KR20060118486A - Therapeutic use of factor xi - Google Patents

Abstract

The present invention 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 XI 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.

Description

Therapeutic use of factor VII {THERAPEUTIC USE OF FACTOR XI}

The present invention relates to the therapeutic use of human factor XI for the prevention and / or treatment of bleeding events, to methods of purifying Factor XI and Factor XI polypeptide from biological fluids, and pharmaceutical formulations.

Human factor XI is a serine protease consisting of two identical subunits each having a molecular mass of about 80 kDa. FXI circulates in plasma as disulfide-linked homodimers with molecular masses of ˜160 kDa. FXI is activated by cleavage of each monomer between Arg369 and Ile370 to form a 50kDa amino-terminal heavy chain and a 35kDa carboxy-terminal light chain, which are disulfide-bonded. This protein is encoded by a 23 kb gene located on chromosome 4 (4q35) 15 exon and 14 introns encoding an mRNA consisting of 2,097 nucleotides, which is then followed by an amino-terminal signal (leader) peptide of 18 amino acids and maturation. The protein encodes 607 amino acids present in each monomer. Exon III-X encodes four serial repeat sequences (Apple domains) that are homologous (58% identical) to similar domains found in human plasma PK. Exon XI-XV encodes a typical trypsin-type catalytic domain, which is activated by proteolytic cleavage of the simogen at the internal Arg369-Ile370 bond, containing heavy and light chains or four Apple domains (369 amino acids) or It provides a catalytic domain (238 amino acids).

One mechanism for initiating coagulation is exposure to circulation of tissue factor (TF) at the wound site, followed by (i) binding of plasma factor VII (FVII) and TF and proteolytic conversion to activating factor VII (FVIIa); (ii) binding of factor X and TF-FVIIa complex and proteolytic conversion to activating factor X (FXa); (iii) proteolytic conversion of prothrombin to thrombin by FXa; And (vi) the generation of tissue factor pathway inhibitor (TFPI) and FXa complex followed by the binding of TFPI: FXa complex with TF-FVIIa, which attenuates FXa activation of thrombin and fluxes of thrombin generated via the TF pathway. Restrict The relatively small amount of thrombin produced during this step results in the activation of FXI to FXIa (which activates Factor IX to FIXa) and activation of Factor V and further activation of Factor X on the platelet surface. These events further promote the formation of sufficient thrombin to convert fibrinogen into fibrin, thereby stabilizing early platelet plugs and leading to adequate hemostasis.

Dimeric FXI is a non-covalent complex with cofactor high molecular weight kininogen (HK) that promotes binding of a negatively charged surface to FXI and activation by its cognate proteases, FXIIa, FXIa, and thrombin. As a gen, it circulates in plasma. HK binding sites with FXI include multiple Apple domains (A1, A2, A4), with the A2 domain being the most important. Complex formation with HK in the presence of Zn ²⁺ ion is known to promote the binding of FXI and activated platelets. The interaction of FXI with the activated platelet surface is known to be mediated by residues Ser248-Val271 in the A3 domain of FXI, and residues Ser248, Arg250, Lys255, Phe260 and Gln263 are also involved in this interaction. The A3 domain of FXI also contained a heparin binding site within residues Thr249-Phe260, and residues Lys252 and Lys253 were involved in binding to platelets. FXI and HK circulate in plasma as non-covalent complexes and HK is known to bind to the surface of activated platelets, but it is clear that the interaction of FXI with platelet surfaces does not require binding of the HK-FXI complex. Instead, the FXI dimer appears to bind directly to a high-affinity, specific site on activated platelets (about 1500 sites / platelet; Kd at about 10 nM). The isolated recombinant A3 domain of FXI binds with the same affinity as the sites on the same number of activated platelets as the FIX dimer.

In addition, the activating enzyme FXIa is known to bind high-affinity, saturation sites on activated platelets (Kd at about 800 pM; 500 sites / platelet) and can activate FIX at rates similar to those observed in solution. . The substrate FIX binding site in FXI includes both the subdomain of the A2 domain (Ala134-Leu172) and the two subdomains in the A3 domain (Ile184-Val192 and Ser259-Ser265). Binding to the platelet surface is mediated by the glycoprotein 1b-V-IX complex using one polypeptide chain of the FXI dimer so that the remaining monomers are present as substrate binding sites for FIX. FIXa development appears to act to localize FIXa-catalytic FX activation to the platelet surface, which also promotes prothrombin activation by FXa.

In addition to the formation of a membrane association complex that results in the local explosive generation of thrombin on the platelet surface, FXIa also exhibits a variety of plasma and other functional properties that have been found to depend on whether FXIa binds to the platelet surface or is free in solution. Regulated by platelet protease inhibitors. As such, many serine protease inhibitors, including α-1-protease inhibitors, antithrombin III, C1 inhibitors, α-2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1), and protein C inhibitors All are known to inactivate FXIa in the plasma compartment. However, the most physiologically relevant inhibitor of FXIa in the context of activated platelets seems to be protease Nexin II (PNII), which is found at very low concentrations in plasma but is secreted from platelet α-granules (1 per 10 ⁸ platelets). -1.5 nM PNII release), which supports a plasma concentration of 3-5 nM under normal physiological conditions. PNII is an potent FXIa inhibitor with a Ki of 300-500pM significantly enhanced in the presence of heparin. Binding of FXI with the platelet surface in the presence of HK and Zn ^{2 +} ions or prothrombin and under the presence of Ca ^{2 +} has is protected from the inactivating by both PNII and α-1- protease inhibitor, which is the FXIa activated platelet surface occurs in While localized to hemostatic thrombus, the regulatory site of FXIa by PNII and other protease inhibitors is shown to occur in solution. In addition, endothelial cells containing heparan sulfate glycosaminoglycan may enhance the inhibition of FXIa on the endothelium by promoting the association of the FXIa / PNII complex.

In addition, the involvement of FXI in thrombin generation on activated platelet surfaces is believed to play a role in inhibiting fibrinolysis by thrombin-activated fibrinolytic inhibitors (TAFIs), which from fibrin plays a role in plasminogen binding and activation. The carboxy-terminal lysine residues are proteolytically removed. It is believed that a complete FXI feedback loop is needed to generate enough thrombin for significant TAFI activation.

In particular, it is clear that platelets and megakaryocytes synthesize a second form of FXI called platelet-derived FXI (pd-FXI), in which exon V, the first exon of the two exons encoding the second Apple domain, Unlike in the circulating form without, in vitro studies indicate that the preferred substrate for platelet factor XIa may be plasma FXI, not FIX. Platelet FXI (Mr 220 kDa) has been shown to be associated with platelet plasma membrane. Platelets contain about 300 molecules of pd-FXI / cell.

FXI defects are autosomal recessive syndromes characterized by variable predisposition to bleed. Even in severe cases, this defect may be clinically asymptomatic until the patient is stimulated by surgical trauma; In some cases, however, bleeding may occur regardless of the severity of this defect. Optimal management of patients with FXI defects requires attention to many features in addition to the FXI level. First, it is important to assess bleeding predisposition in individuals with partial defects and to assess whether additional factors contribute significantly. Such assessment should include measurement of FVIIIC and von Willebrand factor levels, bleeding time and platelet aggregation. The first known cases of FXI defects were treated with fresh frozen plasma, which was the main treatment until the development of FXI concentrates. The main disadvantage of plasma is that it requires a large volume and there is the possibility of allergic reactions and delivery of infectious agents. In addition, the FXI content in this product has been reported to be somewhat variable. Two FXI concentrates are currently available. FXI concentrates from Bio Products Laboratory (BPL; UK) are formulated with high concentrations of antithrombin (average 102iu / ml) and heparin (10 u / ml), which is believed to protect against any remaining FXIa. The second FXI concentrate is produced by Hemoleven (France), which is formulated with 3-5 u / ml heparin, 2-3 iu / ml antithrombin and a C1 inhibitor. Moreover, it has been reported that pasteurization of fresh pooled frozen plasma is possible while preserving 75-95% activity of FXI. Patients with mild FXI defects are generally treated with fresh frozen plasma, while patients with severe FXI defects can be treated with FXI concentrate.

Patients in need of blood transfusion due to excessive bleeding associated with surgery or major trauma (including those suffering from congenital FXI defects) have more complications than patients who have not experienced any bleeding. Even mild bleeding that requires the administration of human blood or blood products (eg, platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation defects, etc.) is not currently known for human viruses (hepatitis, HIV, parvovirus, etc.). Can lead to complications related to the risk of virus transmission. Extensive bleeding that requires large blood transfusions can lead to the development of multiple organ disorders, including impaired lung and kidney function. Once a subject exhibits these serious complications, a cascade of events involving many cytokines and inflammatory reactions begins, making certain treatments extremely difficult or often failing. Thus, the main goal in surgery as well as in the treatment of large tissue damage is to avoid or minimize bleeding. In order to avoid or minimize such bleeding, it is important to ensure the formation of stable, rigid hemostatic plugs that are not readily soluble by fibrinolytic enzymes. Moreover, it is important to ensure fast and effective formation of such plugs or clots.

WO 2003 007983 discloses the use of a combination of Factor VIIa and FXI for the treatment of bleeding events.

Thus, there is a need in the art for improved hemostatic treatment modalities that result in rapid and controlled formation of stable fibrin clots.

Summary of the Invention

The present invention provides methods and compositions for treating bleeding events. The method is performed by administering a formulation comprising a Factor XI (FXI) polypeptide to a patient in need thereof in an amount effective for such treatment. The method of the present invention comprises a method for reducing clotting time in said patient; Enhancement of hemostasis; Increased clotting time; Increased coagulation intensity; And / or an increase in overall coagulation quality (OCQ). In some embodiments, after administration of the 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.

In some embodiments, the FXI polypeptide comprises the sequence of SEQ ID NO: 1, or a fragment thereof that retains at least one FXI-related biological activity. In some embodiments, the FXI polypeptide comprises a sequence of SEQ ID NO: 2, or a fragment thereof that retains at least one FXI-related biological activity. In some embodiments, the FXI polypeptide comprises a chemically modified derivative of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant of SEQ ID NO: 1 or SEQ ID NO: 2 containing one or more amino acid sequence changes Include. In some embodiments, 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 congenital FXI defects. In some embodiments, the bleeding event is accompanied by surgery, dental procedures, trauma, or hemodilution. In some embodiments, the patient suffers from an acquired FXI defect.

The present invention also provides methods and compositions for preventing bleeding events. The method is performed by administering a formulation comprising a FXI polypeptide to a patient in need thereof in an amount effective to prevent bleeding.

In some embodiments, the method of the present invention comprises prior to administration of the FXI polypeptide: (a) obtaining a blood sample from said patient; (b) measuring at least one of FXI concentration, FXIa: FXI ratio, or amount of exogenous FXI required to restore coagulation; And (c) determining said amount of FXI effective for treatment based on the results of step (b).

In one embodiment, the methods of the present invention do not comprise the administration of Factor VII / Factor VIIa coagulants.

As used herein, Factor VII / Factor VIIa coagulants are Factor VII polypeptides or Factor VII-cognate polypeptides described in WO 2003 007983.

The present invention also provides methods and compositions for treating a bleeding event, wherein the patient comprises (i) an agent comprising a first amount of FXI polypeptide and (ii) a second amount of non-factor VII / factor VIIa coagulant The formulation comprising the first and second amounts in combination is administered under conditions effective for such treatment. Non-limiting examples of non-factor VII / factor VIIa coagulants include Factor XII, Phospholipids, Factor XIII; Tissue factor pathway inhibitor (TFPI) inhibitors; Factor IX; Thrombin activated fibrinolytic inhibitor (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitors; Protein S inhibitors; Tissue plasminogen activator (tPA) inhibitors; Prothrombin, factor VIII, fibrinogen, and factor X.

The present invention also provides a pharmaceutical formulation comprising (i) an isolated recombinant FXI polypeptide and (ii) a pharmaceutically acceptable carrier or excipient.

Furthermore, the present invention provides a method for purifying Factor XI polypeptide from a biological material, which method comprises continuation of the material in cation-exchange chromatography material, hydrophobic interaction chromatography material, and hydroxyapatite chromatography material. Chromatography.

1 is a graph showing the effect of increasing the amount of FXI on the overall coagulation quality in blood obtained from patients before and after cardiac surgery.

2 is a graph showing the effect of increasing the amount of FXI on the overall coagulation quality in blood obtained from normal subjects.

3 is a graph showing the biological activity of different FXI formulations after storage at 5 ° C. for 96 days.

4 is a preliminary chromatogram of Factor XI polypeptide-containing fractions from initial cation-exchange chromatography using Obelix ST CIEX (cat no 11-0010) described in Example 7. FIG.

FIG. 5 is a preliminary chromatogram of Factor XI polypeptide-containing fractions from hydrophobic interaction chromatography using Butyl Sepharose High Performance High Substitution (cat no 17-3100) described in Example 8. .

FIG. 6 is a preliminary chromatogram of factor XI-polypeptide containing portions from hydroxyapatite chromatography using CHT hydroxyapatite type I (BioRad cat no 157-0020) described in Example 9. FIG.

Detailed description of the invention

The present invention is based on the surprising finding that external administration of Factor XI (FXI) can be effective as a general hemostatic agent in human blood without administration of Factor VII / Factor VIIa coagulants. Therapeutic use of FXI in accordance with the present invention may provide one or more of reduced coagulation time, tighter coagulation, and increased resistance to fibrinolysis of the formed coagulation and reduction of bleeding-related complications.

The present invention provides methods and compositions useful for the therapeutic use of FXI in human patients for the treatment or prevention of bleeding events, to enhance hemostasis, to increase coagulation time, and / or to increase coagulation intensity. The method is performed by administering to a patient an effective amount of Factor XI to achieve one or more of these desired therapeutic goals. The composition comprises a pharmaceutical formulation for therapeutic use of FXI, including FXI. In one embodiment, the composition comprises a pharmaceutical formulation for therapeutic use of FXI comprising isolated FXI. In one embodiment, the composition comprises a pharmaceutical formulation for therapeutic use of FXI, including recombinant FXI. In one embodiment, the composition comprises a pharmaceutical formulation for therapeutic use of FXI comprising isolated recombinant FXI.

In one series of embodiments, the present invention relates to the administration of FXI to normal human patients. As used herein, a “normal” person is one who does not suffer from birth defects of factor XI (ie, hemophilia C, Seligsohn (1993), Thromb. Haemost. 70: 68-71); A normal person may be a patient with thrombocytopenia (a decrease in the number or activity of platelets), a patient planning or undergoing a surgical or dental procedure, and a trauma or organ injury resulting in a decrease in platelet count and / or fibrinogen, FVIII, And / or patients that may exhibit lowering of other coagulation protein levels. In normal human patients, for example, bleeding, trauma, chemotherapy, liver disease, hemodilution (for example, which may be the result of infusion of a dilator or salt solution to maintain blood volume or prevent shock) , Or patients who have experienced a temporary decrease in plasma levels of FXI (or any other coagulation-related protein or factor) due to any other environment not directly related to a birth defect of the FXI gene.

In another set of embodiments, the present invention relates to the administration of isolated and / or recombinant FXI to human patients suffering from birth FXI defects.

In another set of embodiments, the present invention relates to the administration of isolated and / or recombinant FXI to human patients suffering from acquired FXI defects.

In the practice of the present invention, any FXI polypeptide effective for preventing or treating bleeding can be used. This includes FXI polypeptides derived from blood or plasma or from those produced by recombinant means in platelets or any suitable host organism or cell. Also included are FXI polypeptides in uncleaved (simogen) form, as well as those that have been hydrolyzed to obtain their respective biologically active forms (called FXIa).

As used herein, FXI polypeptides include, without limitation, FXI as well as FXI-cognate polypeptides. The term “FXI” is described, for example, in Fujikawa et al., Biochem. In addition to wild type human plasma FXI described in 25: 2417 (1986), polypeptides having the amino acid sequence of wild type FXI derived from other species, such as, for example, bovine, swine, dog, rat, rabbit, and salmon FXI It is intended to be included without. In order to reduce the risk of inducing an immune response, it is generally desirable to use FXI proteins that are synonymous with the subject. Preparation and characterization of non-human FXI has been described, for example, by Gailani (1997), Blood 90: 1055. The present invention also encompasses the use of such factor XI proteins in veterinary procedures.

In some embodiments, the FXI polypeptide is wild type human plasma FXI (SEQ ID NO: 1). In another embodiment, FXI is described, for example, in Hsu et al. (1998), J. Biol. Chem. Platelet-derived FXI (pd-FXI) (SEQ ID NO: 2) described in 273: 13787-93.

Further, FXI polypeptides include natural allelic variants of FXI which may exist from one individual to another and may be different. In addition, the degree and location of glycosylation or other post-translational modification may vary in certain circumstances depending on the sequence of the FXI-encoding nucleic acid, the host cell in which the FXI is produced, and the conditions under which the FXI-producing cell is maintained.

FXI-cognate polypeptides include, without limitation, FXI polypeptides (ie, FXI derivatives) chemically modified for human FXI and / or FXI polypeptides (ie, FXI variants) containing one or more amino acid sequence changes for human FXI. . Such FXI-cognate polypeptides include, without limitation, altered stability, altered phospholipid binding, altered specific enzyme activity, altered immunogenicity, altered bioavailability, altered binding with one or more FXI binding partners, altered binding with FXI inhibitors, and the like. , One or more embodiments of biological activity associated with human FXI. FXI-cognate polypeptides include those that have been proteolytically processed to obtain their respective biologically active forms, as well as uncut (zymogen) forms of such polypeptides, which are "FXIa-cognate polypeptides" or "active FXI-cognate polypeptides." "I can say.

Non-limiting examples of FXI derivatives include wild type FXI or FXI variants modified by phosphorylation, sulfidation, FEGylation or the action of one or more glycosyltransferases and / or glycosidases (eg, Ekdahl et al. (1999), Thromb. Haemost. 82: 1283-8).

