acylated (e.g. acetylated) derivatives), C-terminal functionalised arginines (e.g. C-amidated, C-alkylamidated, and C- alkylated derivatives), and combinations thereof.
  • Other parts of the protease inhibitor may also be important, in particular with respect to optimisation of the stabilising effect and the tolerance by the patient.
  • the radical Y is typically selected in order to improve the efficiency of the stabilising effect.
  • the molecular weight of the protease inhibitor is typically at the most 1000 Da, such as at the most 500 Da.
  • the compounds of the present invention may have one or more asymmetric centres and unless otherwise indicated it is intended that stereoisomers (optical isomers), as separated, pure or partially purified stereoisomers or racemic mixtures thereof are included in the scope of the invention.
  • the concentration of the protease inhibitor (or inhibitors) is typically at least 1 ⁇ M.
  • the desirable (or necessary) concentration typically depends on the selected protease inhibitor (or inhibitors), more specifically on the binding affinity of the selected protease inhibitor to an activated FXI polypeptide.
  • the protease inhibitor is present in a concentration of at least 5 ⁇ M, at least 10 ⁇ M, at least 20 ⁇ M, at least 50 ⁇ M, at least 100 ⁇ M, at least 150 ⁇ M, at least 250 ⁇ M, at least 500 ⁇ M, at least 1 mM, at least 2 mM, at least 4 mM, at least 5 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 20 mM, such as, e.g., in the range of 1 -10000 ⁇ M , 10-10000 ⁇ M, 20-10000 ⁇ M, 50-10000 ⁇ M, 10-5000 ⁇ M, 10- 2000 ⁇ M, 20-5000 ⁇ M, 20-2000 ⁇ M, 50-5000 ⁇ M, 0.1-100 mM, 0.1 -75 mM, 0.1 -50 mM, 0.1 - 10 mM, 0.2-75 mM,
  • the protease inhibitor is benzamidine and the concentration of said inhibitor is at least 1 mM, such as, e.g., at least 2 mM, although it is envisaged that substituted benzamidines may be more potent for what reason they can be added in lower concentrations.
  • the protease inhibitor is arginine and the concentration of said inhibitor is at least 10 mM, such as, e.g., at least 50 mM.
  • the protease inhibitor is p-amino-benzamidine and the concentra-tion of said inhibitor is at least 0.001 mM
  • the protease inhibitor is S-2-[3-(4-Carbamimidoylphenyl)- ureido]-N-[1 -(3-methoxyphenyl)-ethyl]-acetamide with the formula and the concentration of said inhibitor is at least 0.001 mM.
  • the protease inhibitor is S-2-[3-(4-Carbamimidoylphenyl)- ureido]-N-(1 -phenylethyl)-acetamide with the formula
  • the concentration of said inhibitor is at least 0.001 mM.
  • the protease inhibitor is N-(3-Bromobenzyl)-2-[3-(4- carbamimidoylphenyl)-ureido]-acetarnide with the formula
  • the concentration of said inhibitor is at least 0.001 mM.
  • the pharmaceutical formulation comprises a FXI or FXI-related polypeptide in its uncleaved zymogen form in a total amount of about 0.5-50 mg, such as about 0.5-25 mg, such as about 0.5-10 mg, such as about 0.5-5 mg. In one series of embodiments the pharmaceutical formulation comprises a FXI or
  • the pharmaceutical formulation has a pH is in the range of about 4-10, such as in the range of about 4-8, such as in the range of about 4-7, such as in the range of about 5-7, such as in the range of about 5.4-6.5.
  • the pharmaceutical formulation further comprises a surfactant in a concentration of about 0.01 mg/ml to about 50 mg/ml.
  • the pharmaceutical formulation comprises a surfactant selected from the list consisting of sorbitan fatty acid esters, polyoxypropylene- polyoxyethylene block polymers, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives, phospholipids, CHAPS (3-[(3-cholamidopropyl) dimethylammonio] 1 -propanesulfonate), and polyoxyethylene castor oil derivatives.
  • the pharmaceutical formulation further comprises an anti-oxidative agent.
  • anti-oxidative agent means an agent which is able to prevent or inhibit oxidation of at least one methionine.
  • the above mentioned anti-oxidative agent is selected from dithiotreitol, mercapto-ethanol, sulphites, rongalite, erythorbic acid, ascorbic acid, cysteine, methionine, thioglycerol, butylhydroxyanisol, butylhydroxytoluen, dibutylhydroxytoluene, ascorbyl palmitate, tocopherole, alpha-tocopherol, ascorbic acid, ascorbylpalmitate, ascorbylstearate, propyl gallate, octylgallate, dodecylgallate, lecithines, lactate, citric acid, tartaric acid, phosphoric acid, malate, metatartaric acid, adipic acid, succinic acid, calcium disodium EDTA, salts of any thereof, and combinations of any thereof.
  • the above mentioned anti-oxidative agent is selected from dithiotreitol, mercapto-ethanol, sulphites, tocopherols, such as ⁇ -tocopherol (Vitamine E), ascorbic acid, salts of any thereof, and combinations thereof.
  • the above mentioned anti-oxidative agent is approved for use in foodstuffs and beverages for mammalian (e.g., human) consumption.
  • mammalian e.g., human
  • the above mentioned anti-oxidative agent is selected from the group of anti-oxidative agents E-300 to E-399, which is the group of anti-oxidative agents approved as additives in foodstuffs and beverages in Denmark (according to The Danish Veterinary and Food Administration).
  • These anti-oxidative agents have the advantage that they not only are effective in the inhibition or in the prevention of the formation of methionine- oxidised forms, but also are non-toxic and thus do not pose problems in the final drug product.
  • the pharmaceutical formulation is in lyophilized form.
  • the pre-lyophilized FXI pharmaceutical formulations comprises a cryoprotectant and/or a lyoprotectant in sufficient amount to protect said FXI or FXI-related polypeptide against freeze induced stress and/or stress during removal of water during lyophilization.
  • the cryoprotectants is selected from sucrose, trehalose, glucose, raffinose, glycerol, sorbitol, inositol, dextran, polyethyleneglycol, and polyvinylpyrrolidone.
  • the lyoprotectants is selected from saccharides, such as di- and trisaccharides, and histidine.
  • the pre-lyophilized FXI pharmaceutical formulations further comprises one or more of (i) a bulking agents, such as glycine or mannitol, (ii) a buffer that control the pH, and (iii) non-ionic surfactants.
  • a bulking agents such as glycine or mannitol
  • a buffer that control the pH such as glycine or mannitol
  • non-ionic surfactants such as citric acid, acetic acid, histidine, malic acid, phosphoric acid, tartaric acid, succinic acid, MES, HEPES, PIPES, imidazole, TRIS, lactic acid, glutaric acid and glycylglycine; or any salt thereof.