Non-limiting examples of FXI variants include FXI with one or more N-linked or O-linked glycosylation consensus sites modified, short chain FXI (ie, FXI in which monomer polypeptide is not hydrolyzed in chains as in wild type). And cysteine variants in which one or more cysteine residues have been removed or rearranged, including but not limited to changes in the disulfide bond pattern of the monomer or dimer. In one embodiment, Cys11 (which is believed not to participate in intermolecular or intramolecular disulfide bonds) is removed or substituted.

In one series of embodiments, the FXI variant has a reduced half-life in plasma relative to wild type human FXI. In one embodiment, the FXI variant has a half life of less than 50 hours. In one embodiment, the FXI variant has a half life of less than 24 hours. In one embodiment, the FXI variant has a half life of less than 12 hours. In one embodiment, the FXI variant has a half life of less than 6 hours. In one embodiment, the FXI variant has a half life of less than 3 hours.

In one set of embodiments, the FXI variant is characterized in that N-linked glycosylation at one or more sites is modified by modification of cognate N-linked glycosylation consensus sites, eg, N72, N108, N335, N432, N473, or those described above. Polypeptides that are destroyed by independent substitution with any of the combinations. 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. In addition, disruption of N-linked glycosylation at one or more sites is, for example, independent of (i) any of residues 72-74, 108-110, 335-337, 432-434, and 473-475 (ie, One or more residues are deleted at each site and may not be substituted by any other amino acid); (ii) independent substitution of the N + 2 residue (eg, substitution of T74 with any residue other than S, substitution of S110 with any residue other than T, substitution of S337 with any residue other than S) , Substitution of any residue other than T of S434, substitution of any residue other than T of T475); (iii) substitution of the N + 1 residues with glycosylation-destroying amino acids (not limited but exemplified by proline (P)). It will be appreciated that any combination of the above means can be used to independently destroy glycosylation at different sites in the FXI polypeptide.

In addition, the present invention includes chimeric or fusion polypeptides between all or part of an FXI sequence and other heterologous peptide sequences. For example, one or more of the four Apple domains may be substituted by a similar Apple domain from another polypeptide (see, eg, Gailani et al. (1999) Blood 94: 621a), or one or more of the Apple domains This can be entirely fruitful. In other embodiments, factor IXa is known to contribute to interaction with LRP, such as LDL receptor-associated protein (LRP) (eg, peptides comprising residues Phe342-Asn346 of factor IXa, Rohlena et al. (2003), J Binding site for Biol. Chem. 278: 9394) is attached to the sequence of the FXI polypeptide to modify its pharmacokinetic properties.

In addition, the dimeric nature of the active form of FXI (and the asymmetrical function of the two monomers in platelet binding and FXI activation, for example) is an FXI heterodimer, i.e. two unequal FXI (or FXI-glyco) monomer polypeptides. Formulations used in the present invention, including combinations of The only requirement is to exhibit one or more advantageous embodiments of FXI biological activity.

FXI polypeptides used in the present invention include, without limitation, polypeptides that exhibit substantially the same or improved biological activity with respect to wild type human FXI, and polypeptides whose FXI biological activity is substantially modified or reduced with respect to the activity of wild type human FXI.

In the practice of the present invention, useful compositions comprising the FXIa or FXIa-cognate polypeptides, including variants, have FXI activity when the wild type FXI equivalents are from the same source or are generated in the same cell type, and the activity comparison is the same When done by parallel testing in the assay, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60 of the specific activity of a composition comprising only wild type FXI alone %, 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%. As used herein, the terms "activity" and "specific activity" apply to any aspect or aspects of FXI biological activity, respectively or collectively.

In some embodiments, the ratio of the specific proteolytic activity of the FXI-cognate polypeptide to the proteolytic activity of the wild-type human FXI is at least about 1.25 when tested by the FXI amidolytic assay; In other embodiments, this ratio is at least about 2.0; In further embodiments, this ratio is at least about 4.0.

FXI  Biological Activity:

In the practice of the present invention, one or more different aspects of FXI biological activity can be quantified, such as (i) selecting a suitable FXI composition for therapeutic administration, formulation, FXI production or purification methods, and the like; And / or (ii) assess the efficacy of different therapeutic modalities. It will be appreciated that the "specific activity" of FXIa for any of these embodiments of biological activity is expressed as units of activity per unit mass of FXIa polypeptide. These aspects include the following:

I. Proteolysis  activation

(a) Ekdahl et al. (1999), Thromb. Haemost. As described in 82: 1283-8, amidohydrolytic activity can be quantified in vitro using, for example, a suitable chromosomal substrate such as S2355 (Chromogenix). The activity measured is compared to a standard FXIa formulation with defined specific activity (Enzyme Research Laboratories), which values are expressed as AU of FXIa activity.

(b) As described, for example, in Gailani et al. (2001), Blood 97: 3117-3122, FXI activation activity can be quantified directly in vitro by measuring proteolytic conversion of factor IX to IXa.

II. Combination of Activity and Inhibitors

Wild-type human FXI has many binding partners, including prekallikrein (PK), high molecular weight kininogen (HK), thrombin / prothrombin, factor IX (FIX), and platelet-associated FXI receptors named GP1b-V-IX Have them. In the practice of the present invention, any conventional binding assay can be used to quantify the affinity of the FXI polypeptide for any of these (or other) binding partners. Such binding assays include, but are not limited to, competitive binding assays in which either binding partner is labeled.

FXI polypeptide binding partners also include FXI active site inhibitors including, without limitation, antithrombin III, C1 inhibitors, α-2-antitrypsin, PAI-1, protein C inhibitors and protease nexin II (PNII). The affinity of these compounds for FXI polypeptides can be quantified using conventional binding assays; Alternatively, the inhibitory activity of such compounds on the proteolytic activity of a particular FXI polypeptide formulation can be measured using amidohydrolysis or FIX-activation assays.

III. Coagulation  parameter

Coagulation time, coagulation lysis time, and coagulation intensity are clinical parameters used to analyze the condition of the patient's hemostatic system. Blood samples are taken from the patient at appropriate intervals after administration of the FXI polypeptide and one or more of these parameters are analyzed. Alternatively, the FXI polypeptide or agent may be used for in vitro / in vitro treatment of blood drawn from a human subject.

Coagulation time can be analyzed by standard PT or aPTT assay.

Coagulation dissolution time and coagulation intensity are described, for example, in Vig et al. (2001) Blood coagulation & fibrinolysis, Vol. 12 (7) pp.555-561. And by thromboelastograpy, as described by Sorensen (2003) Throm Haemost 1: 551-558. Alternatively, coagulation intensity is described by Carr et al. (1991), Am. J. Med. Sci. 302: may be analyzed as described by 13-8.

One parameter that reflects the coagulation activity of FXI as measured by thromboelastomography is "Comprehensive Coagulation Quality" (OCQ). Once clotting is initiated (t = 0), coagulation intensity is measured as a function of time to determine the maximum rate of clot formation (max vel) and the time required to reach the maximum rate (t _{max vel} ). Tissue plasminogen activator (tPA) is then added to determine fibrinogen and derive the time (t _{min vel} ) required to reach the maximum rate of fibrinolysis. OCQ is calculated as follows:

(Max vel / t _{max vel} ) X (t _{min vel} -t _{max vel} )

IV. Pharmacokinetic Parameters

Wild-type human FXI is believed to have a plasma half-life of about 50 hours, which is mediated at least in part upon its interaction with HK. In the practice of the present invention, FXI polypeptides exhibiting pharmacokinetic properties different from native FXI can 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 modified by PEGylation, and FXI polypeptides that exhibit altered interaction with HK. . In the practice of the present invention, pharmacokinetic properties can be calculated using, for example, WinNonlin Professional version 3.1 (Pharsight Inc., Mountain View, CA, USA). If more than one value is present, the calculation is performed using the average concentration value at each time point.

Can use the following expression for the following pharmacokinetic parameters of can be _{calculated: AUC, AUC% Extrap, C} max, t max, γ z, t 1/2, CL, and V _z:

AUC: Area under the plasma concentration-time curve from time 0 to infinity. Extrapolated to infinity and calculated using linear / log trapezoidal rule.

The linear trapezoid quadrature is used from time 0 to t _max :

The log trapezoid quadrature is used from time t _max to the final time point t:

Extrapolation to infinity is performed using the following equation:

AUC _{% Extrap} : Percentage of AUC due to extrapolation from final concentration to infinity

C _max : maximum extrapolated plasma concentration up to time 0

CL: Total Body Clearance

t _max : time at which the maximum plasma concentration was observed

t _1/2: Half-Life

γ _z : final velocity constant. Calculated by log-linear regression of (average) concentration versus time

V _z : Distribution volume based on the final stage

FXI Generation and Purification of:

FXI polypeptides used in the present invention can be prepared from plasma or from recombinant sources using any suitable method known in the art. As used herein, the term “isolated” refers to an FXI polypeptide isolated from the cells to which they are synthesized or from the medium in which they are found in their natural state (eg, plasma or blood).

Isolation of the polypeptide from the cells of origin may include removal of cell culture medium containing the desired product from the adherent cell culture; Centrifugation or filtration to remove non-adherent cells; It may be achieved by any method known in the art, including without limitation, and the like. Optionally, the FXI polypeptide can be further purified. Purification includes, for example, an anti-FXI antibody column or a peptide affinity column (non-limiting examples of which are heparin, blue, red, L-arginine, benzamidine peptides, other dyes, or RP-chromatography). Affinity chromatography such as; Hydrophobic interaction chromatography; Ion exchange chromatography; Size exclusion chromatography; Electrophoretic processes (eg, preliminary isopotential focusing (IEF), differential solubility (eg, any precipitation or crystallization using salts, pH, ammonium sulfate, or other additives), or extraction, etc. Can be achieved using any method known in the art, these have been described in more detail above After the purification, the formulation is preferably less than about 10% by weight, more preferably less than about 5% by weight, most preferably Contains less than about 1% by weight of non-FXI polypeptide derived from a host cell.

Purification of FXI from plasma is described in Koide et al. (1977), Biochem. 16: 2279 and Bouma et al. (1977), J. Biol. Chem. It may also be accomplished by known methods, including without limitation those disclosed by 252: 6432. See, eg, Kemball-Cook et al. (1994), Gene 139: 275, Fujikawa et al. (1986), Biochem. 25: 2417, and Meijers et al. (1992), Blood 79: 1435. FXIa is also commercially available from Enzyme Research Laboratories (South Bend, India).

Further, the present invention relates to a method for purifying FXI polypeptides, such as recombinant FXI, from other biological materials, which comprises chromatography the material in a cation-exchange chromatography material.

Further, the present invention relates to a method for purifying FXI polypeptides, such as recombinant FXI, from other biological materials, which comprises chromatography the material in hydrophobic interaction chromatography materials.

Further, the present invention relates to a method for purifying FXI polypeptides, such as recombinant FXI, from other biological materials, which comprises chromatography the material in a hydroxyapatite chromatography material.

Further, the present invention relates to a method for purifying FXI polypeptides, such as recombinant FXI, from other biological materials, which methods are used in cation-exchange chromatography materials, hydrophobic interaction chromatography materials and hydroxyapatite chromatography materials. Continuous chromatography. It should be understood that continuous chromatography is performed in the order described.

As used herein, the term “hydroxyapatite chromatography material” means any hydroxyapatite chromatography material known in the art that can bind to FXI polypeptides, such as hydroxyapatite matrices.

As used herein, the term “cation-exchange chromatography material” means any cation-exchange chromatography material known in the art capable of binding to an FXI polypeptide, such as a cation-exchange matrix.

As used herein, the term “hydrophobic interaction chromatography material” means any hydrophobic interaction chromatography material known in the art capable of binding to an FXI polypeptide, such as a hydrophobic interaction matrix.

In one embodiment, the invention is directed to a method of purifying a FXI polypeptide from a biological material, the method comprising chromatography on a first cation-exchange chromatography material a biological material comprising the FXI polypeptide, wherein the chromatography Photography involves the following steps:

(i) applying the biological material to the original cation-exchange chromatography material;

(ii) eluting the unbound material from the original cation-exchange chromatography material using buffer A, wherein buffer A is suitable for eluting material not bound to the original cation-exchange chromatography material; And

(iii) eluting unbound material from the original cation-exchange chromatography material using buffer A ', wherein buffer A' is suitable for eluting material not bound to the original cation-exchange chromatography material; And

(vi) eluting the FXI polypeptide from the original cation-exchange chromatography material by elution with buffer A '', wherein buffer A '' is suitable for eluting the FXI polypeptide from the original cation-exchange chromatography material. Do.

In one embodiment, the invention is directed to a method for purifying a FXI polypeptide from a biological material, the method comprising hydrolyzing a fluid prepared using an eluate from step (iv) or an eluate from step (iv). Chromatography using a functional chromatography material, the chromatography comprising the following steps:

(v) applying the fluid prepared using the eluate from step (iv) or the eluate from step (iv) to the hydrophobic interaction chromatography material;

(vi) eluting unbound material from the hydrophobic interaction chromatography material using buffer B, wherein buffer B is suitable for eluting material not bound with the hydrophobic interaction chromatography material; And

(vii) eluting the FXI polypeptide from the hydrophobic interaction chromatography material by gradient-elution with buffer B ', wherein buffer B' is suitable for eluting the FXI polypeptide from the hydrophobic interaction chromatography material.

In one embodiment, the present invention is directed to a method of purifying a FXI polypeptide from a biological material, which method comprises hydroxy treating a fluid prepared using an eluate from step (vii) or an eluate from step (vii). Chromatography using an apatite chromatography material, the chromatography comprising the following steps:

(viii) applying to the hydroxyapatite chromatography material a fluid prepared using the eluate from step (vii) or the eluate from step (vii);

(ix) eluting the unbound material from the hydroxyapatite chromatography material using buffer C, wherein buffer C is suitable for eluting material not bound with the hydroxyapatite chromatography material; And

(x) eluting the FXI polypeptide from the hydroxyapatite chromatography material by gradient-elution with buffer C ', wherein buffer C' is suitable for eluting the FXI polypeptide from the hydroxyapatite chromatography material.

In one embodiment, the invention is directed to a method of purifying a FXI polypeptide from a biological material, the method comprising the following steps:

(a) chromatography a biological material comprising a FXI polypeptide on an original cation-exchange chromatography material, the chromatography comprising the following steps:

(i) applying the biological material to the original cation-exchange chromatography material;

(ii) eluting the unbound material from the original cation-exchange chromatography material using buffer A, wherein buffer A is suitable for eluting material not bound to the original cation-exchange chromatography material; And

(iii) eluting unbound material from the original cation-exchange chromatography material using buffer A ', wherein buffer A' is suitable for eluting material not bound to the original cation-exchange chromatography material; And

(vi) eluting the FXI polypeptide from the original cation-exchange chromatography material by elution with buffer A '', wherein buffer A '' is suitable for eluting the FXI polypeptide from the original cation-exchange chromatography material. Do.

(b) chromatography the fluid prepared using the eluate from step (iv) or the eluate from step (iv) with a hydrophobic interaction chromatography material, the chromatography comprising the following steps: :

(v) applying the fluid prepared using the eluate from step (iv) or the eluate from step (iv) to the hydrophobic interaction chromatography material;

(vi) eluting unbound material from the hydrophobic interaction chromatography material using buffer B, wherein buffer B is suitable for eluting material not bound with the hydrophobic interaction chromatography material; And

(vii) eluting the FXI polypeptide from the hydrophobic interaction chromatography material by gradient-elution with buffer B ', wherein buffer B' is suitable for eluting the FXI polypeptide from the hydrophobic interaction chromatography material.

(c) chromatography the fluid prepared using the eluate from step (vii) or the eluate from step (vii) using a hydroxyapatite chromatography material, the chromatography comprising the following steps: :

(viii) applying to the hydroxyapatite chromatography material a fluid prepared using the eluate from step (vii) or the eluate from step (vii);

(ix) eluting the unbound material from the hydroxyapatite chromatography material using buffer C, wherein buffer C is suitable for eluting material not bound with the hydroxyapatite chromatography material; And

(x) eluting the FXI polypeptide from the hydroxyapatite chromatography material by gradient-elution with buffer C ', wherein buffer C' is suitable for eluting the FXI polypeptide from the hydroxyapatite chromatography material.

Purification of the FXI polypeptide is the process of increasing the concentration of the FXI polypeptide relative to the other components of the sample, resulting in increased purity of the FXI polypeptide. It should be understood that the concentration of the FXI polypeptide for the other components of the sample in the sample is not the same as the concentration of the FXI polypeptide in the sample. After increasing the purity of the FXI polypeptide, such as, for example, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), HPLC (high performance liquid chromatography) or Dade Behring Diagnostics, or coagulation activity analysis, Measurements can be followed using methods known in the art.

The biological substance can be any substance derived from or containing a cell, cellular component or cell product. The biological material may be a biological fluid.

The biological fluid can be any fluid derived from or containing a cell, cellular component or cell product. Biological fluids include, but are not limited to, cell culture, cell culture supernatant, cell lysate, clear cell lysate, cell extract, tissue extract, blood, plasma, serum, all of which may also be homogenates and filtrates, For example, their fractions collected by chromatography of undifferentiated biological fluids.

FXI polypeptides can be purified from a wide variety of biological materials, including cell culture supernatants, which naturally produce FXI polypeptides, but are also mammalian cells (eg, CHO cells) that have been transformed with DNA encoding the FXI polypeptides. As such, their cells may be genetically modified to produce FXI polypeptides.

Biological materials can be treated using many methods prior to application to the original cation-exchange chromatography material. Such methods include, but are not limited to, centrifugation, filtration. In one embodiment, the biological material is a biological fluid. In one embodiment of the invention, the biological fluid is a supernatant of yeast cell lysate.

In one embodiment of the invention, the FXI polypeptide is purified from cell culture, such as the mammalian cell culture described above. Mammalian cells may be separated from the cell culture supernatant by centrifugation and / or filtration prior to chromatography in step (a). Inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCl may be included before chromatography in step (a).

A buffer is a solution that contains a substance that can primarily maintain the original acidity or basicity of the solution by adding a small amount of acid or base to prevent significant changes in solution pH. The buffer usually contains a weak acid or weak base with its salt.

The pH of the biological fluid can be adjusted to the pH of Buffer A prior to chromatography in step (a), for example using 1M HCl or 1M NaOH or by other means known in the art.

The original cation-exchange chromatography material is any cation known in the art capable of binding to and releasing FXI polypeptide under a set of conditions, such as a cation-exchange chromatography material comprising a sulfopropyl group. -Exchange chromatography material. Non-limiting examples of further cation-exchange chromatography materials include derivatized dextran, agarose, cellulose, polyacrylamide, and specialty silicas such as carboxymethyl. Suitable cation-exchange chromatography materials include chromatography of biological fluids, including FXI polypeptides, in selected cation-exchange chromatography materials, collecting fractions, for example SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrolysis). Electrophoresis), HPLC (high performance liquid chromatography), coagulation activity or Dare Behring Diagnostics to determine the purity and content of fractions, by monitoring the absorbance of the eluate at 280 nm, and other known in the art. This can be confirmed using the method. Examples of suitable cation-exchange chromatography 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 cat no 17-5266), S-Support Unosphere (BioRad cat no. 156-0113) or Toyopearl SP-550C (Toso Haas cat no. 14028). In one embodiment, Obelix ST CIEX is used.

The original cation-exchange chromatography material may be pre-equilibrated with buffer A prior to application of the biological material.

Buffer A may include protease inhibitors such as EDTA (ethylenediamine tetraacetic acid) and benzamidineHCl, although other commercially available protease inhibitors may also be used.

In one embodiment of the invention, the pH of buffer A is 6.5-9. In further embodiments, the pH of buffer A is 7-9. In further embodiments, the pH of buffer A is about 8.

In one embodiment of the invention, the conductivity of buffer A is less than about 40 mS / cm.

Buffer A '' is used for the elution of FXI. Typically, the concentration of one or more of the components of the buffer, in this case buffers A and A ', used for washing in step (ii) is increased or decreased during the elution process, or a new component is added to the buffer and The concentration is increased during the elution process. This increase or decrease may occur in succession or in separate steps as is known in the art. For elution of the material combined with the cation-exchange chromatography material, it is customary to add a salt, for example NaCl, to Buffer A. This specific cation exchanger may also be used as a hydrophobic interaction chromatography resin, in which it is common to make buffer A 'by adding propanediol / glycerol to buffer A. FXI may elute if both NaCl and propanediol / glycerol are added to buffer A. For example, to exclude unwanted impurities eluting at the beginning or end of FXI polypeptide elution, it is within the knowledge of those skilled in the art to determine whether further fractions containing the FXI polypeptide will be collected for further processing. Similarly, general techniques for performing cation-exchange chromatography are well known, for example with regard to pre-equilibration, elution time, washing, recovery of cation-exchange chromatography material, and the like.

After elution of the FXI polypeptide in step (iv), an eluate containing an FXI polypeptide protease inhibitor such as EDTA (ethylenediamine tetraacetic acid) and benzamidine is added followed by step (v). This eluate can also be maintained at 4 ° C., for example at −80 ° C., for example for at least 24 hours.

The hydrophobic interaction chromatography material used in step (b) binds to the FXI polypeptide under a set of conditions and differs from the set of conditions, such as a hydrophobic interaction chromatography material derivatized with a phenyl, butyl or octyl group, or polyacrylic resin. It may be any hydrophobic interaction chromatography material known in the art that can release it under the conditions of. Non-limiting examples of suitable hydrophobic interaction chromatography materials include

Amberchrom ^TM CG 71 from Tosoh Bioscience,

^{Phenyl Sepharose TM High Performance (Amersham,} cat no 17-1082),

Phenyl Sepharose ^™ 6 Fast Flow High Substitution (Amersham, cat no 17-0973),

Butyl 650(Tosoh Bioscience), Toyopearl

Butyl 650 (Tosoh Bioscience),

Phenyl(Tosoh Bioscience), Toyopearl

Phenyl (Tosoh Bioscience),

Source ^TM 15 Phe (Amersham, cat no 17-0147),

Butyl Sepharose ^TM High Performance High Substitution (Amersham, cat no 17-3100),

Octyl Sepharose ^™ (Amersham, cat no 17-0946), and

Phenyl Sepharose ^™ High Performance High Substitution (Amersham). In one embodiment of the invention, the hydrophobic interaction chromatography material uses butyl as the ligand.

Buffer B and NaCl may be added to the fluid prepared using the eluate from step (iv) or the eluate from step (iv) prior to chromatography in step (b) in an amount of at least about 1-2 volumes Or buffer B in concentrated form, which contains the same ingredients as buffer B, but has a concentration twice, for example, in an amount corresponding to the strength of the concentration buffer (twice concentrated buffer is added in an amount of 1.5 volumes). Eluate from (iv) or eluate from step (iv) is added to the fluid prepared.

Buffer B may have a pH of about 5 to about 9, for example about 8. In one embodiment of the invention, buffer B has a conductivity of at least 25 mS / cm, for example at least 70 mS / cm. This can be achieved, for example, by the use of phosphate buffers or by other means known in the art, for example NaCl. In one embodiment of the present invention, the conductivity of the fluid prepared using the eluate from step (iv) or the eluate from step (iv) is adjusted to a conductivity of at least about 60 mS / cm.

Buffer B 'is used to elute the FXI polypeptide by gradient elution. In gradient elution, the composition of buffer B 'changes during the elution process. Typically, the concentration of one or more of the components of the buffer, in this case buffer B, used for washing in step (vi) is increased or decreased during the elution process, or a new component is added to the buffer and the concentration of this component Is increased during the process. This increase or decrease can occur either continuously or in separate steps as is well known in the art. For elution of materials bound with hydrophobic interaction chromatography materials, it is common to dilute the wash buffer with water until at least a large portion of the bound FXI polypeptide is eluted. For example, to exclude unwanted impurities eluting at the beginning or end of FXI polypeptide elution, the determination of whether to collect fractions containing the FXI polypeptide for further processing is within the knowledge of those skilled in the art. Similarly, general techniques for performing hydrophobic interaction chromatography are well known, for example with regard to pre-equilibration, elution time, washing, recovery of hydrophobic interaction chromatography material, and the like.

In one embodiment of the present invention, the fluid prepared using the eluate from step (vii) or the eluate from step (vii) is treated using a method comprising the following steps:

(1) adding one or more stabilizers that can increase the stability of the FXI polypeptide in an amount effective to significantly improve its stability, and / or

(2) adjusting the pH of the fluid prepared using the eluate from step (vii) or the eluate from step (vii) to a pH of about 7 to about 9.

This step and other post-treatment optional steps known in the art can be performed alone or in combination, the order in which the steps are performed is not critical. Those skilled in the art will be able to determine how and when to perform these steps.

In one embodiment of the invention, stabilizers that can increase the physical and / or chemical stability of the FXI polypeptide are added to the fraction containing FXI.

As used herein, the term “physical stability” for FXI polypeptides is biologically inert as a result of exposure of proteins to thermo-mechanical stress and / or interaction with unstable interfaces and surfaces such as hydrophobic surfaces and interfaces. Refers to the potential tendency of a protein to form agglomerates or multimers of phosphorus and / or insoluble proteins. The physical stability of the FXI polypeptide when present in buffer A is determined by exposing the formulations filled in suitable containers (eg cartridges or vials) to mechanical / physical stress (eg, agitation) at different temperatures for various time periods. It can be evaluated by visual inspection and / or turbidity measurement.

Visual examination of the FXI polypeptide when present in the buffer can be performed in strong spotlight of the cancerous background. The turbidity of the composition can be characterized, for example, by a visual score that gives a degree of turbidity on a scale of 0-3 (a composition that does not exhibit turbidity corresponds to a visual score of 0 and exhibits visual turbidity under daylight). Corresponds to visual score 3). When the composition exhibits visual haze in sunlight, the composition is classified as physically unstable with regard to protein aggregation. Alternatively, the turbidity of the composition can be assessed by simple turbidity measurements well known to those skilled in the art, for example by measuring the optical density of the solution at a wavelength of ₄₀₅ nm (OD ₄₀₅ ). In addition, the physical stability of the aqueous protein composition can be assessed using spectroscopic agents or probes for the conformational state of the protein. The probe is preferably a small molecule that preferentially binds to non-autogenous isoforms of the protein. One example of small molecule spectroscopic props for protein structure is Thioflavin T. Thioflavin T is a fluorescent dye widely used for amyloid fibril detection. In the presence of fibril, and possibly also in the presence of other protein conformations, thioflavin T, when combined with the fibrillar protein form, produces a new excited state peak at about 450 nm and enhanced emission at about 482 nm. Unbound thioflavin T is not inherently fluorescent at these wavelengths.

Other small molecules can be used as probes for changes in protein structure from the native to non-native states. For example, a "hydrophobic patch" is a probe that preferentially binds to an exposed hydrophobic patch of a protein. Hydrophobic patches generally hide within the tertiary structure of a protein in its native state, but are exposed as the protein begins to fold or denature. Examples of these small molecules, spectroscopic probes are aromatic, hydrophobic dyes such as anthracene, acridine, phenanthroline and the like. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids such as phenylalanine, leucine, isoleucine, methionine and valine and the like.

As used herein, the term “chemical stability” for FXI polypeptides refers to protein structures that result in the formation of chemical denaturation products with potentially lower biological efficacy and / or potentially increased immunogenic properties when compared to native protein structures. Changes in chemical covalent states at Various chemically modified products may be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Removal of chemical denaturation is largely probably not completely avoided, and an increase in the amount of chemical denaturation products is mainly seen during storage and use of protein compositions as is well known to those skilled in the art. Most proteins have a tendency to deamidate, which is the process by which side chain amide groups in glutaminel or asparaginyl residues are hydrolyzed to form free carboxylic acids. Other denaturation pathways involve the formation of high molecular weight conversion products, wherein two or more protein molecules are covalently bonded to each other by transamidation and / or disulfide interactions, resulting in the formation of covalent dimers, oligomers, and polymer denaturation products. (Stability of Protein Pharmaceuticals, Ahern. TJ & Manning MC Plenum Press, New York 1992). As another variant of chemical denaturation, oxidation (eg of methionine residues) can be mentioned. The chemical stability of the FXI polypeptide when present in buffer B 'can be assessed by measuring the amount of chemical denaturation product at various times after exposure to different environmental conditions; The formation of the denaturing product can be accelerated mainly, for example by increasing the temperature. The amount of each individual denaturation product is determined primarily by the separation of the denaturation products according to molecular size and / or charge using various chromatographic techniques (eg SEC-HPLC and / or RP-HPLC).

Any agent that can significantly improve the physical and / or chemical stability (eg, determined by measuring turbidity at OD ₄₀₅ over a period of time) of the FXI polypeptide when present in buffer B 'can be used as a stabilizer.

Formulations suitable for use as stabilizers include, for example, salts (eg sodium chloride), sugars, alcohols (such as C4-C8 alcohols), alditol, amino acids (eg glycine, histidine, arginine, lysine, isoleucine , Aspartic acid, tryptophan or threonine), polyethyleneglycol (eg PEG400), or mixtures of one or more thereof. Monosaccharides, disaccharides, or any sugar such as polysaccharides or water soluble glucan can be used. Alditol is a polyalcohol of HOCH _2- [CH (OH)] _n -CH ₂ OH structure, where n is 1, 2, 3... Non-limiting examples of substances that are sugars, alcohols or altitols 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), propane-1,2-diol (propylene glycol), propane -1,3-diol, and butane-1,3-diol. The above-mentioned sugars, alcohols and alditols can be used individually or in combination. The amount used is not limited so long as this substance is soluble in liquid formulations and improves the physical stability of the FXI polypeptide in solution. In this regard, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In one embodiment of the invention, one or more polyalcohol type stabilizers are added.

In one embodiment of the invention, glycerol (propane-1,2,3-triol), propylene glycol (propane-1,2-diol), propane-1,3-diol, propyl alcohol (1-propanol) and At least one stabilizer selected from the group consisting of isopropyl alcohol (2-propanol) is added. In one embodiment of the invention, one or more stabilizers selected from the group consisting of glycerol, propylene glycol and propane-1,3-diol are added.

In further embodiments of the invention, when the stabilizer is a liquid alcohol or a liquid polyalcohol [eg, glycerol, propylene glycol, propane-1,3-diol, propyl alcohol or isopropyl alcohol], the stabilizer is about 5 volumes It is present at a concentration of% (v / v) to about 50% (v / v). In further embodiments, the liquid alcohol or liquid polyalcohol type stabilizer is present at a concentration of about 10% (v / v) to about 50% (v / v). In further embodiments, the liquid alcohol or liquid polyalcohol type stabilizer is present at a concentration of about 10% (v / v) to about 20% (v / v). In further embodiments, the liquid alcohol or liquid polyalcohol type stabilizer is present at a concentration of about 10% (v / v). In still further embodiments, the stabilizer of the liquid alcohol or liquid polyalcohol type is present at a concentration of about 20% (v / v).

The stabilizers mentioned should be able to increase the above mentioned physical and / or chemical stability of the FXI polypeptide. Any agent that can significantly improve the physical and / or chemical stability of the FXI polypeptide (eg, determined by measuring turbidity at OD ₄₀₅ over a period of time) can be used as a stabilizer.

The eluate from step (vii) can be used for the preparation of the pharmaceutical composition. This may involve altering the buffer, and / or adjusting the conductivity and / or pH to physiological values, and / or allowing the eluate to be used in a mammal, such as a human: Means for making use of are well known in the art. In addition, the eluate may be maintained at 4 ° C., for example at −80 ° C., for example for at least 24 hours.

In one embodiment of the invention, the method further comprises chromatography in a hydroxyapatite chromatography material a fluid prepared using the eluate from step (vii) or the eluate from step (vii); , The chromatography comprises the following steps:

(viii) applying to the hydroxyapatite chromatography material a fluid prepared using the eluate from step (vii) or the eluate from step (vii);

(ix) eluting the unbound material from the hydroxyapatite chromatography material using buffer C, wherein buffer C is suitable for eluting material not bound with the hydroxyapatite chromatography material; And

(x) eluting the FXI polypeptide from the hydroxyapatite chromatography material using buffer C ', wherein buffer C' is suitable for eluting the FXI polypeptide bound with the hydroxyapatite chromatography material in step (ix).

Fluids prepared using the eluate from step (vii) can be prepared, for example, prior to application.

In one embodiment of the present invention, the conductivity of the fluid prepared using the eluate from step (vii) or the eluate from step (vii) is adjusted to less than about 20 mS / cm by adding water. pH is adjusted to 5.8-9. In one embodiment, the pH is adjusted to 6.0.

The components of Buffer C and Buffer C 'may be selected in consideration of the desired final pharmaceutical composition of the FXI polypeptide. Such considerations are within the knowledge of one of ordinary skill in the art.

In one embodiment of the invention, buffer C comprises one or more stabilizers, which stabilizers can increase the physical and / or chemical stability described above of the FXI polypeptide. When present in buffer C, any agent capable of significantly improving the physical and / or chemical stability of the FXI polypeptide (eg, determined by measuring turbidity at OD ₄₀₅ over a period of time) is either buffer C or buffer C '. Can be used as a stabilizer.

Formulations suitable for use as stabilizers in buffer C are, for example, salts (eg sodium chloride), sugars, alcohols (such as C4-C8 alcohols), alditol, amino acids (eg glycine, histidine, arginine, lysine). , Isoleucine, aspartic acid, tryptophan or threonine), polyethylene glycol (eg PEG400), or mixtures of one or more thereof. Monosaccharides, disaccharides, or any sugar such as polysaccharides or water soluble glucan can be used. Non-limiting examples of substances that are sugars, alcohols or altitols 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), propane-1,2-diol (propylene glycol), propane -1,3-diol, and butane-1,3-diol. The above-mentioned sugars, alcohols and alditols can be used individually or in combination. The amount used is not limited as long as it is soluble in liquid formulations and improves the physical stability of the FXI polypeptide in solution. In this regard, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In one embodiment of the invention, buffer C comprises at least one stabilizer of polyalcohol type.

In one embodiment of the invention, buffer C comprises NaCl.

Glycerol (propane-1,2,3-triol), propylene glycol (propane-1,2-diol), propane-1,3-diol, propyl alcohol (1-propanol) and isopropyl alcohol (2-propanol) One or more stabilizers selected from the group consisting of are added to the fluid from (x).

In one embodiment of the invention, one or more stabilizers selected from the group consisting of glycerol, propylene glycol and propane-1,3-diol are added. In one embodiment of the invention, propylene glycol is added.

In further embodiments of the invention, when the stabilizer is a liquid alcohol or a liquid polyalcohol, the stabilizer is present at a concentration of about 5% (v / v) to about 50% (v / v). In further embodiments, the liquid alcohol or liquid polyalcohol type stabilizer is present at a concentration of about 10% (v / v) to about 50% (v / v). In a further embodiment, the liquid alcohol or liquid polyalcohol type stabilizer used is present at a concentration of about 10% (v / v) to about 20% (v / v). In further embodiments, the liquid alcohol or liquid polyalcohol type stabilizer is present at a concentration of about 10% (v / v).