  • the moisture content of the lyophilized FXI pharmaceutical formulation is not more than 3.0 % w/w upon completion of lyophilization.
  • the composition comprises (i) FXI, and (ii) a further compound selected from mannitol, sucrose, methionine, Poloxamer 188, histidine, acetic acid, trimethylamine, and Tween 80.
  • the composition comprises FXI, and mannitol, sucrose, and Tween 80.
  • the composition comprises FXI, mannitol, sucrose, methionine and Tween 80.
  • the composition comprises FXI, mannitol, sucrose, and Poloxamer 188. In one embodiment, the composition (i) comprises FXI, and (ii) has a moisture content of not more than 3.0 % w/w.
  • the composition (i) comprises FXI, and (ii) has a pH in the range 5.0 to 7.0 when the composition is dissolved in water.
  • the composition (i) comprises FXI, mannitol, sucrose, and Tween 80, and (ii) has a pH in the range 5.0 to 7.0 when the composition is dissolved in water.
  • the composition (i) comprises FXI, mannitol, sucrose, and Tween 80, (ii) has a moisture content of not more than 3.0 % w/w, and (iii) has a pH in the range 5.0 to 7.0 when the composition is dissolved in water.
  • the composition (i) comprises FXI, mannitol, sucrose, methionine and Tween 80, (ii) has a moisture content of not more than 3.0 % w/w, and (iii) has a pH in the range 5.0 to 7.0 when the composition is dissolved in water.
  • the composition (i) comprises FXI, mannitol, sucrose, and Poloxamer 188, (ii) has a moisture content of not more than 3.0 % w/w, and (iii) has a pH in the range 5.0 to 7.0 when the composition is dissolved in water.
  • the composition (i) comprises FXI, mannitol, sucrose, histidine, methionine, and Poloxamer 188, (ii) has a moisture content of not more than 3.0 % w/w, and (iii) has a pH in the range 5.0 to 7.0 when the composition is dissolved in water.
  • composition (i) comprises FXI and a volatile buffer system such as trimethylamine and acetic acid.
  • the composition comprises 1mg/ml FXI, 25 mM acetic acid and 25 mM trimethylamine, pH 5.4.
  • the composition further comprises one or more components selected from NaCI and/or glycylglycine.
  • compositions are as shown in Table A below.
  • compositions are as shown in Table B below. Table B.
  • the present invention provides for the prevention and treatment of bleeding using FXI.
  • Bleeding refers to extravasation of blood from any component of the circulatory system.
  • a bleeding episode encompasses 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.
  • Bleedings may occur as a spontaneous events, such as intra-cerebral hemorrhage (ICH). 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.
  • ICH intra-cerebral hemorrhage
  • the bleedings may be likened to bleedings caused by haemophilia because the haemostatic system, as in haemophilia, lacks or has abnormal essential clotting "compounds" (e.g., platelets or von Willebrand factor protein).
  • compounds e.g., platelets or von Willebrand factor protein.
  • 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 (i.e., 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
  • Such therapy may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K-antagonists, inhibitors of coagulation proteins, as well as aspirin and other platelet aggregation inhibitors, such as, e.g., antibodies or other inhibitors of GP llb/llla activity.
  • the bleeding may also be due to so-called thrombolytic therapy which comprises combined treatment with an antiplatelet agent (e.g., acetylsalicylic acid), an anticoagulant (e.g., heparin), and a fibrinolytic agent (e.g., tissue plasminogen activator, tPA).
  • an antiplatelet agent e.g., acetylsalicylic acid
  • an anticoagulant e.g., heparin
  • a fibrinolytic agent e.g., 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, (e.g., muscular, retroperitoneal, sublingual and retropharyngeal), bleedings in other tissue, haematuria (bleeding from the renal tract), cerebral haemorrhage, surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings (e.g., UGI bleeds).
  • acute haemarthroses bleedings in joints
  • chronic haemophilic arthropathy haematomas, (e.g., muscular, retroperitoneal, sublingual and retropharyngeal)
  • bleedings in other tissue e.g., haematuria (bleeding from the renal tract), cerebral haemorrhage, surgery (e.g., hepatectomy), dental extraction, and gastrointestinal bleedings (e.
  • 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 Xl; thrombocytopenia; deficiency of von Willebrand factor (von Willebrand's disease); severe tissue damage; severe trauma; surgery; laparoscopic surgery; acidosis, hemodilution, consumption coagulopathies, hyperfibrinolysis, hyopthermia, 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.
  • a 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, e.g., 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 150,000 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.
  • Aspects of platelet activity include, without limitation, aggregation, adhesion, and coagulant activity of the platelets. Decreased activity may be due, e.g., 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.
  • treatment encompasses both prevention of bleeding, including, without limitation, prevention of an expected bleeding, such as, for example, might be expected to occur during or consequent to a surgical procedure, as well as regulation of an already occurring bleeding, such as, for example, in trauma, with the purpose of inhibiting or minimizing the bleeding.
  • the bleeding may be at an identified site or may be at an undetermined site. Prophylactic administration of a preparation comprising a FXI polypeptide is thus included in treatment.
  • a normal human patient i.e., one not suffering from a congenital deficiency of FXI
  • FXI and/or a FXI-related polypeptide at a dosage that corresponds to about 0.05 mg to about 500 mg of wild-type FXI per day or per bleeding episode, e.g., from about 1 mg to about 200 mg, or, e.g., from about 1 mg to about 175 mg per day or per bleeding episode 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.
  • blood is drawn from a patient in need of treatment with a FXI polypeptide and an assay is performed (prior to FXI polypeptide administration) to assess one or more of: (i) the plasma level of FXI; (ii) the ratio of activated:zymogen FXI; and/or (iii) the concentration of FXI needed to be added exogenously in order to restore effective coagulation; based on the results of the assay, an appropriate amount of FXI polypeptide is administered using a predetermined regimen. Any suitable assay may be used for these determinations, including, e.g., an ELISA or a gel-based method.
  • Appropriate calibration standards are used in order to allow the comparison of the measured level with the usual level of FXI in human plasma (about 30 nM). Typically, it will be desired to replenish FXI levels to at least about 5 nM, such as about 10 nM, such as about 15 nM, such as about 20 nM, and such as at least about 30 nM FXI, such as 60 nM, such as 120 nM.
  • the FXI-related polypeptide When a FXI-related polypeptide is being used to replenish FXI activity in a patient, the FXI-related polypeptide will exhibit a particular level of at least one FXI bioactivity and the goal of the treatment is to provide an amount of that bioactivity that corresponds to a predetermined amount of wild-type FXI (i.e., an "effective FXI plasma concentration").