Buffer C 'is used to elute the FXI polypeptide by gradient elution, where the composition of the buffer C' changes during the elution process. Typically, the concentration of one or more of the components of the buffer, in this case buffer C, used for washing in step (ix) is increased or decreased during the elution process, or a new component is added to the buffer and the concentration of this component is eluted. Is increased during the process. This increase or decrease can occur either continuously or in separate steps as is well known in the art. For elution of materials bound with hydroxyapatite chromatography materials, it is common to add salts, such as KPO ₄ or NaCl, to buffer C, and then the concentration of salt until at least a large portion of the bound FXI polypeptide is eluted. To increase. It is within the knowledge of one of ordinary skill in the art to determine whether to collect fractions containing FXI polypeptides for further processing, for example to exclude unwanted impurities eluting at the beginning or end of FXI polypeptide elution. Likewise, general techniques for performing hydroxyapatite chromatography are well known, for example with regard to pre-equilibration, elution time, washing, recovery of cation-exchange chromatography material, and the like.

In a series of embodiments, the use of one or more stabilizers in any or all of the solutions used in the purification of FXI may involve the physical and / or chemical of the control (ie, FXI with the same treatment but without stabilizers). Increase the physical and / or chemical stability of FXI by at least 10%, 25%, 50% or 100% relative to stability. In another set of embodiments, the use of one or more stabilizers in any or all of the solutions used in the purification of FXI may involve the physical and / or chemical of the control (i.e., FXI with the same treatment but without stabilizers). Increase the physical and / or chemical stability of FXI by at least 2, 5, 10, or 20 times relative to stability.

FXI Activation of:

Wild-type human FXI is normally activated by proteolytic cleavage between Arg360 and Ile370, which can be catalyzed by FXIa, FXIIa, or thrombin. If desired, activation of FXI used in the present invention can be accomplished using FXIa or FXIIa (both from Enzyme Research Laboratories, South Bend, India) or thrombin (Sigma). See, eg, 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 present invention to activate FXI polypeptides, particularly FXI-cognate polypeptides, using other proteases.

The present invention includes methods and compositions for the therapeutic administration of FXI using agents having different levels of FXI activation. In some embodiments, the methods and compositions use FXI polypeptides that have not undergone any activation process. In some embodiments, the formulation of the FXI or FXI-cognate polypeptide can be from about 1:99 to about 99: 1, for example from 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 an activation: simogen FXI or FXI-cognate polypeptide ratio (mass) of about 50:50. In some embodiments, the formulation comprises about 5% or less relative to the total FXI on a molar basis; More preferably about 2.5% or less, even more preferably about 1% or less, most preferably about 0.5% or 0.1% or less of FXIa. In some embodiments, the formulation contains up to about 0.01-0.05% FXIa on a molar basis. In some embodiments, the formulation contains up to about 0.01-0.04% FXIa on a molar basis. In some embodiments, the formulation contains up to about 0.01-0.03% FXIa on a molar basis.

In addition, the present invention provides FXI-cognate polypeptides that exhibit differential ability to be activated against wild-type FXI, such as FXI-cognate polypeptides that are more readily activated by FXIIa than by thrombin; Polypeptides that are constitutively activated without proteolytic cleavage; One monomer (by mutation or chemical modification) is a heterodimer that cannot be proteolytically activated; Etc.

Moreover, the present invention also relates to variants in which the ratio of the activation rate by FXI-cognate polypeptides resistant to self activation, ie thrombin (and / or FXIIa) to activation rate by FXIa, is higher than for wild type FXI.

In addition, the methods and compositions of the present invention may utilize treatment, pretreatment, storage, or co-administration of additional agents that inhibit and / or promote activation with FXI polypeptides. Non-limiting examples of agents that inhibit activation include C1 esterase inhibitors (C1Inb), α-2-antiplasmin (α2AP), α-1-antitrypsin (α1AT), protease nexin II, benzamidine, Heparin, and antithrombin III; Non-limiting examples of agents that promote activation include FXIa, FXIIa, and thrombin.

FXI Pharmaceutical formulations comprising:

The present invention includes pharmaceutical compositions comprising a formulation of an FXI or FXI-glycopetic polypeptide for prophylactic and / or therapeutic treatment.

The pharmaceutical composition or formulation according to the invention comprises the FXI polypeptide, for example at a concentration of 0.001-100 mg / ml, which is preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent. In brief, pharmaceutical compositions suitable for use in accordance with the present invention are preferably prepared by mixing a formulation comprising a FXI and / or FXI-glycopetic polypeptide in purified form with a suitable adjuvant and a suitable carrier or diluent. Various aqueous carriers can be used such as water, buffered water, 0.4% saline, 0.3% glycine, sugars, detergents, salts, buffers, glycerol, preservatives, protease inhibitors, glycols and the like. The formulations of the invention can also be formulated using non-aqueous carriers, for example in the form of gels or as liposome formulations for delivery or targeting to the wound site. Liposomal formulations are generally described in US Pat. 4,837,028, 4,501, 728, and 4,975,282. The composition can be sterilized by conventional well known sterilization techniques. The resulting aqueous solution is packaged or filtered for use under sterile conditions and lyophilized, and the lyophilized formulation is combined with a sterile aqueous solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliaries or adjuvants, which include, without limitation, pH adjusting and buffering agents, tonicity adjusting agents, preservatives, stabilizers, surfactants, chelating agents and the like. Those skilled in the art can formulate the compositions of the present invention in a suitable manner and according to approved practices such as those disclosed in Remington's Pharmaceutical Sciences (Gennaro edited by Mack Publishing Co., Easton, Pennsylvania, 1990).

In one embodiment of the invention, the pharmaceutical composition comprising a formulation of an FXI or FXI-glycopeptide polypeptide further comprises a pH adjuster and a buffer. In one embodiment of the invention, the pharmaceutical composition comprising a formulation of an FXI or FXI-glycopetic polypeptide further comprises an enteric modulator. In one embodiment of the invention, the pharmaceutical composition comprising a formulation of an FXI or FXI-glycopetic polypeptide further comprises a preservative. In one embodiment of the invention, the pharmaceutical composition comprising a formulation of an FXI or FXI-glycopetic polypeptide further comprises a stabilizer. In one embodiment of the invention, the pharmaceutical composition comprising a formulation of an FXI or FXI-glycopetic polypeptide further comprises a surfactant. In one embodiment of the invention, the pharmaceutical composition comprising a formulation of an FXI or FXI-glycopetic polypeptide further comprises a chelating agent.

Non-limiting examples of suitable buffers include acetate buffer, carbonate buffer, citrate buffer, glycylglycine buffer, histidine buffer, glycine buffer, lysine buffer, arginine buffer, phosphate buffer (e.g. sodium dihydrogen phosphate, disodium Containing hydrogen phosphate or trisodium phosphate), TRIS [tris (hydroxymethyl) aminomethane] buffer, bisine buffer, tricine buffer, maleate buffer, succinate buffer, maleate buffer, fumarate buffer, tartrate Buffers, aspartate buffers, and mixtures thereof.

Non-limiting examples of pharmaceutically acceptable preservatives include phenol, o-cresol, m-cresol, p-cresol, chlorocresol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydrate Hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, 2-phenylethanol, benzyl alcohol, chlorobutanol, thiomeroseal, bronopol, benzoic acid, imideurea, chlorohexidine, sodium dihydro Acetates, benzetonium chloride, chlorphenesin (3-p-chlorphenoxypropane-1,2-diol), benzamidine, and mixtures thereof. In further embodiments of the invention, the preservative is present at a concentration of 0.1 mg / ml to 20 mg / ml. In one further embodiment of the invention, the preservative is present at a concentration of 0.1 mg / ml to 5 mg / ml. In another further embodiment of the invention, the preservative is present at a concentration of 5 mg / ml to 10 mg / ml. In another further embodiment of the invention, the preservative is present at a concentration of 10 mg / ml to 20 mg / ml. The use of preservatives in pharmaceutical compositions is well known to those skilled in the art (see, eg, Remington: The Science and Practice of Pharmacy, 19th edition, 1995,).

Non-limiting examples of tonicity modifiers (usually included for the purpose of making the formulation substantially isotonic) include salts (eg, sodium chloride), sugars, alcohols (such as C4-C8 alcohols), alditol, amino acids ( For example glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), polyethyleneglycol (eg PEG400), and mixtures thereof. Monosaccharides, disaccharides, or any sugar such as polysaccharides or water soluble glucan can be used. Non-limiting examples of substances that are sugars, alcohols or altitols 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), propane-1,2-diol (propylene glycol), propane -1,3-diol and butane-1,3-diol. The above-mentioned sugars, alcohols and alditols can be used individually or in combination. The amount used is not limited as long as this substance is soluble in liquid formulations.

In one embodiment, the tonicity modifier is present at a concentration of about 1 mg / ml to about 150 mg / ml. In a further embodiment of the invention, the tonicity modifier is present at a concentration of about 1 mg / ml to about 50 mg / ml. In one embodiment, the tonicity modifier is NaCl. In one embodiment, the tonicity modifier is NaCl present at a concentration of about 1 mg / ml to about 150 mg / ml. In still further embodiments of the invention, the tonicity modifier is NaCl present at a concentration of 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. In some embodiments, the chelating agent is 0.1 mg / ml to 5 mg / ml; 0.1 mg / ml to 2 mg / ml; Or at a concentration of 2 mg / ml to 5 mg / ml.

The pharmaceutical composition of the present invention may comprise as a therapeutically active ingredient a polypeptide which is likely to exhibit clot formation during storage in a liquid pharmaceutical composition. The term “coagulation formation” is intended to refer to the physical application between polypeptide molecules resulting in the formation of oligomers that can maintain solubility, or large visible clots precipitated from solution. The term "during storage" refers to a pharmaceutical composition or formulation of a liquid that is prepared once and is not administered directly to a subject. Rather, after manufacture, it is packaged for storage in liquid form, in frozen form, or in dry form, and later recovered in liquid form or another form suitable for administration to a subject. The term “dry form” refers to freeze drying [ie, lyophilization; For example, Williams and Polli (1984), J. Parenteral Sci. Technol. 38: 48-59], Masters (1991) in Spray- Drying Handbook (5th edition; Longman Scientific and Technical, Essex, UK) pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18: 1169-1206; And Mumenthaler et al. (1994) Pharm. Res. 11: 12-20], or air drying [Carpenter and Crowe (1988), Cryobiology 25: 459-470; And Roser (1991) Biopharm. 4: 47-53] to dry liquid pharmaceutical compositions or formulations. Formation of clots by the polypeptide during storage of the liquid pharmaceutical composition can adversely affect the biological activity of the polypeptide, resulting in a loss of therapeutic efficacy of the pharmaceutical composition. Moreover, clot formation can cause other problems such as blockage of the tube, membrane or pump when the polypeptide-containing pharmaceutical composition is administered using an infusion system.

In one embodiment of the invention, the pharmaceutical composition comprises an amount of amino acid base sufficient to reduce clot formation by the polypeptide during storage of the composition. The term "amino acid base" refers to an amino acid or combination of amino acids in which a given amino acid is present in its free base form or in salt form. When a combination of amino acids is used, all amino acids may be in their free base form, or all amino acids may be in their salt form, or some may be in free base form and others may be in salt form. In one embodiment, the amino acids used to prepare the compositions of the present invention are those with charged side chains such as arginine, lysine, aspartic acid or glutamic acid. As long as a particular amino acid is in its free base form or in the form of a salt, a particular amino acid (eg, glycine, methionine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine, or a mixture of one or more thereof) Stereoisomers of or combinations of these stereoisomers may be present in the pharmaceutical compositions of the present invention. In one embodiment L-stereoisomers are used. In addition, the compositions of the present invention can be formulated using analogs of these amino acids. "Amino acid analogue" means a derivative of naturally occurring amino acids that has the desired effect of reducing clot formation by polypeptides during storage of the liquid pharmaceutical composition of the present invention. Suitable arginine analogs include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogs include ethionine and butionine, and suitable cysteine analogs are S-methyl-L- Contains cysteine. The remaining amino acids are likewise included in the composition in their free base form or in salt form. In addition, imidazole compounds can be considered as amino acid analogs in the context of the present invention. Typically, amino acids or amino acid analogs are used at concentrations sufficient to prevent or delay aggregation of the protein.

In one embodiment, the pharmaceutical formulation is selected from the group consisting of methionine (or other sulfur-) to inhibit such oxidation when the Factor XI polypeptide is a polypeptide comprising at least one methionine residue that is sensitive to oxidation of the methionine residue to the sulfoxide form. Containing amino acids or amino acid analogs). The term “oxidation inhibition” is intended to denote the minimization of accumulation of oxidized species (of methionine) over time. Inhibition of methionine oxidation allows the polypeptide to stay longer in its suitable molecular form. Any stereoisomer (L, D or DL isomer) of methionine or a combination thereof may be used. The amount added should be an amount sufficient to inhibit the oxidation of methionine residues such that the amount of methionine in the sulfoxide form can be approved by the administration. Typically, this means that the composition contains from about 10% up to about 30% of the methionine sulfoxide form. This can generally be accomplished 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, for example 10: 1 to about 100: 1.

Non-limiting examples of stabilizers include high molecular weight polymers or low molecular weight compounds such as polyethylene glycol (eg PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy / hydroxycellulose and their Sulfur-containing substances such as derivatives (HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, monothioglycerols, thioglycolic acid and 2-methylthioethanol, various salts (e.g. sodium chloride), glycerol, Propylene glycol, propane-1,3-diol, propyl alcohol (1-propanol) and isopropyl alcohol (2-propanol).

F68, Poloxamer 188 및 407과 같은 폴록사머, Triton X-100), 폴리옥시에틸렌 소르비탄 지방산 에스테르, 폴리옥시에틸렌 및 폴리에틸렌 유도체, 예로서 알킬화 및 알콕시화 유도체("Tweens", 예를 들어 Tween-20, Tween-40, Tween-80 및 Brij-35), 모노글리세리드 및 그의 에톡실화 유도체, 디글리세리드 및 그의 폴리옥시에틸렌 유도체, 알콜, 글리세롤, 렉틴 및 인지질(예를 들어, 포스파티딜-세린, 포스파티딜-콜린, 포스파티딜-에탄올아 민, 포스파티딜-이노시톨, 디포스파티딜-글리세롤 및 스핑고미엘린), 인지질의 유도체(예를 들어, 디팔미토일-포스파티드산) 및 리소인지질(예를 들어, 팔미토일 리소포스파티딜-L-세린 및 에탄올아민, 콜린, 세린 또는 트레오닌의 1-아실-sn-글리세로-3-포스페이트 에스테르), 및 리소포스파티딜 및 포스파디틸콜린의 알킬-, 알콕시-(알킬에스테르) 및 알콕시-(알킬에테르) 유도체, 예를 들어 리소포스파티딜콜린의 라우로일 및 미리스토일 유도체, 디팔미토일포스파티딜콜린, 및 극성 머리기, 즉 콜린, 에탄올아민, 포스파티드산, 세린, 트레오닌, 글리세롤, 이노시톨, 및 양으로 하전된 DODAC, DOTMA, DCP, BISHOP, 리소포스파티딜세린 및 리소포스파티딜트레오닌의 변형, 및 글리세로포스포리피드(예를 들어, 세팔린), 글리세로글리코리피드(예를 들어, 갈락토피라노시드), 스핑고글리코리피드(예를 들어, 세라마이드, 간글리오시드), 도데실포스포콜린, 계란 리소레시틴, 푸시딘산 유도체(예를 들어, 나트륨 타우로-디히드로푸시데이트 등), 장쇄 지방산[예를 들어, C6-C12 지방산(예를 들어, 올레산 또는 카프릴산)] 및 그의 염, 아실카르니틴 및 그의 유도체, 리신, 아르기닌 및 히스티딘의 N^α-아실화 유도체, 리신 및 아르기닌의 측쇄 아실화 유도체, 리신, 아르기닌 및 히스티딘과 중성 또는 산성 아미노산의 어떤 조합을 포함하는 디펩티드의 N^α-아실화 유도체, 중성 아미노산과 2개의 하전된 아미노산의 어떤 조합을 포함하는 트리펩티드의 N^α-아실화 유도체, DSS(도쿠세이트 나트륨, CAS 레지스트리 no [577-11-7], 도쿠세이트 칼슘, CAS 레지스트리 no [128-49-4], 도쿠세이트 칼륨, CAS 레지스트리 no [7491-09-0]), SDS(나트륨도데실술페이트 또는 나트 륨 라우릴 술페이트), 나트륨 카프릴레이트, 콜산 및 그의 유도체, 담즙산 및 그의 유도체, 및 글리신 또는 타우린 콘쥬게이트, 우르소디옥시콜산, 나트륨 콜레이트, 나트륨 디옥시콜레이트, 나트륨 타우로콜레이트, 나트륨 글리코콜레이트, N-헥사데실-N,N-디메틸-3-암모니오-1-프로판술포네이트, 음이온(알킬-아릴-술포네이트) 1가 계면활성제, 양쪽성 계면활성제(예를 들어, N-알킬-N,N-디메틸암모니오-1-프로판술포네이트, 3-콜아미도-1-프로필디메틸암모니오-1-프로판술포네이트), 양이온 계면활성제(4차 암모늄 염기; 예를 들어, 세틸-트리메틸암모늄 브로마이드, 염화 세틸피리디늄), 비이온 계면활성제(예를 들어, 도데실 β-D-글루코피라노시드), 및 폴록사민(예를 들어, Tetronic's), 즉 에틸렌디아민에 프로필렌 옥시드와 에틸렌 옥시드의 연속첨가에 의해 유도된 4작용성 블록 코폴리머를 포함한다; 또는, 계면활성제는 이미다졸린 유도체, 또는 이들의 혼합물로 구성된 군으로부터 선택될 수 있다. 한 구체예에서 제약학적 제제는 약 0.01mg/ml 내지 약 50mg/ml의 농도로 계면활성제를 포함한다. 한 구체예에서, 제약학적 제제는 Tween-80을 포함한다. 한 구체예에서, 제약학적 제제는 Poloxamer 188을 포함한다.Non-limiting examples of surfactants include detergents, ethoxylated castol oil, polyglycolated glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (e.g., Pluronic

F68, poloxamers such as Poloxamer 188 and 407, Triton X-100), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives ("Tweens", for example Tween-20) , Tween-40, Tween-80 and Brij-35), monoglycerides and ethoxylated derivatives thereof, diglycerides and polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (e.g., phosphatidyl-serine, phosphatidyl-choline Phosphatidyl-ethanolamine, phosphatidyl-inositol, diphosphatidyl-glycerol and sphingomyelin), derivatives of phospholipids (e.g. dipalmitoyl-phosphatidic acid) and lysophospholipids (e.g. palmitoyl lysophosphatidyl- 1-acyl-sn-glycero-3-phosphate esters of L-serine and ethanolamine, choline, serine or threonine, and alkyl-, alkoxy- (alkylesters of lysophosphatidyl and phosphaditylcholine ) And alkoxy- (alkylether) derivatives such as lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and polar hair groups such as choline, ethanolamine, phosphatidic acid, serine, threonine, glycerol , Inositol, and modifications of positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidyl threonine, and glycerophospholipids (eg, cephalins), glyceroglycolipides (eg, Galactopyranoside), sphingoglycolipid (e.g. ceramide, gangliside), dodecylphosphocholine, egg lysolecithin, fusidic acid derivatives (e.g. sodium tau-dihydrofucidate, etc. ), Long chain fatty acids [e.g. C6-C12 fatty acids (e.g. oleic acid or caprylic acid)] and salts thereof, acylcarnitine and derivatives thereof, N ^α -acylated derivatives of lysine, arginine and histidine , Side chain acylated derivatives of lysine and arginine, N ^α -acylated derivatives of dipeptides comprising any combination of lysine, arginine and histidine with neutral or acidic amino acids, any combination of neutral and two charged amino acids N ^α -acylated derivatives of tripeptide, 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 sulfate or sodium lauryl sulfate), sodium caprylate, cholic acid and derivatives thereof, bile acids and derivatives thereof, and glycine or taurine conjugates, ursodioxycholic acids, Sodium cholate, sodium dioxycholate, sodium taurocholate, sodium glycocholate, N-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, anion (alkyl-aryl-sulfonone Monovalent surfactants, amphoteric surfactants (eg, N-alkyl-N, N-dimethylammonio-1-propanesulfonate, 3-colamido-1-propyldimethylammonio-1-propane) Sulfonates), cationic surfactants (quaternary ammonium bases); For example, cetyl-trimethylammonium bromide, cetylpyridinium chloride), nonionic surfactants (eg dodecyl β-D-glucopyranoside), and poloxamines (eg Tetronic's), ie ethylene Diamines include tetrafunctional block copolymers derived by the continuous addition of propylene oxide and ethylene oxide; Alternatively, the surfactant may be selected from the group consisting of imidazoline derivatives, or mixtures thereof. In one embodiment the pharmaceutical formulation comprises a surfactant at a concentration of about 0.01 mg / ml to about 50 mg / ml. In one embodiment, the pharmaceutical formulation comprises Tween-80. In one embodiment, the pharmaceutical formulation comprises Poloxamer 188.

In one embodiment, the pharmaceutical formulation comprises an electrolyte. In one embodiment, the pharmaceutical formulation comprises an electrolyte such as NaCl. In one embodiment, the pharmaceutical formulation comprises an electrolyte such as KCl.

In one embodiment, when Tween 80 is used as a stabilizer, an electrolyte such as for example NaCl at a concentration of 150 mM is used. In one embodiment, aggregation upon storage is avoided.

In a series of embodiments, the use of one or more stabilizers used in pharmaceutical formulations, including formulations of FXI or FXI-glycopolypeptides, is the physical and / or physical of the control (ie, FXI with the same treatment but no stabilizer). Increase the physical and / or chemical stability of FXI by at least 10%, 25%, 50% or 100% relative to chemical stability. In another set of embodiments, the use of one or more stabilizers in a pharmaceutical formulation comprising a formulation of an FXI or FXI-glycopolypeptide is the physical and / or chemical of the control (ie, FXI with the same treatment but no stabilizer). Increase the physical and / or chemical stability of FXI by at least 2, 5, 10, or 20 times relative to stability.

The following table provides non-limiting examples of suitable formulations. When such formulations contained 1.7 mg / ml FXI, no aggregation was observed after at least 1 month at ambient temperature.

ID pH buffer Appearance stabilizator Antimicrobial preservative A1 8.5  50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na na na A2 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 150 mM NaCl na na A3 8.5  50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 0.001% w / v Tween 80 na A4 8.5  50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 0.1% w / v Tween 80 na A5 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 150 mM NaCl 0.1% Tween 80 na A6 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 16.0 mg / ml glycerol na 0.5 w / v% phenol A7 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 5 w / v% hydroxypropyl-beta-cyclodextrin na A8 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 0.1 w / v% human serum albumin na A9 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 0.5 M sucrose na A10 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 0.3 w / v% Poloxamer 188 na A11 8.5 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.5 na 18.6 mg / ml EDTA B1 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na na na B2 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 150 mM NaCl na na B3 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 0.001% w / v Tween 80 na B4 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 0.1% w / v Tween 80 na B5 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 150 mM NaCl 0.1% Tween 80 na  B6 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 16.0 mg / ml glycerol na 0.5 w / v% phenol B7 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 5 w / v% hydroxypropyl-beta-cyclodextrin na B8 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 0.1 w / v% human serum albumin na B9 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 0.5 M sucrose na B10 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 0.3 w / v% Poloxamer 188 na B11 8.0 50 mM Tris (hydroxymethyl) aminomethane (TRIS), pH 8.0 na 18.6 mg / ml EDTA B12 6.1 Histidine 1.36 mg / ml, pH 6.1 40 mg / ml mannitol 0.3 w / v% Poloxamer 188 6 mg / ml phenol C1 7.4 50 mM phosphate, pH 7.4 na na na C2 7.4 50 mM phosphate, pH 7.4 150 mM NaCl na na C3 7.4 50 mM phosphate, pH 7.4 na 0.001% w / v Tween 80 na C4 7.4 50 mM phosphate, pH 7.4 na 0.1% w / v Tween 80 na C5 7.4 50 mM phosphate, pH 7.4 150 mM NaCl 0.1% Tween 80 na C6 7.4 50 mM phosphate, pH 7.4 16.0 mg / ml glycerol na 0.5 w / v% phenol C7 7.4 50 mM phosphate, pH 7.4 na 5 w / v% hydroxypropyl-beta-cyclodextrin na C8 7.4 50 mM phosphate, pH 7.4 na 0.1 w / v% human serum albumin na C9 7.4 50 mM phosphate, pH 7.4 na 0.5 M sucrose na C10 7.4 50 mM phosphate, pH 7.4 na 0.3 w / v% Poloxamer 188 na C11 7.4 50 mM phosphate, pH 7.4 na 18.6 mg / ml EDTA C12 7.7 50 mM phosphate, pH 7.7 14 mg / ml propylene glycol na 5.5 mg / ml phenol D1 6.0 50 mM Citrate, pH 6.0 na na na D2 6.0 50 mM Citrate, pH 6.0 150 mM NaCl na na  D3 6.0 50 mM Citrate, pH 6.0 na 0.001% w / v Tween 80 na D5 6.0 50 mM Citrate, pH 6.0 150 mM NaCl 0.1% Tween 80 na D6 6.0 50 mM Citrate, pH 6.0 16.0 mg / ml glycerol na 0.5 w / v% phenol D7 6.0 50 mM Citrate, pH 6.0 na 5 w / v% hydroxypropyl-beta-cyclodextrin na D8 6.0 50 mM Citrate, pH 6.0 na 0.1 w / v% human serum albumin na D9 6.0 50 mM Citrate, pH 6.0 na 0.5 M sucrose na D10 6.0 50 mM Citrate, pH 6.0 na 0.3 w / v% Poloxamer 188 na D11 6.0 50 mM Citrate, pH 6.0 na 18.6 mg / ml EDTA E2 10.0 50 mM glycine, pH 10.0 150 mM NaCl na na E3 10.0 50 mM glycine, pH 10.0 na 0.001% w / v Tween 80 na E4 10.0 50 mM glycine, pH 10.0 na 0.1% w / v Tween 80 na E5 10.0 50 mM glycine, pH 10.0 150 mM NaCl 0.1% Tween 80 na E6 10.0 50 mM glycine, pH 10.0 16.0 mg / ml glycerol na 0.5 w / v% phenol E7 10.0 50 mM glycine, pH 10.0 na 5 w / v% hydroxypropyl-beta-cyclodextrin na E8 10.0 50 mM glycine, pH 10.0 na 0.1 w / v% human serum albumin na E9 10.0 50 mM glycine, pH 10.0 na 0.5 M sucrose na E10 10.0 50 mM glycine, pH 10.0 na 0.3 w / v% Poloxamer 188 na F1 7.0 50 mM phosphate, pH 7.0 na na na F2 7.0 50 mM phosphate, pH 7.0 150 mM NaCl na na F3 7.0 50 mM phosphate, pH 7.0 na 0.001% w / v Tween 80 na F5 7.0 50 mM phosphate, pH 7.0 150 mM NaCl 0.1% Tween 80 na F6 7.0 50 mM phosphate, pH 7.0 16.0 mg / ml glycerol na 0.5 w / v% phenol  F7 7.0 50 mM phosphate, pH 7.0 na 5 w / v% hydroxypropyl-beta-cyclodextrin na F8 7.0 50 mM phosphate, pH 7.0 na 0.1 w / v% human serum albumin na F9 7.0 50 mM phosphate, pH 7.0 na 0.5 M sucrose na F10 7.0 50 mM phosphate, pH 7.0 na 0.3 w / v% Poloxamer 188 na G2 5.0 50 mM citrate, pH 5.0 150 mM NaCl na na G5 5.0 50 mM citrate, pH 5.0 150 mM NaCl 0.1% Tween 80 na G6 5.0 50 mM citrate, pH 5.0 16.0 mg / ml glycerol na 0.5 w / v% phenol G7 5.0 50 mM citrate, pH 5.0 na 5 w / v% hydroxypropyl-beta-cyclodextrin na G8 5.0 50 mM citrate, pH 5.0 na 0.1 w / v% human serum albumin na G9 5.0 50 mM citrate, pH 5.0 na 0.5 M sucrose na G10 5.0 50 mM citrate, pH 5.0 na 0.3 w / v% Poloxamer 188 na G12 7.6 50 mM glycylglycine, pH 7.6 na 0.05 mg / ml Tween 20 0.21 mg / ml phenol H2 3.0 50 mM citrate, pH 3.0 150 mM NaCl na na H4 3.0 50 mM citrate, pH 3.0 na 0.1% w / v Tween 80 na H5 3.0 50 mM citrate, pH 3.0 150 mM NaCl 0.1% Tween 80 na H6 3.0 50 mM citrate, pH 3.0 16.0 mg / ml glycerol na 0.5 w / v% phenol H7 3.0 50 mM citrate, pH 3.0 na 5 w / v% hydroxypropyl-beta-cyclodextrin na H8 3.0 50 mM citrate, pH 3.0 na 0.1 w / v% human serum albumin na H9 3.0 50 mM citrate, pH 3.0 na 0.5 M sucrose na H10 3.0 50 mM citrate, pH 3.0 na 0.3 w / v% Poloxamer 188 na  H12 7.3 5.7 mM phosphate, pH 7.3 137 mM NaCl 5.4 mM KCl

In a non-limiting embodiment, suitable formulations that allow recovery of active FXI after lyophilization contain:

FXI concentration: 0.2 mg / ml

Buffer: 20 mM buffer (histidine or TRIS) (pH 5.5, 6.5 or 7.4), 25 mg / ml mannitol (bulking agent), 2.5 mg / ml NaCl (bulking agent), 0.01% Tween 80

Preferably, the pharmaceutical composition is administered parenterally, ie by the intravenous, subcutaneous, or intramuscular route, with the intravenous route being most preferred. In addition, they may be administered by continuous or pulsating infusion. It will be appreciated that any effective method of administering an FXI polypeptide can be used, including, for example, using mucosal or inhaled administration.

For example, topical delivery of a formulation of the invention, such as topical application, for example, incorporation into a spray, perfusion, dual balloon catheter, stent, vascular implant or stent, hydrogel used to coat balloon catheter, Incorporation into gauze or other bandage material, or by other well established methods.

The pharmaceutical compositions of the invention can be formulated in a variety of formulations, for example solutions, suspensions, emulsions, microemulsions, multiple emulsions, foams, plasters, pastes, gypsum, ointments, tablets, coated tablets, rinses, capsules (e.g. hard Gelatin capsules or soft gelatin capsules), suppositories, rectal capsules, drops, gels, sprays, powders, aerosols, inhalants, eye drops, eye ointments, anrins, vaginal suppositories, vaginal rings, vaginal ointments, injection solutions, in situ conversion solutions (e.g. For example, in-situ gelation, in-situ curing, in-situ precipitation or in-situ crystallization), as an infusion, or as an implant.

To further enhance the stability of the Factor XI polypeptide, to increase bioavailability, to increase solubility, to reduce side effects, to achieve chronotherapy well known to those skilled in the art, and / or to increase patient compliance. In addition, the pharmaceutical compositions of the present invention may be further compounded in, combined with or conjugated to (eg, by covalent, hydrophobic or electrostatic interaction) within a drug carrier, drug delivery system or advanced drug delivery system. . Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers such as cellulose and derivatives thereof, other polysaccharides (eg dextran and derivatives thereof, starch and derivatives thereof), poly (vinyl alcohol) ), Acrylate and methacrylate polymers, carrier proteins (eg albumin), gels (eg, thermal gelling systems such as block copolymer systems well known to those skilled in the art), micelles, liposomes, microspheres, nano Fine particles, liquid crystals and their dispersions, L2 phases and their dispersions, polymer micelles, multiple emulsions (self-emulsifying and self-microemulsifying), cyclodextrins and their derivatives, well known to those skilled in the art of phase behavior in lipid-water systems , And dendrimers.

Pharmaceutical compositions comprising Factor XI polypeptides prepared using the method according to the invention may be prepared in the form of solids, semisolids, powders and solutions suitable for pulmonary route administration, eg, using a metered dose inhaler, dry powder inhaler or nebulizer. Suitable for use, all of which are well known to those skilled in the art.

Pharmaceutical compositions comprising Factor XI polypeptides prepared using the method according to the invention are suitable for use in the preparation of controlled-release, sustained-release, extended-release, delayed-release or slow-release drug delivery systems. Pharmaceutical compositions comprising a Factor XI polypeptide prepared using the method according to the invention may be parenteral controlled-release of the kind well known to those skilled in the art, for example, in controlled-release and sustained-release systems for subcutaneous administration. And sustained-release systems, both of which result in several orders of magnitude reduction in administration. Without limiting the scope of the invention, examples of useful controlled-release systems and compositions are hydrogels, oily gels, liquid crystals, polymer micelles, microspheres and nanoparticles.

Methods of preparing controlled-release systems useful for pharmaceutical compositions comprising Factor XI polypeptides prepared using the method according to the invention include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion , High pressure homogenization, encapsulation, spray drying, microencapsulation, droplet formation, phase separation, microsphere generation by solvent evaporation, extrusion and supercritical liquid processes. In general, Handbook of Pharmaceutical Controlled Release (Wise, DL Editor, Marcel Dekker, New York, 2000) and Drugs and the Pharma- ceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, EJ, edited by Marcel Dekker, New York, 2000).

Parenteral administration can be carried out by subcutaneous, intramuscular, intraperitoneal or intravenous injection by syringe, eg a syringe in a pen type device. Alternatively, parenteral administration can be performed by an infusion pump. An additional option for administration of the composition in the form of a solution or suspension containing Factor XI polypeptides prepared using the method according to the invention is administration as a nasal or pulmonary spray. As another option, a pharmaceutical composition containing a Factor XI polypeptide prepared using the method according to the invention may be applied, for example, by needleless injection, by patch application (such as an iontophoretic patch) or via mucosal membranes (eg For example, percutaneous administration by cheek) administration.

In one embodiment of the invention, pharmaceutical compositions comprising a Factor XI polypeptide prepared using the method according to the invention are stable upon use for at least 6 weeks and at least 3 years of storage.

In another embodiment of the invention, the pharmaceutical composition comprising a Factor XI polypeptide prepared using the method according to the invention is stable upon use for at least 4 weeks and at least 3 years of storage.

In a further embodiment of the invention, the pharmaceutical composition comprising a Factor XI polypeptide prepared using the method according to the invention is stable upon use for at least 4 weeks and at least 2 years of storage.

In another embodiment of the invention, a pharmaceutical composition comprising a Factor XI polypeptide prepared using the method according to the invention is stable upon use for at least two weeks and at least two years of storage.

In some embodiments, the FXI polypeptide formulation has a pH of about 4.0 to about 10.0. In some embodiments, the FXI polypeptide formulation has a pH of about 4.0 to about 8.0. In some embodiments, the FXI polypeptide formulation has a pH of about 4.0 to about 7.0. In some embodiments, the FXI polypeptide formulation has a pH of about 4.0 to about 6.5. In some embodiments, the FXI polypeptide formulation has a pH of about 4.0 to about 6.0. In some embodiments, the FXI polypeptide formulation has a pH of about 6.5 or less, for example about pH 5.0 to about 6.5; For example about 5.5 to 6.5.

Of factor XI Therapeutic  administration:

The present invention provides for the prevention and treatment of bleeding with FXI.