  • the present invention encompasses therapeutic administration of FXI polypeptide to patients whose plasma level of FXI is below about 3 nM; 5 nM; or 1 O nM.
  • the present invention also encompasses methods and compositions that provide combination therapies in which FXI polypeptide is administered with a non-Factor Vll/Factor Vila coagulation agent.
  • Suitable non-Factor Vll/Factor VIIA coagulation agents include, without limitation, Factor XIII (see, e.g., WO 01/85198); inhibitors of tissue factor pathway inhibitor (TFPI inhibitors) (see, e.g., WO 01/85199); Factor IX (see, e.g., WO 02/062376); thrombin activatable fibrinolysis inhibitor (TAFI) (see, e.g., PCT/DK02/00734; PAI-1 (see, e.g., PCT/DK02/00735; Factor V (see, e.g., PCT/DK02/00736); protein C inhibitors (see, e.g., PCT/DK02/00737); thrombomodulin (
  • PCT/DK02/00740 ⁇ 2-antiplasmin (see, e.g., PCT/DK02/00741); aprotinin (see, e.g., PCT/DK02/00742); tranexamic acid (see, e.g., PCT/DK02/00751); ⁇ -aminocaproic acid (see, e.g., PCT/DK02/00752); prothrombin, thrombin, Factor VII, Factor X, and fibrinogen.
  • Embodiment 1 A method for treating bleeding episodes, said method comprising administering to a patient in need thereof a preparation comprising Factor Xl (FXI) or FXI- related polypeptide, in an amount effective for such treatment.
  • a preparation comprising Factor Xl (FXI) or FXI- related polypeptide, in an amount effective for such treatment.
  • Embodiment 2 A method as defined in embodiment 1 , wherein said administering results in a reduced clotting time in said patient.
  • Embodiment 3 A method as defined in embodiment 1 or embodiment 2, wherein said administering results in an enhancement of hemostasis in said patient.
  • Embodiment 4 A method as defined in any of embodiments 1 to 3, wherein said administering results in an increase in clot lysis time in said patient.
  • Embodiment 5 A method as defined in any of embodiments 1 to 4, wherein said administering results in an increase in clot strength in said patient.
  • Embodiment 6 A method as defined in any of embodiments 1 to 5, wherein said administering results in an increase in overall clot quality (OCQ) in said patient.
  • OCQ overall clot quality
  • Embodiment 7 A method as defined in any of embodiments 1 to 6, wherein, following said administration, said patient exhibits an effective FXI plasma concentration of at least about 5 nM.
  • Embodiment 8 A method as defined in embodiment 7, wherein said effective FXI plasma concentration is at least about 10 nM.
  • Embodiment 9 A method as defined in embodiment 8, wherein said effective FXI plasma concentration is at least about 30 nM, such as at least about 60 nM, such as at least about 12O nM.
  • Embodiment 10 A method as defined in any of embodiments 1 to 9, wherein said FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:1 , or a fragment thereof that retains at least one FXI-associated biological activity.
  • Embodiment 11 A method as defined in any of embodiments 1 to 9, wherein said FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity.
  • Embodiment 12 A method as defined in any of embodiments 1 to 11 , wherein said patient does not suffer from a congenital FXI deficiency.
  • Embodiment 13 A method as defined in any of embodiments 1 to 12, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
  • Embodiment 14 A method as defined in any of embodiments 1 to 13, further comprising, prior to said administering:
  • step (a) obtaining a sample of blood from said patient; (b) determining at least one of: FXI concentration, ratio of FXIa:FXI, or amount of exogenous FXI necessary to restore coagulation; and (c) based on the results of step (b), determining said amount of FXI effective for treatment.
  • Embodiment 15 A method for treating bleeding episodes, said method comprising administering to said patient (i) a first amount of a preparation comprising a FXI polypeptide and (ii) a second amount of a preparation comprising a non-Factor Vll/Factor Vila coagulation agent, wherein said first and second amounts in combination are effective for such treatment.
  • Embodiment 16 A method as defined in embodiment 15, wherein said non-Factor Vll/Factor Vila coagulation agent is selected from the group consisting of: Factor XIII; tissue factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis inhibitor (TAFI); plasminogen activator inhibitor-1 (PAI-1); Factor V; protein C inhibitor; protein S inhibitor; and tissue plasminogen activator (tPA) inhibitor.
  • TFPI tissue factor pathway inhibitor
  • TAFI thrombin activatable fibrinolysis inhibitor
  • PAI-1 plasminogen activator inhibitor-1
  • tPA tissue plasminogen activator
  • Embodiment 17 A method as defined in embodiment 15 or embodiment 16, wherein said administering results in a reduced clotting time in said patient.
  • Embodiment 18 A method as defined in any of embodiments 15 to 17, wherein said administering results in an enhancement of hemostasis in said patient.
  • Embodiment 19 A method as defined in any of embodiments 15 to 18, wherein said administering results in an increase in clot lysis time in said patient.
  • Embodiment 20 A method as defined in any of embodiments 15 to 19, wherein said administering results in an increase in clot strength in said patient.
  • Embodiment 21 A method as defined in any of embodiments 15 to 20, wherein said administering results in an increase in overall clot quality (OCQ) in said patient.
  • OCQ overall clot quality
  • Embodiment 22 A method as defined in any of embodiments 15 to 21 , wherein, following said administration, said patient exhibits an effective FXI plasma concentration of at least about 5 nM.
  • Embodiment 23 A method as defined in embodiment 22, wherein said effective FXI plasma concentration is at least about 10 nM.
  • Embodiment 24 A method as defined in embodiment 23, wherein said effective FXI plasma concentration is at least about 30 nM, such as at least about 60 nM, such as at least about 12O nM.
  • Embodiment 25 A method as defined in any of embodiments 15 to 14, wherein said
  • FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:1 , or a fragment thereof that retains at least one FXI-associated biological activity.
  • Embodiment 26 A method as defined in any of embodiments 15 to 24, wherein said FXI or FXI-related polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity.
  • Embodiment 27 A method as defined in any of embodiments 15 to 26, wherein said patient does not suffer from a congenital FXI deficiency.
  • Embodiment 28 A method as defined in any of embodiments 15 to 27, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
  • Embodiment 29 A method as defined in any of embodiments 15 to 28, further comprising, prior to said administering:
  • step (a) obtaining a sample of blood from said patient; (b) determining at least one of: FXI concentration, ratio of FXIa:FXI, or amount of exogenous FXI necessary to restore coagulation; and (c) based on the results of step (b), determining said amount of FXI effective for treatment.