Bleeding is the extravasation of blood from certain components of the circulatory system. Bleeding events include unwanted, uncontrolled and predominantly excessive bleeding associated with surgery, trauma, or other forms of tissue damage, as well as unwanted bleeding in subjects with bleeding disorders. Bleeding can occur as a spontaneous event, such as intracranial hemorrhage (ICH). Bleeding events can occur in subjects who have basically a coagulation system but who have experienced (temporary) coagulopathy and who have a prior or acquired coagulation or bleeding disorder. In subjects with platelet deficiency, as with hemophilia, bleeding is caused by hemophilia because, as with hemophilia, the hemostatic system lacks or has an abnormal essential coagulation "compound" (eg, platelets or von Willebrand factor protein) Can be compared to For example, in subjects with extensive tissue damage associated with surgery or major trauma, normal hemostatic mechanisms may be overturned by impaired immediate hemostatic action, which is basically normal (pre-traumatic or pre-surgical). Excessive bleeding may occur despite the hemostatic mechanism. Such subjects, who often receive multiple transfusions, develop (temporary) coagulopathy as a result of bleeding and / or transfusion (ie, coagulation protein dilution due to bleeding and / or transfusion, increased fibrinolysis, and decreased platelet count). In addition, bleeding can occur in organs such as the brain, inner ear region and eye; These are areas with limited potential for surgical hemostasis and therefore have problems in achieving satisfactory hemostasis.

Similar problems may occur not only in the process of taking biopsy tissue from various organs (liver, lung, tumor tissue, gastrointestinal tract) but also in peritoneal surgery and posterior periodontal prostatectomy. In all these situations, it is conventionally difficult to provide hemostasis by surgical techniques (sutures, clips, etc.), even when bleeding spreads (eg hemorrhagic gastritis and excessive uterine bleeding). In addition, bleeding can occur in subjects undergoing anticoagulant therapy, in which case a hemostatic defect is induced by a provided therapy; These bleedings are mainly acute and excessive. Anticoagulant therapy is primarily provided to prevent thromboembolic diseases. Such therapies include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K-antagonists, coagulation protein inhibitors, and aspirin and other platelet aggregation inhibitors, such as antibodies to GP IIb / IIIa activity or other inhibitors. In addition, bleeding is the so-called treatment involving combination of antiplatelet agents (eg acetylsalicylic acid), anticoagulants (eg heparin) and fibrinolytic agents (eg tissue plasminogen activator, tPA). It may be due to thrombolytic therapy. In addition, bleeding events may include surgery or trauma in subjects with acute hemorrhagic joint disease (bleeding in the joints), chronic hemophiliac arthrosis, hematomas (eg, muscle, peritoneum, sublingual and pharyngeal), and other tissues. Intended to include, without limitation, uncontrolled bleeding associated with bleeding, hematuria (bleeding from the renal tract), cerebral hemorrhage, surgery (eg, liver resection), extraction, and gastrointestinal bleeding (eg, UGI bleeding). do. Bleeding events include inhibitors for factor VIII; Hemophilia A; Hemophilia A with inhibitors; Hemophilia B; Defects of factor VII; Defect of factor XI; Hypoplatelet; Binding of von Willebrand factor (von Willebrand disease); Severe tissue damage; Severe trauma; Operation; Laparoscopic surgery; Acidosis, hemodilution, wasting coagulopathy, fibrin hyperlysis, hypothermia, hemorrhagic gastritis; Biopsy tissue collection; Anticoagulant therapy, upper gastrointestinal bleeding (UGI); Or stem cell transplantation. The bleeding event may be excessive uterine bleeding; May occur in organs with limited potential for mechanical hemostasis; Can occur in the brain; May occur in the inner ear region; Or in the eye.

Decreased platelet count or activity refers to the number of platelets (thrombosites) present in the subject's plasma and the biological, coagulation-related activity of such platelets. The lowered number may be due to, for example, increased platelet breakdown, reduced platelet production, and retention above the normal rate of platelet in the spleen. Hypothelium is defined as, for example, platelet counts less than 150,000 per microliter; The upper limit of normal platelet count is generally considered to be 150,000 to 450,000 per microliter. Platelet count can be measured by an automatic platelet counter; This is a method well known to those skilled in the art. Syndromes due to reduced platelet count include without limitation thrombocytopenia, coagulopathy. Aspects of platelet activity include, without limitation, aggregation, adhesion, and coagulation activity of platelets. Reduced activity may be due to, for example, glycoprotein abnormalities, abnormal membrane-cell framework interactions, abnormalities of platelet granules, abnormalities of platelet coagulation activity, abnormalities in signal transduction and secretion. Platelet activity, including aggregation, adhesion and coagulation activity, is measured by standard methods known to those skilled in the art, for example Platelets. A Practical Approach, edited by S.P. Watson & K.S. Authi: Clinical Aspects of Platelet Disorders (K. J. Clemetson) 15: 299-318, 1996, Oxford University Press; See Williams Hematology, 6th edition, compilation, Beutler, Lichtman, Coller, Kipps & Seligsohn, 2001, McGraw-Hill. Syndromes due to degraded platelet activity include, without limitation, glansmann thrombocytopenia, Bernard-Sieli syndrome, storage diseases, anticoagulant therapy and thrombolytic therapy.

In the present invention, treatment is intended to prevent or minimize bleeding, as well as to prevent or reduce bleeding, including but not limited to the prevention of anticipated bleeding, such as may be expected to occur during or following a surgical procedure, for example. It includes all of the control of bleeding that has already occurred, for example, for trauma. The bleeding may be at a site identified or at an unidentified site. As such, prophylactic administration of an agent comprising a FXI polypeptide is included in the treatment.

In some embodiments, a normal human patient, ie, a patient not suffering from a congenital FXI defect, for a 70 kg subject as a loading and maintenance dose, per day or per bleeding event, depending on the subject's weight, condition, and severity of condition. FXI and / or FXI-cognate at a dose corresponding to wild type FXI of about 0.05 mg to about 500 mg, for example about 1 mg to about 200 mg per day or per bleeding event, or for example about 1 mg to about 175 mg Polypeptides can be administered.

In some embodiments, blood is drawn from a patient in need of treatment with a FXI polypeptide and comprises (i) plasma levels of FXI; (ii) ratio of activation: simogen FXI; And / or (iii) an assay is performed to assess one or more of the concentrations of FXI that need to be added exogenously to restore effective coagulation (prior to administration of the FXI polypeptide); Based on the results of the assay, an appropriate amount of FXI polypeptide is administered using the prescribed therapy. For these measurements any suitable assay can be used, including, for example, ELISA or gel-based methods. Suitable assay standards are used to compare measured levels with typical FXI levels (about 30 nM) in human plasma. Typically, supplementing FXI levels to at least about 5 nM, for example about 10 nM, for example about 15 nM, for example about 20 nM, and for example at least about 30 nM FXI, for example 60 nM, for example 120 nM Would be preferred.

If an FXI-cognate polypeptide is used to supplement FXI activity in a patient, the FXI-cognate polypeptide will exhibit at least one FXI biological activity at a certain level, and the goal of treatment is that biological activity corresponding to a given amount of wild type FXI. To provide an amount (ie, "effective FXI plasma concentration").

In some embodiments, the present invention has a plasma level of FXI of about 3 nM; 5 nM; Or therapeutic administration of a FXI polypeptide to a patient that is 10 nM or less.

Combination therapy:

The invention also includes methods and compositions that provide a combination therapy wherein the FXI polypeptide is administered with a non-factor VII / factor VIIa coagulant. Suitable non-factor VII / factor VIIa coagulants include Factor XIII (see, eg, WO 01/85198); Inhibitors of tissue factor pathway inhibitors (TFPI inhibitors) (see, eg, WO 01/85199); Factor XI (see, eg, WO 02/062376); Thrombin activated fibrinolytic inhibitor (TAFI) (see, eg, PCT / DK02 / 00734); PAI-1 (see, eg, PCT / DK02 / 00735); Factor V (see, eg, PCT / DK02 / 00736); Protein C inhibitors (see, eg, PCT / DK02 / 00737); Thrombomodulin (see, eg, PCT / DK02 / 00738); Protein S inhibitors (see, eg, PCT / DK02 / 00739); Tissue plasminogen activator inhibitors (see eg PCT / DK02 / 00740); α-2-antiplasmin (see, eg, PCT / DK02 / 00741); Aprotinin (see, eg, PCT / DK02 / 00742); Tranexamic acid (see, eg, PCT / DK02 / 00751); ε-aminocaproic acid (see, eg, PCT / DK02 / 00752); Prothrombin, thrombin, factor VII, factor X, and fibrinogen are included without limitation.

The following is a list of embodiments of the present invention.

Embodiment 1: A method of treating a bleeding event comprising administering to a patient in need thereof an agent comprising a Factor XI (FXI) or FXI-cognate polypeptide in an amount effective for such treatment.

Embodiment 2: The method of embodiment 1, wherein the administering results in reduced clotting time in the patient.

Embodiment 3: The method of embodiment 1 or 2, wherein said administration results in enhanced hemostasis in said patient.

Embodiment 4: The method of any one of embodiments 1 to 3, wherein said administration results in an increase in clotting dissolution time in said patient.

Embodiment 5: The method of any one of embodiments 1 to 4, wherein the administration results in an increase in coagulation intensity in the patient.

Embodiment 6: The method of any of embodiments 1-5, wherein the administering results in an increase in comprehensive coagulation quality (OCQ) in the patient.

Embodiment 7: The method of any one of embodiments 1 to 6, wherein after the administration, the patient exhibits an effective FXI plasma concentration of at least about 5 nM.

Embodiment 8: The method of embodiment 7, wherein the effective FXI plasma concentration is at least about 10 nM.

Embodiment 9: The method of embodiment 8, wherein the effective FXI plasma concentration is at least about 30 nM, for example at least about 60 nM, for example at least about 120 nM.

Embodiment 10: The method of any of embodiments 1 to 9, wherein the FXI or FXI-cognate polypeptide comprises a sequence of SEQ ID NO: 1, or a fragment thereof that retains at least one FXI-related biological activity How to.

Embodiment 11: The method of any of embodiments 1-9, wherein the FXI or FXI-cognate polypeptide comprises the sequence of SEQ ID NO: 2, or a fragment thereof that retains at least one FXI-related biological activity How to.

Embodiment 12 The method according to any one of embodiments 1 to 11, wherein the patient does not suffer from congenital FXI defects.

Embodiment 13: The method of any of embodiments 1-12, wherein the bleeding event is accompanied by a condition selected from the group consisting of surgery, dental treatment, trauma, or hemodilution.

Embodiment 14: The method of any of embodiments 1 to 13, wherein prior to the administration,

(a) obtaining a blood sample from the patient; (b) measuring at least one of FXI concentration, FXIa: FXI ratio, or amount of exogenous FXI required to restore coagulation; And (c) determining said amount of FXI effective for treatment based on the results of step (b).

Embodiment 15: administering to said patient an agent comprising (i) an agent comprising a first amount of FXI polypeptide and (ii) a second agent comprising a non-factor VII / factor VIIa coagulant A method for treating a bleeding event, characterized in that the first and second amounts are effective for the treatment of the bleeding event.

Embodiment 16: The compound of embodiment 15, wherein the non-factor VII / factor VIIa coagulant is Factor XIII; Tissue factor pathway inhibitor (TFPI) inhibitors; Factor IX; Thrombin activated fibrinolytic inhibitor (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitors; Protein S inhibitors; And a tissue plasminogen activator (tPA) inhibitor.

Embodiment 17: The method of embodiment 15 or 16, wherein the administering results in reduced clotting time in the patient.

Embodiment 18: The method of any of embodiments 15 to 17, wherein said administering results in enhanced hemostasis in said patient.

Embodiment 19 The method according to any one of embodiments 15 to 18, wherein said administration results in an increase in the time for coagulation lysis in said patient.

Embodiment 20: The method of any of embodiments 15 to 19, wherein the administering results in an increase in coagulation intensity in the patient.

Embodiment 21 The method according to any one of embodiments 15 to 20, wherein said administration results in an increase in overall coagulation quality (OCQ) in said patient.

Embodiment 22: The method of any of embodiments 15 to 21, wherein after the administration, the patient exhibits an effective FXI plasma concentration of at least about 5 nM.

Embodiment 23: The method of embodiment 22, wherein the effective FXI plasma concentration is at least about 10 nM.

Embodiment 24: The method of embodiment 23, wherein the effective FXI plasma concentration is at least about 30 nM, for example at least about 60 nM, for example at least about 120 nM.

Embodiment 25: The method of any of embodiments 15 to 24, wherein the FXI or FXI-cognate polypeptide comprises a sequence of SEQ ID NO: 1, or a fragment thereof that retains at least one FXI-related biological activity How to.

Embodiment 26: The method of any of embodiments 15 to 24, wherein the FXI or FXI-cognate polypeptide comprises the sequence of SEQ ID NO: 2, or a fragment thereof that retains at least one FXI-related biological activity How to.

Embodiment 27: The method of any of embodiments 15 to 26, wherein the patient does not suffer from congenital FXI defects.

Embodiment 28 The method of any one of embodiments 15 to 27, wherein the bleeding event is accompanied by a condition selected from the group consisting of surgery, dental treatment, trauma, or hemodilution.

Embodiment 29: The method of any of embodiments 15 to 28, wherein prior to the administration,

(a) obtaining a blood sample from the patient; (b) measuring at least one of FXI concentration, FXIa: FXI ratio, or amount of exogenous FXI required to restore coagulation; And (c) determining said amount of FXI effective for treatment based on the results of step (b).

Embodiment 30: The method of embodiment 1, wherein the method does not comprise administration of Factor VII / Factor VIIa coagulant.

Embodiment 31: A pharmaceutical formulation comprising (i) an isolated recombinant FXI polypeptide and (ii) a pharmaceutically acceptable carrier or excipient.

Embodiment 32: Use of FXI Polypeptide for the Treatment of Bleeding Events.

Embodiment 33 The use of embodiment 32, wherein the bleeding event is accompanied by a condition selected from the group consisting of surgery, dental treatment, trauma, or hemodilution.

Embodiment 34 The use of embodiment 32 or embodiment 33, wherein the bleeding event is not treated with Factor VII / Factor VIIa coagulant.

Embodiment 35: Use of FXI polypeptide for enhancing hemostasis in a patient in need thereof.

Embodiment 36: Use of an FXI polypeptide to increase clotting lysis time in a patient in need thereof.

Embodiment 37: Use of FXI polypeptide to increase coagulation intensity in a patient in need thereof.

Embodiment 38: Use of FXI polypeptide to increase comprehensive clotting quality (OCQ) in a patient in need thereof.

Embodiment 39: Use of an FXI polypeptide to reduce clotting time in a patient in need thereof.

Embodiment 40: The use of any one of embodiments 32 to 39, wherein the effective FXI plasma concentration in the patient is increased to at least about 5 nM.

Embodiment 41: The use of embodiment 40, wherein the effective FXI plasma concentration is increased to at least about 10 nM.

Embodiment 42: The use of embodiment 41, wherein the effective FXI plasma concentration is increased to at least about 30 nM, for example at least about 60 nM, for example at least about 120 nM.

Embodiment 43: The use according to any of embodiments 32 to 42, wherein the patient to be treated is not treated with Factor VII / Factor VIIa coagulant.

Embodiment 44: Use of an FXI polypeptide in the manufacture of a pharmaceutical formulation for treating a bleeding event.

Embodiment 45: The use of embodiment 44, wherein the bleeding event is accompanied by a condition selected from the group consisting of surgery, dental treatment, trauma, or hemodilution.

Embodiment 46: The use of embodiment 44 or 45, wherein the bleeding event is not treated with Factor VII / Factor VIIa coagulant.

Embodiment 47: Use of a FXI polypeptide in the manufacture of a pharmaceutical formulation for the promotion of hemostasis in a patient in need thereof.

Embodiment 48 Use of FXI polypeptide in the manufacture of a pharmaceutical formulation for increasing coagulation lysis time in a patient in need thereof.

Embodiment 49: Use of an FXI polypeptide in the manufacture of a pharmaceutical formulation for increasing coagulation intensity in a patient in need thereof.

Embodiment 50: Use of a FXI polypeptide in the manufacture of a pharmaceutical formulation for increasing comprehensive clotting quality (OCQ) in a patient in need thereof.

Embodiment 51: Use of an FXI polypeptide in the manufacture of a pharmaceutical formulation for reducing clotting time in a patient in need thereof.

Embodiment 52: The use of 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: The use of embodiment 52, wherein the effective FXI plasma concentration is increased to at least about 10 nM.

Embodiment 54: The use of embodiment 53, wherein the effective FXI plasma concentration is increased to at least about 30 nM, for example at least about 60 nM, for example at least about 120 nM.

Embodiment 55: The use of any of embodiments 44 to 54, wherein the patient to be treated is not treated with Factor VII / Factor VIIa coagulant.

Embodiment 56 The use according to any of embodiments 32 to 55, wherein the patient to be treated does not suffer from congenital FXI defects.

Embodiment 57: The use of any of embodiments 32 to 56, wherein the FXI polypeptide comprises a sequence of SEQ ID NO: 1, or a fragment thereof that retains at least one FXI-related biological activity.

Embodiment 57: The use of any of embodiments 32 to 56, wherein the FXI polypeptide comprises a sequence of SEQ ID NO: 2, or a fragment thereof that retains at least one FXI-related biological activity.

Embodiment 58: The use of any one of embodiments 32 to 57, wherein the FXI polypeptide is administered with a non-factor VII / factor VIIa coagulant.

Embodiment 59: The compound of embodiment 58, wherein the non-factor VII / factor VIIa coagulant is Factor XIII; Tissue factor pathway inhibitor (TFPI) inhibitors; Factor IX; Thrombin activated fibrinolytic inhibitor (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C billion preparations; Protein S inhibitors; And a tissue plasminogen activator (tPA) inhibitor.