  • Embodiment 30 A method as defined in embodiment 1 , wherein said method does not comprise administration of a Factor Vll/Factor Vila coagulation agent.
  • Embodiment 31 A pharmaceutical formulation comprising (i) isolated recombinant a FXI polypeptide and (ii) a pharmaceutically acceptable carrier or excipient.
  • Embodiment 32 Use of a FXl polypeptide for treating bleeding episodes.
  • Embodiment 33 Use according to embodiment 32, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
  • Embodiment 34 Use according to embodiment 32 or embodiment 33, wherein said bleeding episodes are not treated with a Factor Vl I/Factor Vila coagulation agent.
  • Embodiment 35 Use of a FXI polypeptide for enhancement of hemostasis in a patient in need thereof .
  • Embodiment 36 Use of a FXI polypeptide for increasing clot lysis time in a patient in need thereof.
  • Embodiment 37 Use of a FXI polypeptide for increasing clot strength in a patient in need thereof.
  • Embodiment 38 Use of a FXI polypeptide for increasing overall clot quality (OCQ) in a patient in need thereof.
  • Embodiment 39 Use of a FXI polypeptide for reducing clotting time in a patient in need thereof.
  • Embodiment 40 Use according to any of embodiments 32 to 39, wherein the effective FXI plasma concentration in the patient is increased to at least about 5 nM.
  • Embodiment 41 Use according to embodiment 40, wherein the effective FXI plasma concentration is increased to at least about 10 nM.
  • Embodiment 42 Use according to embodiment 41 , wherein the effective FXI plasma concentration is increased to at least about 30 nM, such as at least about 60 nM, such as at least about 120 nM.
  • Embodiment 43 Use according to any of embodiments 32 to 42, wherein the patient to be treated is not treated with a Factor Vll/Factor Vila coagulation agent.
  • Embodiment 44 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for treating bleeding episodes.
  • Embodiment 45 Use according to embodiment 44, wherein said bleeding episodes are secondary to a condition selected from the group consisting of: surgery, a dental procedure, trauma, or hemodilution.
  • Embodiment 46 Use according to embodiment 44 or embodiment 45, wherein said bleeding episodes are not being treated with a Factor Vll/Factor Vila coagulation agent.
  • Embodiment 47 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for enhancement of hemostasis in a patient in need thereof .
  • Embodiment 48 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for increasing clot lysis time in a patient in need thereof.
  • Embodiment 49 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for increasing clot strength in a patient in need thereof.
  • Embodiment 50 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for increasing overall clot quality (OCQ) in a patient in need thereof.
  • OCQ overall clot quality
  • Embodiment 51 Use of a FXI polypeptide for preparation of a pharmaceutical formulation for reducing clotting time in a patient in need thereof..
  • Embodiment 52 Use according to any of embodiments 47 to 51 , wherein the effective FXI plasma concentration in the patient is increased to at least about 5 nM.
  • Embodiment 53 Use according to embodiment 52, wherein the effective FXI plasma concentration is increased to at least about 10 nM.
  • Embodiment 54 Use according to embodiment 53, wherein the effective FXI plasma concentration is increased to at least about 30 nM, such as at least about 60 nM, such as at least about 120 nM.
  • Embodiment 55 Use according to any of embodiments 44 to 54, wherein the patient to be treated is not treated with a Factor Vll/Factor Vila coagulation agent.
  • Embodiment 56 Use according to any of embodiments 32 to 55, wherein the patient to be treated does not suffer from a congenital FXI deficiency.
  • Embodiment 57 Use according to any of embodiments 32 to 56, wherein said FXI polypeptide comprises the sequence of SEQ ID NO:1 , or a fragment thereof that retains at least one FXI-associated biological activity.
  • Embodiment 57 Use according to any of embodiments 32 to 56, wherein said FXI polypeptide comprises the sequence of SEQ ID NO:2, or a fragment thereof that retains at least one FXI-associated biological activity.
  • Embodiment 58 Use according to any of embodiments 32 to 57, wherein said FXI polypeptide is to be administered in combination with a non-Factor Vll/Factor Vila coagulation agent.
  • Embodiment 59 Use according to embodiment 58, wherein said non-Factor Vll/Factor Vila coagulation agent is selected from the group consisting of: Factor XIII; tissue factor pathway inhibitor (TFPI) inhibitor; Factor IX; thrombin activatable fibrinolysis inhibitor (TAFI); plasminogen activator inhibitor-1 (PAI-1 ); Factor V; protein C inhibitor; protein S inhibitor; and tissue plasminogen activator (tPA) inhibitor.
  • TFPI tissue factor pathway inhibitor
  • TAFI thrombin activatable fibrinolysis inhibitor
  • PAI-1 plasminogen activator inhibitor-1
  • Factor V protein C inhibitor
  • protein S inhibitor protein S inhibitor
  • tPA tissue plasminogen activator
  • Embodiment 60 A method for purifying a FXI polypeptide from a biological material, the method comprising subjecting the material to sequential chromatography on an cation- exchange chromatographic material, a hydrophobic interaction chromatographic material and a hydroxyapatite chromatographic material.
  • Embodiment 61 A method according to embodiment 60, wherein the FXI polypeptide is a recombinant FXI.
  • Embodiment 62 A method according to embodiment 60 or embodiment 61 , wherein the FXI polypeptide is human FXI.
  • Embodiment 63 A method according to embodiment 60 or embodiment 61 , wherein the FXI polypeptide is a dimer.
  • Embodiment 64 A method according to embodiment 63, wherein the FXI polypeptide is a dimer of human subunits.
  • Embodiment 65 A method according to any of embodiments 60 to 64, wherein the biological material is a biological fluid.
  • Embodiment 66 A method according to embodiment 65, wherein the biological fluid is the supernatant of a mammalian cell.
  • Embodiment 67 A method according to embodiment 66, wherein the biological fluid is the supernatant of a CHO culture.
  • Embodiment 68 A method according to any of embodiments 60 to 67, wherein the method comprises the steps of:
  • step (b) subjecting the eluate from step (iv), or a fluid prepared by use of the eluate from step (iv), to chromatography using a hydrophobic interaction chromatographic material, said chromatography comprising: (v) applying the eluate from step (iv), or a fluid prepared by use of the eluate from step (iv), to said hydrophobic interaction chromatographic material;
  • Embodiment 69 A method according to embodiment 68, wherein buffer A comprises one or more stabilizing agents which are capable of increasing the stability of the FXI polypeptide.
  • Embodiment 70 A method according to embodiment 69, wherein buffer A comprises a stabilizing agent, which stabilizing agent is a sugar, an alcohol or an alditol.
  • Embodiment 71 A method according to embodiment 70, wherein buffer A comprises a stabilizing agent, which stabilizing agent is a sugar, a C ⁇ -Cs-alcohol or an alditol.
  • buffer A comprises a stabilizing agent, which stabilizing agent is a sugar, a C ⁇ -Cs-alcohol or an alditol.
  • Embodiment 72 A method according to embodiment 71 , wherein buffer A comprises a stabilizing agent, which stabilizing agent is a polyalcohol.
  • Embodiment 73 A method according to embodiment 72, wherein buffer A comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol.
  • buffer A comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol.
  • Embodiment 74 A method according to embodiment 73, wherein buffer A comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol.
  • buffer A comprises a stabilizing agent selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol.
  • Embodiment 75 A method according to any of embodiments 72 to 74, wherein said stabilizing agent is present in a concentration of from about 5% (v/v) to about 50% (v/v).
  • Embodiment 76 A method according to embodiment 75, wherein said stabilizing agent is present in a concentration of from about 10% (v/v) to about 50% (v/v).
  • Embodiment 77 A method according to embodiment 76, wherein said stabilizing agent is present in a concentration of from about 10% (v/v) to about 20% (v/v).
  • Embodiment 78 A method according to embodiment 77, wherein said stabilizing agent is present in a concentration of about 10% (v/v).
  • Embodiment 79 A method according to embodiment 78, wherein said stabilizing agent is present in a concentration of about 20% (v/v).
  • Embodiment 80 A method according to any of embodiments 68 to 79, wherein the pH of buffer A is between about 6.5 and about 9.
  • Embodiment 81 A method according to embodiment 80, wherein the pH of buffer A is between about 7 and about 9.
  • Embodiment 82 A method according to embodiment 81 , wherein the pH of buffer A is about 8.
  • Embodiment 83 A method according to any of embodiments 68 to 82, wherein buffer A has a conductivity of less than about 50 mS/cm.
  • Embodiment 84 A method according to any of embodiments 60 to 83, wherein the hydrophobic interaction chromatographic material uses butyl or phenyl as the ligand.
  • Embodiment 85 A method according to embodiment 84, wherein the hydrophobic interaction chromatographic material is Phenyl Sepharose High Performance High Substitution.
  • Embodiment 86 A method according to embodiment 84, wherein the hydrophobic interaction chromatographic material is Butyl Sepharose High Performance High Substitution.
  • Embodiment 87 A method according to any of embodiments 68 to 86, wherein the pH of buffer B is from about 6 to about 9.
  • Embodiment 88 A method according to embodiment 87, wherein the pH of buffer B is about 8.
  • Embodiment 89 A method according to any of embodiments 68 to 88, wherein buffer B has a conductivity of more than 50 ' mS/cm.
  • Embodiment 90 A method according to embodiment 89, wherein buffer B has a conductivity of more than 70 mS/cm.
  • Embodiment 91 A method according to any of embodiments 68 to 90, wherein the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), is treated by use of a method comprising a step of
  • Embodiment 92 A method according to embodiment 91 , wherein the stabilizing agent used in step (1 ) is a sugar, an alcohol or an alditol.
  • Embodiment 93 A method according to embodiment 92, wherein the stabilizing agent used in step (1) is a sugar, a C 4 -C 8 -alcohol or an alditol.
  • Embodiment 94 A method according to embodiment 93, wherein the stabilizing agent used in step (1 ) is a polyalcohol.
  • Embodiment 95 A method according to embodiment 94, wherein the stabilizing agent used in step (1 ) is selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol.
  • Embodiment 96 A method according to embodiment 95, wherein the stabilizing agent used in step (1 ) is selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol.
  • Embodiment 97 A method according to any of embodiments 94 to 96, wherein the stabilizing agent used in step (1 ) is added to a concentration of from about 5% (v/v) to about 50% (v/v).
  • Embodiment 98. A method according to embodiment 97, wherein the stabilizing agent used in step (1 ) is added to a concentration of from about 10% (v/v) to about 50% (v/v).
  • Embodiment 99 A method according to embodiment 98, wherein the stabilizing agent used in step (1 ) is added to a concentration of from about 10% (v/v) to about 20% (v/v).
  • Embodiment 100 A method according to any of embodiments 60 to 99, wherein the method further comprises a step of subjecting the eluate from the hydrophobic interaction chromatography, or a material prepared by use of the eluate from the hydrophobic interaction chromatography, to chromatography on a Hydroxyapatite chromatographic material.
  • Embodiment 101 A method according to any of embodiments 60 to 99, wherein the method further comprises a step of subjecting the eluate from the hydrophobic interaction chromatography, or a material prepared by use of the eluate from the hydrophobic interaction chromatography, to chromatography on a Hydroxyapatite chromatographic material.
  • a method according to any of embodiments 68 to 100, wherein the method further comprises a step of: subjecting the eluate from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to chromatography on a hydroxyapatite chromatographic material, said chromatography comprising: (viii) applying the eluate (diluted and pH adjusted) from stage (vii), or a fluid prepared by use of the eluate from stage (vii), to said hydroxyapatite chromatographic material;
  • buffer C eluting said FXI polypeptide from the hydroxyapatite chromatographic material with buffer C, wherein buffer C is suitable for eluting FXI polypeptides which bind to the hydroxyapatite chromatographic material in step (viii).
  • Embodiment 102 A method according to embodiment 101 , wherein buffer C and/or buffer C comprises one or more stabilizing agents which are capable of increasing the stability of the FXI polypeptide.
  • Embodiment 103 A method according to embodiment 101 , wherein a stabilizing agent is added to the fXI containing fractions, which stabilizing agent is a sugar, an alcohol or an alditol.
  • Embodiment 104 A method according to embodiment 103, wherein a stabilizing agent is added, which stabilizing agent is a sugar, a C 4 -C 8 -alcohol or an alditol.
  • Embodiment 105 A method according to embodiment 104, wherein a stabilizing agent is added, which stabilizing agent is a polyalcohol.
  • Embodiment 106 A method according to embodiment 105 wherein a stabilizing agent selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol is added.
  • a stabilizing agent selected from the group consisting of glycerol, propylene glycol, propan-1 ,3-diol, propyl alcohol and isopropyl alcohol is added.
  • Embodiment 107 A method according to embodiment 106, wherein a stabilizing agent selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol is added.
  • a stabilizing agent selected from the group consisting of glycerol, propylene glycol and propan-1 ,3-diol is added.
  • Embodiment 108 A method according to any of embodiments 105 to 107, wherein said stabilizing agent is added to a concentration of from about 5% (v/v) to about 50% (v/v).
  • Embodiment 109 A method according to embodiment 108, wherein said stabilizing agent is adde to a concentration of from about 10% (v/v) to about 50% (v/v).