Embodiment 60: A method of purifying a FXI polypeptide from a biological material, comprising continuously chromatography the biological material in a cation-exchange chromatography material, a hydrophobic interaction chromatography material, and a hydroxyapatite chromatography material.

Embodiment 61: The method of embodiment 60, wherein the FXI polypeptide is recombinant FXI.

Embodiment 62: The method of embodiment 60 or 61, wherein the FXI polypeptide is human FXI.

Embodiment 63: The method of embodiment 60 or 61, wherein the FXI polypeptide is a dimer.

Embodiment 64: The method of embodiment 63, wherein the FXI polypeptide is a dimer of a human subunit.

Embodiment 65 The method according to any one of embodiments 60 to 64, wherein the biological material is a biological fluid.

Embodiment 66: The method of embodiment 65, wherein the biological fluid is a supernatant of mammalian cells.

Embodiment 67: The method of embodiment 66, wherein the biological fluid is a supernatant of a CHO culture.

Embodiment 68 The method of any one of embodiments 60 to 67, comprising the following steps.

(a) chromatography a biological material comprising a FXI polypeptide on an original cation-exchange chromatography material, the chromatography comprising the following steps:

(i) applying the biological material to the original cation-exchange chromatography material;

(ii) eluting the unbound material from the original cation-exchange chromatography material using buffer A, wherein buffer A is suitable for eluting material not bound to the original cation-exchange chromatography material; And

(iii) eluting unbound material from the original cation-exchange chromatography material using buffer A ', wherein buffer A' is suitable for eluting material not bound to the original cation-exchange chromatography material; And

(vi) eluting the FXI polypeptide from the original cation-exchange chromatography material by elution with buffer A '', wherein buffer A '' is suitable for eluting the FXI polypeptide from the original cation-exchange chromatography material. Do.

(b) chromatography the fluid prepared using the eluate from step (iv) or the eluate from step (iv) with a hydrophobic interaction chromatography material, the chromatography comprising the following steps: :

(v) applying the fluid prepared using the eluate from step (iv) or the eluate from step (iv) to the hydrophobic interaction chromatography material;

(vi) eluting unbound material from the chromatography material using buffer B, wherein buffer B is suitable for eluting material not bound with the hydrophobic interaction chromatography material; And

(vii) eluting the FXI polypeptide from the chromatography material by gradient-elution with buffer B ', wherein buffer B' is suitable for eluting the FXI polypeptide from the hydrophobic interaction chromatography material.

Embodiment 69: The method of embodiment 68, wherein buffer A comprises one or more stabilizers capable of increasing the stability of the FXI polypeptide.

Embodiment 70: The method of embodiment 69, wherein buffer A comprises a stabilizer and the stabilizer is a sugar, alcohol, or altitol.

Embodiment 71: The method of embodiment 70, wherein buffer A comprises a stabilizer and the stabilizer is a sugar, C4-C8-alcohol or alditol.

Embodiment 72: The method of embodiment 71, wherein buffer A comprises a stabilizer and the stabilizer is polyalcohol.

Embodiment 73: The method of embodiment 72, wherein buffer A comprises a stabilizer selected from the group consisting of glycerol, propylene glycol, propane-1,3-diol, propyl alcohol and isopropyl alcohol.

Embodiment 74: The method of embodiment 73, wherein buffer A comprises a stabilizer selected from the group consisting of glycerol, propylene glycol and propane-1,3-diol.

Embodiment 75: The method of any of embodiments 72 to 74, wherein the stabilizer is present at a concentration of about 5% (v / v) to about 50% (v / v).

Embodiment 76: The method of embodiment 75, wherein the stabilizer is present at a concentration of about 10% (v / v) to about 50% (v / v).

Embodiment 77 The method of embodiment 76, wherein the stabilizer is present at a concentration of about 10% (v / v) to about 20% (v / v).

Embodiment 78: The method of embodiment 77, wherein the stabilizer is present at a concentration of about 10% (v / v).

Embodiment 79: The method of embodiment 78, wherein the stabilizer is present at a concentration of about 20% (v / v).

Embodiment 80: The method of any of embodiments 68 to 79, wherein the pH of buffer A is about 6.5 to about 9.

Embodiment 81: The method of embodiment 80, wherein the pH of buffer A is about 7 to about 9.

Embodiment 82: The method of embodiment 81, wherein the pH of buffer A is about 8.

Embodiment 83: The method of any of embodiments 68 to 82, wherein buffer A has a conductivity of less than about 50 mS / cm.

Embodiment 84: The method of any of embodiments 60 to 83, wherein the hydrophobic interaction chromatography material uses butyl or phenyl as ligand.

Embodiment 85: The method of embodiment 84, wherein the hydrophobic interaction chromatography material is phenyl sepharose high performance high substitution.

Embodiment 86 The method of embodiment 84, wherein the hydrophobic interaction chromatography material is butyl sepharose high performance high substitution.

Embodiment 87: The method of any of embodiments 68 to 86, wherein the pH of buffer B is about 6 to about 9.

Embodiment 88: The method of embodiment 87, wherein the pH of buffer B is about 8.

Embodiment 89: The method of any of embodiments 68 to 88, wherein buffer B has a conductivity of at least 50 mS / cm.

Embodiment 90: The method of embodiment 89, wherein buffer B has a conductivity of at least 70 mS / cm.

Embodiment 91: The fluid of any of embodiments 68 to 90, wherein the fluid prepared using the eluate from step (vii), or the eluate from step (vii),

(1) adding one or more stabilizers that can increase the stability of the FXI polypeptide in an amount effective to significantly improve its stability, and / or

(2) adjusting the pH of the fluid prepared using the eluate from step (vii) or the eluate from step (vii) to a pH of about 7 to about 9

Process using a method comprising a.

Embodiment 92 The method of embodiment 91, wherein the stabilizer used in step (1) is sugar, alcohol or alditol.

Embodiment 93 The method of embodiment 92, wherein the stabilizer used in step (1) is sugar, C4-C8-alcohol or alditol.

Embodiment 94 The method of embodiment 93, wherein the stabilizer used in step (1) is polyalcohol.

Embodiment 95: The method of embodiment 94, wherein the stabilizer used in step (1) is selected from the group consisting of glycerol, propylene glycol, propane-1,3-diol, propyl alcohol and isopropyl alcohol .

Embodiment 96 The method of embodiment 95, wherein the stabilizer used in step (1) is selected from the group consisting of glycerol, propylene glycol and propane-1,3-diol.

Embodiment 97: The method of any one of embodiments 94 to 96, wherein the stabilizer used in step (1) is added at a concentration of about 5% (v / v) to about 50% (v / v). .

Embodiment 98: The method of embodiment 97, wherein the stabilizer used in step (1) is added at a concentration of about 10% (v / v) to about 50% (v / v).

Embodiment 99: The method of embodiment 98, wherein the stabilizer used in step (1) is added at a concentration of about 10% (v / v) to about 20% (v / v).

Embodiment 100: The method of any of embodiments 60-99, wherein the method comprises elution from hydrophobic interaction chromatography, or a material prepared using an eluate from hydrophobic interaction chromatography in a hydroxyapatite chromatography material. The method further comprises the step of chromatography.

Embodiment 101: The method of any of embodiments 68-100, wherein the method comprises chromatography of a fluid prepared using the eluate from step (vii), or the eluate from step (vii), on a hydroxyapatite chromatography material. The method further comprises the step of: said chromatography comprising the following steps.

(viii) applying to the hydroxyapatite chromatography material a fluid prepared using the eluate from step (vii) or the eluate from step (vii);

(ix) eluting the unbound material from the hydroxyapatite chromatography material using buffer C, wherein buffer C is suitable for eluting material not bound with the hydroxyapatite chromatography material; And

(x) eluting the FXI polypeptide from the hydroxyapatite chromatography material using buffer C ', wherein buffer C' is suitable for eluting the FXI polypeptide bound with the hydroxyapatite chromatography material in step (viii).

Embodiment 102: The method of embodiment 101, wherein buffer C and / or buffer C 'comprises one or more stabilizers capable of increasing the stability of the FXI polypeptide.

Embodiment 103 The method of embodiment 101, wherein the stabilizer is added to the FXI containing fractions and the stabilizer is a sugar, alcohol or alditol.

Embodiment 104 The method of embodiment 103, wherein the stabilizer is added and the stabilizer is sugar, C4-C8-alcohol or alditol.

Embodiment 105: The method of embodiment 104, wherein the stabilizer is added and the stabilizer is polyalcohol.

Embodiment 106: The method of embodiment 105, wherein a stabilizer selected from the group consisting of glycerol, propylene glycol, propane-1,3-diol, propyl alcohol and isopropyl alcohol is added.

Embodiment 107 The method of embodiment 106, wherein a stabilizer selected from the group consisting of glycerol, propylene glycol and propane-1,3-diol is added.

Embodiment 108 The method of any one of embodiments 105 to 107, wherein the stabilizer is added at a concentration of about 5% (v / v) to about 50% (v / v).

Embodiment 109: The method of embodiment 108, wherein the stabilizer is added at a concentration of about 10% (v / v) to about 50% (v / v).

Embodiment 110: The method of embodiment 109, wherein the stabilizer is added at a concentration of about 10% (v / v) to about 20% (v / v).

Embodiment 111: The method of embodiment 110, wherein the stabilizer is added at a concentration of about 10% (v / v).

Embodiment 112: The method of any of embodiments 101 to 111, wherein buffer C and / or buffer C 'has a pH of about 5.8 to about 7.8.

Embodiment 113: The method of 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 using the method according to any of embodiments 60-113.

The following are non-limiting embodiments of the invention.

Example  1: heart disease In the patient  For hemostasis FXI Effect

Blood was drawn from five patients who underwent cardiac bypass surgery before and after surgery. The effect of FXI on clot formation and stability was assessed using ROTEC (cyclic thromboelastomography), which uses the method of Vig et al. (2001), Blood Coagulation & Fibrinolysis 12: 555. In brief, Innovin (final dilution: 1: 50,0000) (Dade Behring) and CaCl ₂ (final concentration: 15nM), with or without FXI (2.5, 10, or 25 nM) (HTI / Enzyme Research Laboratories, Essen) Solidification was initiated. Fibrinolysis was initiated by adding 4 nM tPA (American Diagnostica). ROTEG-04 Whole Blood Hemostatic System Circulation was measured using a thromboelastography instrument (Pentapharm GmBH). Comprehensive coagulation quality (OCQ) was calculated as follows:

(Max vel / t _{max vel} ) X (t _{min vel} -t _{max vel} )

OCQ was then normalized to a control sample (incubated without any hemostasis).

The results are shown in FIG. FXI significantly improved the overall clotting quality and coagulation formed in the presence of FXI increased resistance to fibrinolysis.

Example  2: for hemostasis in normal blood FXI Effect

Blood was taken from four normal subjects and the effect of FXI on clot formation was evaluated by ROTEG described in Example 1.

2 illustrates that the dose-dependent increase caused by FXI is OCQ in normal blood.

Example  3: Glycosylation -Destruction FXI  Activity of polypeptide

FXI variants containing the following substituents were constructed using standard methods and expressed after transfection into HEK293 cells. Crude cell culture supernatants were collected from the grown cells at 37 ° C. for 96 hours. FXI activity was measured by ROTEG described in Example 1.

The results are shown in the following table.

protein % Expected FXI activity  NHP (normal human plasma) (31nM FXI) 106  FXI N72Q-1.2 nM 42  FXI N108Q-1.3 nM 62  FXI N335Q-0.4 nM 75  FXI N432Q-1.2 nM 33  FXI N473Q-0.6 nM 83

Example  4: FXI  Storage stability of the formulation

Following FXI solution was prepared and stored at 5 ° C. for 5 weeks, FXI activity was measured as described in Example 1.

1.384 nM FXI, 4 mM acetate, 150 mM NaCl, pH 5.4

2. 190 nM FXI, 50 mM acetate buffer, 150 mM NaCl, pH 5.4

3. 190 nM FXI, 50 mM acetate buffer, 150 mM NaCl, pH 5.4, 1 mM CaCl ₂

4. 190 nM FXI, 50 mM acetate buffer, 75 mM NaCl, pH 5.4, 300 mg / ml sucrose

5.190 nM FXI, 50 mM MES buffer, pH 6.5, 150 mM NaCl

6.190 nM FXI, 50 mM MES buffer, pH 6.5, 150 mM NaCl, 1 mM CaCl ₂

7.190 nM FXI, 50 mM MES buffer, pH 6.5, 75 mM NaCl, 300 mg / ml sucrose

The results are shown in FIG.

Example  5: FXI Binding Peptides for

The following experiment was performed to identify peptides that bind to FXI.

1. Synthesis of peptide library:

The following library was synthesized using Fmoc solid phase peptide synthesis on Tentagel resin beads from Rapp Polymere (Germany). Three different peptide bead libraries were used for screening. They are named BL121, BL122 and BL123.

The format of library BL121 is:

O ₁ -O ₂ -O ₃ -O ₄ -O ₅ -O ₆ -O ₇ -O ₈ -O ₉ -O ₁₀ -O ₁₁ -O ₁₂ -O ₁₃ -O ₁₄ -Tentagel Resin, where On is L -Amino acids, n = 1, 2 and 11, 12 can be any propenogenic L-amino acids except methionine and cysteine, n = 4, 5 and 7, 8 and 10, 11 and 13, 14 are methionine And any protenogenic L-amino acid except cysteine and deletion, and n = 3, 6, 9, 12 can be Phe, Trp, Tyr, Leu.

The format of library BL122 is:

O ₁ -O ₂ -O ₃ -O ₄ -O ₅ -O ₆ -O ₇ -O ₈ -O ₉ -O ₁₀ -O ₁₁ -O ₁₂ -Tentagel resin, where On is L-amino acid, n = 1-12 can be any protenogenic L-amino acid except methionine and cysteine.

The format of library BL124 is:

O ₁ -O ₂ -O ₃ -O ₄ -O ₅ -O ₆ -O ₇ -Asp-Phe-Pro-O ₈ -O ₉ -O ₁₀ -O ₁₁ -Tentagel resin, where On is L-amino acid , n = 1-11 can be any protenogenic L-amino acid except methionine and cysteine.

2. Peptide Bead Library Screening:

Recombinant factor XI was purchased from Heamatologic Technologies and biotinylated according to standard laboratory protocols. Next, 5 ul of Factor XI (1.2 uM) and 1 ul of streptoavidin-alkaline phosphate (1 mg / ml, Sigma) were added to three synthetic peptide bead libraries BL121, BL122 and BL124, respectively, and incubated for about 2-3 hours. Incubation buffers were 15 mM TRIS-HCl, pH = 7,4, 0,15M NaCl, 0,5% bovine serum albumin (BSA) and 0.05% Tween 20. After washing with wash buffer (M TRIS-HCl, pH = 7,4, 0,15M NaCl, and 0.05% Tween 20), BCIP and NBT were color buffered (50 mM TRIS-HVl pH = 8.8, 0.15M NaCl, 0.05% Tween 20 and 15 mM MgCl ₂ ) were added and the coloring proceeded from 30 minutes to 1.5 hours.

3. Sequence determination:

Active blue beads were taken from the library and sequenced with Edman sequencer (Procise, Applied Biosystems).

result:

Specific factor XI and factor XI-like binding peptides found in libraries BL121, BL122 and BL123 according to the present invention include peptides comprising the amino acid sequences outlined below:

Sequence found in BL121

SEQ ID NO: 03: SRWPWSVFPDFPD

SEQ ID NO: 04: DVWDYVVFDDFPS

SEQ ID NO: 05: QRWVPYDDFPSLRS

SEQ ID NO: 06: RHFHVFPDFPFVH

SEQ ID NO: 07: HHFPPFSHFPDLPQ

SEQ ID NO: 08: RRLPLSRLPDFP

SEQ ID NO: 09: HPFFRGYPDFPD

SEQ ID NO: 10: HPWHLVYPDFPS

SEQ ID NO: 11: HDWLVRWPDFPS

SEQ ID NO: 12: SHFWRQWPDFSD

SEQ ID NO: 13: PQLRWHDFPDFGS

SEQ ID NO: 14: VVWRHWQDFDQFVV

SEQ ID NO: 15: VDWQWSRFDDFPS

SEQ ID NO: 16: HPWFDDFPHLFQ

Sequence from library BL122

SEQ ID NO: 17: YKWIHHDDFPLV

SEQ ID NO: 18: FDRKRVHPDFPH

SEQ ID NO: 19: DVWDYVVFDDFPS

SEQ ID NO: 20: QQPIQRFPDFP

SEQ ID NO: 21: QAIFTRFPDFPN

SEQ ID NO: 22: EWFPDFPEGSDG

SEQ ID NO: 23: HTHAFPDFPPH

SEQ ID NO: 24: LVKGFPDFPNHN

SEQ ID NO: 25: GPFPYAYEDFPE

SEQ ID NO: 26: FYLKTRYYDFPE

SEQ ID NO: 27: FQARHTIGDFPA

SEQ ID NO: 28: RIKDFPSDSNTV

SEQ ID NO: 29: IWESHKVIEDFP

SEQ ID NO: 30: QWFSVSRYQDFD

SEQ ID NO: 31: QKDFHWRILPDF

SEQ ID NO: 32: KIVKFPHTFPDL

SEQ ID NO: 33: HLYDFDLDNEY

SEQ ID NO: 34: KTILGDVDFDI

SEQ ID NO: 35: RQLHPFHHFHG

SEQ ID NO: 36: RSWLRYGYGH

SEQ ID NO: 37: FNWNNVDEYYDW

SEQ ID NO: 38: DQWDWEDYDEAW

SEQ ID NO: 39: YDIYDDYEIWA

Sequence found in BL124

SEQ ID NO: 40: YPKHIYADFPSTRL

SEQ ID NO: 41: YPRHIYPDFPTDTT

SEQ ID NO: 42: YLKHAWPDFPKLQQ

SEQ ID NO: 43: YVRHRFEDFPTALP

SEQ ID NO: 44: FPWHKYEDFPSPRT

SEQ ID NO: 45: QPAHRYPDFPRNNH

SEQ ID NO: 46: LPKTRFLDFPHVSF

SEQ ID NO: 47: LPPARYPDFPAAKK

SEQ ID NO: 48: IPKNRFSDFPDAQG

SEQ ID NO: 49: LPSFRFPDFPATKT

SEQ ID NO: 50: RVLNRYPDFPTTNQ

SEQ ID NO: 51: FFKKTYADFPTSQT

SEQ ID NO: 52: IFKKTYEDFPRFVY

SEQ ID NO: 53: VLHNKYDDFPRVKK

SEQ ID NO: 54: KVKHRFNDFPVWGN

Useful FXI-binding peptides are inferred to include those having the central amino acid motif of Asp-Phe-Pro.