  • Embodiment 110 A method according to embodiment 109, wherein said stabilizing agent is added to a concentration of from about 10% (v/v) to about 20% (v/v).
  • Embodiment 111 A method according to embodiment 110, wherein said stabilizing agent is added to a concentration of about 10% (v/v).
  • Embodiment 112. A method according to any of embodiments 101 to 111 , wherein buffer C and/or buffer C has a pH from about 5,8 to about 7,8.
  • Embodiment 113 A method according to any of embodiments 101 to 112, wherein buffer C and/or buffer C has a pH of about 6,0.
  • Embodiment 114 A pharmaceutical composition comprising a FXI polypeptide prepared by use of a method according to any of embodiments 60 to 113.
  • Embodiment 115 A pharmaceutical formulation comprising: (i) isolated recombinant FXI or FXI-related polypeptide in its uncleaved zymogen form, and (ii) a pharmaceutically acceptable carrier or excipient, wherein said formulation exhibits less than about 1% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 1 month at 5 9 C.
  • the pharmaceutical formulation according to embodiment 115 wherein said formulation exhibits less than about 1% conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 2 month at 5 9 C, such as less than about 1 % conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 6 month at 5 Q C, such as less than about 1 % conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 12 month at 5 Q C, such as less than about 1 % conversion of the uncleaved zymogen form to the cleaved activated form when stored for at least 18 month at 5 Q C.
  • Embodiment 117 The pharmaceutical formulation according to to any one of embodiments 115-116, which is a liquid formulation.
  • Embodiment 118 The pharmaceutical formulation according to any one of embodiments 115-117, which comprises an activated FXI polypeptide in a ratio by mass of activated :zymogen FXI or FXI-related polypeptide of below 1 :99, such as between about 1 :9999 to about 1 :99, such as below 1 :999, such as between about 1 :9999 to about 1 :999.
  • Embodiment 119 Embodiment 119.
  • the pharmaceutical formulation according to any one of embodiments 115-118 wherein the formulation comprises an activated FXI polypeptide in a concentration of less than about 0.05 mg/ml, such as less than about 0.025 mg/ml, such as less than about 0.02 mg/ml, such as less than about 0.01 mg/ml, such as less than about 0.005 mg/ml, such as less than about 0.0001 mg/ml.
  • Embodiment 120 The pharmaceutical formulation according to any one of embodiments 115-119, wherein said pharmaceutical formulation comprises an additional agent that inhibit activation of said FXI or FXI-related polypeptide in its uncleaved zymogen form.
  • Embodiment 121 The pharmaceutical formulation according to embodiment 6, wherein said additional agent is a protease inhibitor selected from the list consisting of: serine protease inhibitors such as ⁇ 1 -antitrypsin ( ⁇ 1AT), ⁇ 1 -antichymotrypsin, ⁇ -2 antiplasmin ( ⁇ 2AP), Complement 1 -inhibitor, Neuroserpin, Plasminogen activator inhibitor-1 and 2, Protein Z-related protease inhibitor; C1 esterase inhibitor (C1 Inb), Protease Nexin-I, Protease Nexin-ll, amidine compounds, benzamidine and analogs of ben ⁇ amidine, heparin, and Antithrombins !-Vl, such as Antithrombin III; storage proteins such as ovalbumin, carriage proteins such as thyroxine-binding globulin and steroid-binding globulin, hormone precursors, such as angiotensinogen, and aprotinin.
  • Embodiment 122 The pharmaceutical formulation according to any one of embodiments 115-121 , wherein said FXI or FXI-related polypeptide in its uncleaved zymogen form is in a total amount of about 0.5-50 mg, such as about 0.5-25 mg, such as about 0.5-10 mg, such as about 0.5-5 mg.
  • Embodiment 123 The pharmaceutical formulation according to any one of embodiments 115-122, wherein said FXI or FXI-related polypeptide in its uncleaved zymogen form is in a concentration of at least about 0.5-10 mg/ml, such as at least about 0.5-5 mg/ml, such as at least about 1 -5 mg/ml, such as at least about 2-5 mg/ml.
  • Embodiment 124 The pharmaceutical formulation according to any one of embodiments 115-123, wherein the pH is in the range of about 4-10, such as in the range of about 4-8, such as in the range of about 4-7, such as in the range of about 5-7, such as in the range of about 5.4-6.5.
  • Embodiment 125 The pharmaceutical formulation according to any one of embodiments 115-124, wherein said formulation further comprises a surfactant in a concentration of about 0.01 mg/ml to about 50 mg/ml.
  • Embodiment 126 The pharmaceutical formulation according to embodiment 11 , wherein said surfactant is selected from the list consisting of sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives, phospholipids, CHAPS (3-[(3-cholamidopropyl) dimethylammonio] 1 - propanesulfonate), and polyoxyethylene castor oil derivatives.
  • Embodiment 127 The pharmaceutical formulation according to any one of embodiments 115-124, wherein said formulation further comprises an anti-oxidative agent.
  • Embodiment 128 The pharmaceutical formulation according to embodiment 1 , which is in lyophilized form.
  • Embodiment 129 The pharmaceutical formulation according to embodiment 128, wherein the pre-lyophilized FXI pharmaceutical formulations comprises a cryoprotectant and/or a lyoprotectant in sufficient amount to protect said FXI or FXI-related polypeptide against freeze induced stress and/or stress during removal of water during lyophilization.
  • Embodiment 130 The pharmaceutical formulation according to embodiment 129, wherein the cryoprotectants is selected from sucrose, trehalose, glucose, raffinose, glycerol, sorbitol, inositol, dextran, polyethyleneglycol, and polyvinylpyrrolidone.
  • cryoprotectants is selected from sucrose, trehalose, glucose, raffinose, glycerol, sorbitol, inositol, dextran, polyethyleneglycol, and polyvinylpyrrolidone.
  • Embodiment 131 The pharmaceutical formulation according to embodiment 129, wherein the lyo protectants is selected from saccharides, such as di- and trisaccharides, and histidine.
  • Embodiment 132 The pharmaceutical formulation according to any one of embodiments 128-131 , wherein the pre-lyophilized formulations further comprises one or more of (i) a bulking agents, such as glycine or mannitol, (ii) a buffer that control the pH, and (iii) non-ionic surfactants.
  • a bulking agents such as glycine or mannitol
  • a buffer that control the pH such as glycine or mannitol
  • non-ionic surfactants such as glycine or mannitol
  • Embodiment 133 The pharmaceutical formulation according to embodiment 132, wherein the buffer is selected from citric acid, acetic acid, histidine, malic acid, phosphoric acid, tartaric acid, succinic acid, MES, HEPES, PIPES, imidazole, TRIS, lactic acid, glutaric acid and glycylglycine; or any salt thereof.
  • the buffer is selected from citric acid, acetic acid, histidine, malic acid, phosphoric acid, tartaric acid, succinic acid, MES, HEPES, PIPES, imidazole, TRIS, lactic acid, glutaric acid and glycylglycine; or any salt thereof.
  • Embodiment 134 The pharmaceutical formulation according to any one of embodiments 128-133, wherein the moisture content is not more than 3.0 % w/w upon completion of lyophilization.
  • Max vel 11 max ve l) X (t mi ⁇ V ei - 1 max ve i) OCQ is then normalized to the control sample (incubated in the absence of any hemostatic agents.
  • Example 2 Effect of FXI on hemostasis in normal blood Blood was obtained from 4 normal subjects, and the effect of FXI on clot formation was evaluated by ROTEG as described in Example 1.
  • Figure 2 illustrates that FXI caused a dose-dependent increase is OCQ in normal blood.
  • FXI variant containing the following substitutions were constructed using standard methodologies and were expressed after transfection in HEK293 cells. Crude cell culture supematants were collected from cells grown for 96 h at 37 0 C. FXI activity was measured by ROTEG as described in Example 1.
  • the format of the library BL121 is:
  • the format of the library BL122 is
  • Oi-O 2 -O 3 -0 4 -O 5 -O 6 -O 7 -O 8 -O9-O 10 -Oir0i 2 -Tentagel resin, where On is a L-amino acid and n 1 -12 can be any proteinogenic L-amino acid except methionine and cysteine.
  • the format of the library BL124 is:
  • Recombinant factor Xl from Heamatologic Technologies was purchased and biotinylated according to standard laboratory protocols. Then 5 ul of factor Xl (1 ,2 uM) and 1 ul streptavidin-alkaline phosphatase (1 mg/ml, Sigma) were added to three synthetic peptide bead libraries, BL121 , BL122 and BL124, respectively, and allowed to incubate for about 2-3 hours.
  • Specific factor Xl and factor Xl-like binding peptides found in library BL121 , BL122 andBL123 according to the invention include peptides comprising amino acid sequences are outlined below: Sequences found in BL121
  • SEQIDNO:12 SHFWRQWPDFSD
  • SEQ ID NO:36 RSWLRYGYGH
  • SEQ ID NO:40 YPKHIYADFPSTRL
  • SEQ ID NO:48 I P KN RFS DFP DAQG
  • SEQ ID NO:49 LPSFRFPDFPATKT
  • FXI-binding peptides include those having a core amino acid motif of Asp-Phe-Pro.
  • Example 6 The cells from mammalian cell culture was separated from the supernatant by centrifugation or filtration. Benzamidine and EDTA was added to final concentration 1 mM.
  • Example 7 The cells from mammalian cell culture was separated from the supernatant by centrifugation or filtration. Benzamidine and EDTA was added to final concentration 1 mM.
  • a Obelix matrix was equilibrated with 5 column volumes (cv) of buffer A, and a load corresponding to 150ml supernatant pr ml packed column was applied to the column.
  • the column was washed with 4 cv of buffer A (3OmM Tris pH 8,0) and then with 5 cv of buffer A' (5OmM Tris 50%Glycerol 87% pH 9,0) . Elution was then performed with 5 cv buffer A" (5OmM Tris 50%GIycerol 87% 1 M NaCI pH 9,0). Flowrate was 16 cv/h, temperature was 0- 10 Q C.
  • the column was regenerated with 1 M NaOH.
  • Fractions were collected from at about 50% of peak height, the first peak eluting is discarded but the next main peak contains FXI.
  • Analysis of FXI polypeptide-containing fractions was performed by HPLC (vide infra) using C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x250 mm and by SDS-PAGE on a NUpage 4-12% Bis/Tris GeI (Invitrogen) with MOPS running buffer under reductive conditions. Benzamidine and EDTA were added (to 1 mM) to the fractions containing FXI polypeptide and kept at approx. 4°C in a refrigerator, or frozen at -80 3 C, until further use.
  • Alternative to Obelix ST CIEX is Streamline Direct CST Amersham cat no 17-
  • the pH of the pool of FXI polypeptide-containing fractions from Example 8 was adjusted to 6,0 and 1 volume water was added to a conductivity of below 20 mS/cm.
  • a Hydroxyapatite Type I 20 ⁇ m matrix was equilibrated with 6 cv of buffer C, and then a load corresponding to 5 mg/ml gel was applied to the column.
  • the column was then washed with 15 cv of buffer C (2OmM K-PO4 pH 6,0), and a buffer containing 95% buffer C and 5% elution buffer C (2OmM K-PO4 2M NaCI pH 6,0) was performed as a washing step.
  • a gradient elution from 5%C to 100%C was performed and used to elute the FXI polypeptide in small fractions.
  • the conductivity of the pool containing the FXI polypeptide fractions was about 60 mS/cm and the pH about 6,0.
  • Analysis of FXI polypeptide-containing fractions was performed by HPLC ( vide infra) using C4 Jupiter Phenomonex cat no OOG-4167-EO,
  • High-Performance Liquid Chromatography (HPLC; referred to in Examples 7-9, above) was performed using C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x250 mm and employing buffers as follows: Buffer I: 0,1%TFA in H 2 O
  • Elution of the column took place using a gradient going from 75% Buffer I / 25% Buffer Il to 39% Buffer I / 61% Buffer Il over a period of 18 minutes (flow rate 1 ml/min.). Regeneration of the column was performed by washing with 100% Buffer Il for 2 minutes, (flow rate 0.5 ml/min.). The detection wavelength employed was 214 nm. Temperature was 50 Q C. Samples of from 2 to 50 ⁇ g were loaded onto the column.
  • Reagent A Dilute Eu-labeled anti-FXI (1 ⁇ g/mL) in Assay Buffer. Eu-cryptate (Eu-TBP-K,
  • Reagent B Dilute biotinylated anti-FXI (1 ⁇ g/mL) and Streptavidin-XL665 (2 ⁇ g/mL) in Assay Buffer. Biotinamidocaproate N-hydroxysuccinimide ester and a second anti-FXI is labeled in the molar ratio of 15:1. Streptavidin-XL665 is purchased from Cisbio International.
  • the described assay provides a method for measuring FXI i human plasma and for the determination of dose to replace the aquired FXI deficiency.

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Abstract

La présente invention concerne des méthodes et des préparations pour le traitement de saignements. Lesdites méthodes consistent à administrer à un patient qui le nécessite une préparation comprenant un polypeptide de facteur XI, dans des quantités efficaces sur le plan thérapeutique. Les méthodes selon l'invention aboutissent à l'un ou plusieurs des résultats suivants : temps de coagulation réduit ; amélioration de l'hémostase ; augmentation du temps de lyse du coagulat ; augmentation de la résistance du coagulat ; et/ou augmentation de la qualité globale de coagulation (OCQ, Overall Clot Quality) chez ledit patient.
PCT/EP2005/052511 2005-06-01 2005-06-01 Formule pharmaceutique de facteur xi WO2006128497A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2222315A4 (fr) * 2007-12-21 2011-03-02 Inspiration Biopharmaceuticals Inc Formulations stabilisées de facteur ix contenant du tréhalose
WO2012066036A1 (fr) * 2010-11-16 2012-05-24 Octapharma Ag Procédé de réduction et/ou d'extraction de fxi et de fxia de solutions contenant lesdits facteurs de coagulation
US8450275B2 (en) 2010-03-19 2013-05-28 Baxter International Inc. TFPI inhibitors and methods of use
US8466108B2 (en) 2008-12-19 2013-06-18 Baxter International Inc. TFPI inhibitors and methods of use
US8962563B2 (en) 2009-12-21 2015-02-24 Baxter International, Inc. TFPI inhibitors and methods of use
EP2845606A1 (fr) * 2014-07-10 2015-03-11 Symrise AG Médicament pour le traitement de la toux et la prophylaxie d'infections respiratiores
US9453045B2 (en) 2010-03-30 2016-09-27 Octapharma Ag Process for the purification of a growth factor protein
WO2018024873A1 (fr) * 2016-08-05 2018-02-08 Csl Behring Gmbh Formulations pharmaceutiques de l'inhibiteur d'estérase c1
CN113848332A (zh) * 2021-09-17 2021-12-28 广州徕西姆医学诊断技术有限公司 一种血栓弹力图检测试剂及其制备方法和应用
CN115524503A (zh) * 2022-09-30 2022-12-27 上海太阳生物技术有限公司 一种异常凝血质控品及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5252217A (en) * 1991-05-07 1993-10-12 Association Pour L'essor De La Transfusion Sanguine Dans La Region Du Nord Blood coagulation factor XI concentrate having high specific activity, suitable for therapeutic use, and process for preparing same
WO2003007983A1 (fr) * 2001-07-20 2003-01-30 Novo Nordisk Health Care Ag Composition pharmaceutique contenant des polypeptides du facteur vii et des polypeptides du facteur xi
WO2005049070A1 (fr) * 2003-11-20 2005-06-02 Novo Nordisk Health Care Ag Utilisation therapeutique du facteur xi

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5252217A (en) * 1991-05-07 1993-10-12 Association Pour L'essor De La Transfusion Sanguine Dans La Region Du Nord Blood coagulation factor XI concentrate having high specific activity, suitable for therapeutic use, and process for preparing same
WO2003007983A1 (fr) * 2001-07-20 2003-01-30 Novo Nordisk Health Care Ag Composition pharmaceutique contenant des polypeptides du facteur vii et des polypeptides du facteur xi
WO2005049070A1 (fr) * 2003-11-20 2005-06-02 Novo Nordisk Health Care Ag Utilisation therapeutique du facteur xi

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2633860A1 (fr) * 2007-12-21 2013-09-04 Inspiration Biopharmaceuticals, Inc. Formulations stabilisées de facteur IX contenant du tréhalose
EP2222315A4 (fr) * 2007-12-21 2011-03-02 Inspiration Biopharmaceuticals Inc Formulations stabilisées de facteur ix contenant du tréhalose
RU2481823C2 (ru) * 2007-12-21 2013-05-20 Инспирейшн Биофармасьютикалс, Инк. Стабилизированные составы, содержащие фактор ix и трегалозу
US11001613B2 (en) 2008-12-19 2021-05-11 Takeda Pharmaceutical Company Limited TFPI inhibitors and methods of use
US8466108B2 (en) 2008-12-19 2013-06-18 Baxter International Inc. TFPI inhibitors and methods of use
US9777051B2 (en) 2008-12-19 2017-10-03 Baxalta GmbH TFPI inhibitors and methods of use
US9873720B2 (en) 2008-12-19 2018-01-23 Baxalta GmbH TFPI inhibitors and methods of use
US8962563B2 (en) 2009-12-21 2015-02-24 Baxter International, Inc. TFPI inhibitors and methods of use
US10201586B2 (en) 2010-03-19 2019-02-12 Baxalta GmbH TFPI inhibitors and methods of use
US8450275B2 (en) 2010-03-19 2013-05-28 Baxter International Inc. TFPI inhibitors and methods of use
US11793855B2 (en) 2010-03-19 2023-10-24 Takeda Pharmaceutical Company Limited TFPI inhibitors and methods of use
US9018167B2 (en) 2010-03-19 2015-04-28 Baxter International Inc. TFPI inhibitors and methods of use
US9556230B2 (en) 2010-03-19 2017-01-31 Baxalta GmbH TFPI inhibitors and methods of use
US9453045B2 (en) 2010-03-30 2016-09-27 Octapharma Ag Process for the purification of a growth factor protein
US10214575B2 (en) 2010-03-30 2019-02-26 Octapharma Ag Process for the purification of a growth factor protein
WO2012066036A1 (fr) * 2010-11-16 2012-05-24 Octapharma Ag Procédé de réduction et/ou d'extraction de fxi et de fxia de solutions contenant lesdits facteurs de coagulation
US10800816B2 (en) 2012-03-21 2020-10-13 Baxalta GmbH TFPI inhibitors and methods of use
EP2845606A1 (fr) * 2014-07-10 2015-03-11 Symrise AG Médicament pour le traitement de la toux et la prophylaxie d'infections respiratiores
WO2018024873A1 (fr) * 2016-08-05 2018-02-08 Csl Behring Gmbh Formulations pharmaceutiques de l'inhibiteur d'estérase c1
EP3493832A1 (fr) * 2016-08-05 2019-06-12 CSL Behring GmbH Formulations pharmaceutiques de l'inhibiteur d'estérase c1
US11554156B2 (en) 2016-08-05 2023-01-17 Csl Behring Gmbh Pharmaceutical formulations of C1 esterase inhibitor
CN113848332A (zh) * 2021-09-17 2021-12-28 广州徕西姆医学诊断技术有限公司 一种血栓弹力图检测试剂及其制备方法和应用
CN113848332B (zh) * 2021-09-17 2024-04-19 广州徕西姆医学诊断技术有限公司 一种血栓弹力图检测试剂及其制备方法和应用
CN115524503A (zh) * 2022-09-30 2022-12-27 上海太阳生物技术有限公司 一种异常凝血质控品及其应用

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