Example 6

Cells from mammalian cell cultures were separated from the supernatant by centrifugation or filtration. Benzamidine and EDTA were added to a final concentration of 1 mM.

Example  7

Obelix  ST CIEX cation-exchange chromatography using (cat no 11-0010)

The Obelix matrix was equilibrated using Buffer A in 5 column volumes (cv) and a load corresponding to 150 ml supernatant per ml column fill was applied to the column. The column was washed with 4cv of buffer A (30mM Tris pH 8.0) followed by 5cv of buffer A '(50mM Tris 50% glycerol _{87 %} pH 9.0). Elution was then performed using 5cv of buffer A '' (50mM Tris 50% glycerol _{87 %} 1M NaCl pH 9.0). The flow rate was 16 cv / h and the temperature was 0-10 ° C. The column was regenerated using 1M NaOH. Fractions were collected from about 50% of the peak height and the first peak elution was discarded but the next week peak contained FXI. Analysis of FXI polypeptide-containing fractions was performed by HPLC using C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x50 mm (see below), and under reduced conditions MOPS on NUpage 4-12% Bis / Tris Gel (Invitrogen). Performed by SDS-PAGE using running buffer. Benzamidine and EDTA were added (up to 1 mM) to the fractions containing the FXI polypeptide and kept at about 4 ° C. in the refrigerator until use, or frozen at −80 ° C.

An alternative to Obelix ST CIEX is Streamline Direct CST (Amersham cat no 17-5266-03). (FIG. 4, preparative chromatogram)

Example  8

Butyl Sepharose  High performance High substitution (cat no 17-3100)  Hydrophobic Interaction Chromatography

1.5 volumes of buffer containing 2M NaCl 40 mM Tris pH 8 were added to the combined fractions containing the FXI polypeptide from Example 7 and the pH was adjusted to 8.2 if the pH was not between 8.0 and 8.4. The Butyl Sepharose High Performance High Substitution Matrix was equilibrated with Buffer B (1M NaCl 20 mM Tris pH 8.0) 3cv and a load corresponding to about 1 mg / ml was applied to the column. Next, the column was washed with buffer B at 2cv, then proceeded at least 20cv from buffer B to 100% elution buffer B '(20 mM Tris pH 8.0) followed by gradient elution with 2cv of 100% elution buffer B'. . The flow rate was 12cv / h and the temperature was 0-10 degreeC. Fractions were collected until about 15 cv after elution of about 10 cv. Analysis of FXI polypeptide-containing fractions was performed by HPLC using C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x250 mm (see below), and under reduced conditions MOPS on NUpage 4-12% Bis / Tris Gel (Invitrogen). Performed by SDS-PAGE using running buffer. 1/4 volume of propylene glycol was added directly to the pool of FXI polypeptide-containing fractions at a final concentration of 20% (v / v) propylene glycol and maintained or frozen at about 4 ° C. until the resulting pool was used.

The substitute for butyl sepharose high performance high substitution is phenyl sepharose high performance high substitution. The matrix of this substitute leads to later elution. (FIG. 5, preparative chromatogram)

Example  9

CHT Hydroxyapatite  Type I ( BioRad  cat no 157-0020) Hydroxyapatite  Chromatography

The pH of the pool of FXI polypeptide-containing fractions from Example 8 was adjusted to 6.0 and 1 volume of water was added to make a conductivity of 20 mS / cm. The 20 μm hydroxyapatite type I matrix was equilibrated with 6 C of buffer C, and then a load corresponding to a 5 mg / ml gel was applied to the column. The column was then washed with 15cv of buffer C (20mM KPO ₄ pH 6.0) and a wash step was performed with a buffer containing 95% buffer C and 5% elution buffer C '(20mM KPO ₄ 2M NaCl pH 6.0). Gradient elution from 5% C ′ to 100% C ′ was performed, which was used to elute the FXI polypeptide in small fractions. The conductivity of the pools containing FXI polypeptide fractions was about 60 mS / cm and pH about 6.0. Analysis of FXI polypeptide-containing fractions was performed by HPLC using C4 Jupiter Phenomonex cat no OOG-4167-EO, 4.6x250 mm (see below), and under reduced conditions MOPS on NUpage 4-12% Bis / Tris Gel (Invitrogen). Performed by SDS-PAGE using running buffer. HPLC purity was> 97%, and the concentration of FXI was about 1.2 mg / ml.

The high purity FXI containing fractions were collected and propylene glycol was added to the final 10% v / v and stored below -20 ° C. (FIG. 6, preparative chromatogram)

Example  10

HPLC  Analytical Process

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 using the following buffer:

Buffer I: 0.1% TFA aqueous solution

Buffer II: 0.07% TFA CH ₃ CN Solution

Equalization of the column was carried out for 5 minutes using a mixture of 75% (v / v) buffer I and 25% (v / v) buffer II (flow rate 1 ml / min).

The column was eluted using a gradient from 18% buffer I / 25% buffer II to 39% buffer I / 61% buffer II over 18 minutes (flow rate 1 ml / min).

Regeneration of the column was performed by washing with 100% buffer II for 2 minutes. The detection wavelength used was 214 nm. The temperature was 50 ° C. 2-50 μg of sample was loaded into the column.

All patents, patent applications, and references mentioned herein are incorporated herein by reference in their entirety.

Many variations of the invention will be suggested to those skilled in the art in light of the above detailed description. Such obvious modifications are within the full intended scope of the appended claims.

<110> Novo Nordisk Health Care AG <120> Therapeutic Use of Factor XI <150> DK PA 2003 01721 <151> 2003-11-20 <150> DK PA 2004 01141 <151> 2004-07-23 <160> 54 <170> KopatentIn 1.71 <210> 1 <211> 607 <212> PRT <213> Homo Sapiens <400> 1 Glu Cys Val Thr Gln Leu Leu Lys Asp Thr Cys Phe Glu Gly Gly Asp   1 5 10 15 Ile Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gln Val Val Cys              20 25 30 Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu Ser Pro          35 40 45 Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp Ser Val      50 55 60 Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala Ile Ser Gly Tyr  65 70 75 80 Ser Phe Lys Gln Cys Ser His Gln Ile Ser Ala Cys Asn Lys Asp Ile                  85 90 95 Tyr Val Asp Leu Asp Met Lys Gly Ile Asn Tyr Asn Ser Ser Val Ala             100 105 110 Lys Ser Ala Gln Glu Cys Gln Glu Arg Cys Thr Asp Asp Val His Cys         115 120 125 His Phe Phe Thr Tyr Ala Thr Arg Gln Phe Pro Ser Leu Glu His Arg     130 135 140 Asn Ile Cys Leu Leu Lys His Thr Gln Thr Gly Thr Pro Thr Arg Ile 145 150 155 160 Thr Lys Leu Asp Lys Val Val Ser Gly Phe Ser Leu Lys Ser Cys Ala                 165 170 175 Leu Ser Asn Leu Ala Cys Ile Arg Asp Ile Phe Pro Asn Thr Val Phe             180 185 190 Ala Asp Ser Asn Ile Asp Ser Val Met Ala Pro Asp Ala Phe Val Cys         195 200 205 Gly Arg Ile Cys Thr His His Pro Gly Cys Leu Phe Phe Thr Phe Phe     210 215 220 Ser Gln Glu Trp Pro Lys Glu Ser Gln Arg Asn Leu Cys Leu Leu Lys 225 230 235 240 Thr Ser Glu Ser Gly Leu Pro Ser Thr Arg Ile Lys Lys Ser Lys Ala                 245 250 255 Leu Ser Gly Phe Ser Leu Gln Ser Cys Arg His Ser Ile Pro Val Phe             260 265 270 Cys His Ser Ser Phe Tyr His Asp Thr Asp Phe Leu Gly Glu Glu Leu         275 280 285 Asp Ile Val Ala Ala Lys Ser His Glu Ala Cys Gln Lys Leu Cys Thr     290 295 300 Asn Ala Val Arg Cys Gln Phe Phe Thr Tyr Thr Pro Ala Gln Ala Ser 305 310 315 320 Cys Asn Glu Gly Lys Gly Lys Cys Tyr Leu Lys Leu Ser Ser Asn Gly                 325 330 335 Ser Pro Thr Lys Ile Leu His Gly Arg Gly Gly Ile Ser Gly Tyr Thr             340 345 350 Leu Arg Leu Cys Lys Met Asp Asn Glu Cys Thr Thr Lys Ile Lys Pro         355 360 365 Arg Ile Val Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro Trp Gln     370 375 380 Val Thr Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys Gly Gly 385 390 395 400 Ser Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys Phe Tyr                 405 410 415 Gly Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile Leu Asn             420 425 430 Gln Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln Glu Ile         435 440 445 Ile Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly Tyr Asp Ile Ala     450 455 460 Leu Leu Lys Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gln Arg Pro 465 470 475 480 Ile Cys Leu Pro Ser Lys Gly Asp Arg Asn Val Ile Tyr Thr Asp Cys                 485 490 495 Trp Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile Gln Asn             500 505 510 Thr Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu Cys Gln         515 520 525 Lys Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys Ala Gly     530 535 540 Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro 545 550 555 560 Leu Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly Ile Thr Ser                 565 570 575 Trp Gly Glu Gly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr Thr Asn             580 585 590 Val Val Glu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala Val         595 600 605 <210> 2 <211> 553 <212> PRT <213> Homo Sapiens <400> 2 Glu Cys Val Thr Gln Leu Leu Lys Asp Thr Cys Phe Glu Gly Gly Asp   1 5 10 15 Ile Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gln Val Val Cys              20 25 30 Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu Ser Pro          35 40 45 Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp Ser Val      50 55 60 Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala Ile Ser Gly Tyr  65 70 75 80 Ser Phe Lys Gln Cys Ser His Gln Ile Ser Asn Ile Cys Leu Leu Lys                  85 90 95 His Thr Gln Thr Gly Thr Pro Thr Arg Ile Thr Lys Leu Asp Lys Val             100 105 110 Val Ser Gly Phe Ser Leu Lys Ser Cys Ala Leu Ser Asn Leu Ala Cys         115 120 125 Ile Arg Asp Ile Phe Pro Asn Thr Val Phe Ala Asp Ser Asn Ile Asp     130 135 140 Ser Val Met Ala Pro Asp Ala Phe Val Cys Gly Arg Ile Cys Thr His 145 150 155 160 His Pro Gly Cys Leu Phe Phe Thr Phe Phe Ser Gln Glu Trp Pro Lys                 165 170 175 Glu Ser Gln Arg Asn Leu Cys Leu Leu Lys Thr Ser Glu Ser Gly Leu             180 185 190 Pro Ser Thr Arg Ile Lys Lys Ser Lys Ala Leu Ser Gly Phe Ser Leu         195 200 205 Gln Ser Cys Arg His Ser Ile Pro Val Phe Cys His Ser Ser Phe Tyr     210 215 220 His Asp Thr Asp Phe Leu Gly Glu Glu Leu Asp Ile Val Ala Ala Lys 225 230 235 240 Ser His Glu Ala Cys Gln Lys Leu Cys Thr Asn Ala Val Arg Cys Gln                 245 250 255 Phe Phe Thr Tyr Thr Pro Ala Gln Ala Ser Cys Asn Glu Gly Lys Gly             260 265 270 Lys Cys Tyr Leu Lys Leu Ser Ser Asn Gly Ser Pro Thr Lys Ile Leu         275 280 285 His Gly Arg Gly Gly Ile Ser Gly Tyr Thr Leu Arg Leu Cys Lys Met     290 295 300 Asp Asn Glu Cys Thr Thr Lys Ile Lys Pro Arg Ile Val Gly Gly Thr 305 310 315 320 Ala Ser Val Arg Gly Glu Trp Pro Trp Gln Val Thr Leu His Thr Thr                 325 330 335 Ser Pro Thr Gln Arg His Leu Cys Gly Gly Ser Ile Ile Gly Asn Gln             340 345 350 Trp Ile Leu Thr Ala Ala His Cys Phe Tyr Gly Val Glu Ser Pro Lys         355 360 365 Ile Leu Arg Val Tyr Ser Gly Ile Leu Asn Gln Ser Glu Ile Lys Glu     370 375 380 Asp Thr Ser Phe Phe Gly Val Gln Glu Ile Ile Ile His Asp Gln Tyr 385 390 395 400 Lys Met Ala Glu Ser Gly Tyr Asp Ile Ala Leu Leu Lys Leu Glu Thr                 405 410 415 Thr Val Asn Tyr Thr Asp Ser Gln Arg Pro Ile Cys Leu Pro Ser Lys             420 425 430 Gly Asp Arg Asn Val Ile Tyr Thr Asp Cys Trp Val Thr Gly Trp Gly         435 440 445 Tyr Arg Lys Leu Arg Asp Lys Ile Gln Asn Thr Leu Gln Lys Ala Lys     450 455 460 Ile Pro Leu Val Thr Asn Glu Glu Cys Gln Lys Arg Tyr Arg Gly His 465 470 475 480 Lys Ile Thr His Lys Met Ile Cys Ala Gly Tyr Arg Glu Gly Gly Lys                 485 490 495 Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Ser Cys Lys His Asn             500 505 510 Glu Val Trp His Leu Val Gly Ile Thr Ser Trp Gly Glu Gly Cys Ala         515 520 525 Gln Arg Glu Arg Pro Gly Val Tyr Thr Asn Val Val Glu Tyr Val Asp     530 535 540 Trp Ile Leu Glu Lys Thr Gln Ala Val 545 550 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Specific Binding Peptide <400> 3 Ser Arg Trp Pro Trp Ser Val Phe Pro Asp Phe Pro Asp   1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 4 Asp Val Trp Asp Tyr Val Val Phe Asp Asp Phe Pro Ser   1 5 10 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 5 Gln Arg Trp Val Pro Tyr Asp Asp Phe Pro Ser Leu Arg Ser   1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 6 Arg His Phe His Val Phe Pro Asp Phe Pro Phe Val His   1 5 10 <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 7 His His Phe Pro Pro Phe Ser His Phe Pro Asp Leu Pro Gln   1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 8 Arg Arg Leu Pro Leu Ser Arg Leu Pro Asp Phe Pro   1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 9 His Pro Phe Phe Arg Gly Tyr Pro Asp Phe Pro Asp   1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 10 His Pro Trp His Leu Val Tyr Pro Asp Phe Pro Ser   1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 11 His Asp Trp Leu Val Arg Trp Pro Asp Phe Pro Ser   1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 12 Ser His Phe Trp Arg Gln Trp Pro Asp Phe Ser Asp   1 5 10 <210> 13 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 13 Pro Gln Leu Arg Trp His Asp Phe Pro Asp Phe Gly Ser   1 5 10 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 14 Val Val Trp Arg His Trp Gln Asp Phe Asp Gln Phe Val Val   1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 15 Val Asp Trp Gln Trp Ser Arg Phe Asp Asp Phe Pro Ser   1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 16 His Pro Trp Phe Asp Asp Phe Pro His Leu Phe Gln   1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 17 Tyr Lys Trp Ile His His Asp Asp Phe Pro Leu Val   1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 18 Phe Asp Arg Lys Arg Val His Pro Asp Phe Pro His   1 5 10 <210> 19 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 19 Asp Val Trp Asp Tyr Val Val Phe Asp Asp Phe Pro Ser   1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 20 Gln Gln Pro Ile Gln Arg Phe Pro Asp Phe Pro   1 5 10 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 21 Gln Ala Ile Phe Thr Arg Phe Pro Asp Phe Pro Asn   1 5 10 <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 22 Glu Trp Phe Pro Asp Phe Pro Glu Gly Ser Asp Gly   1 5 10 <210> 23 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 23 His Thr His Ala Phe Pro Asp Phe Pro Pro His   1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 24 Leu Val Lys Gly Phe Pro Asp Phe Pro Asn His Asn   1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 25 Gly Pro Phe Pro Tyr Ala Tyr Glu Asp Phe Pro Glu   1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 26 Phe Tyr Leu Lys Thr Arg Tyr Tyr Asp Phe Pro Glu   1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 27 Phe Gln Ala Arg His Thr Ile Gly Asp Phe Pro Ala   1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 28 Arg Ile Lys Asp Phe Pro Ser Asp Ser Asn Thr Val   1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 29 Ile Trp Glu Ser His Lys Val Ile Glu Asp Phe Pro   1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 30 Gln Trp Phe Ser Val Ser Arg Tyr Gln Asp Phe Asp   1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 31 Gln Lys Asp Phe His Trp Arg Ile Leu Pro Asp Phe   1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 32 Lys Ile Val Lys Phe Pro His Thr Phe Pro Asp Leu   1 5 10 <210> 33 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 33 His Leu Tyr Asp Phe Asp Leu Asp Asn Glu Tyr   1 5 10 <210> 34 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 34 Lys Thr Ile Leu Gly Asp Val Asp Phe Asp Ile   1 5 10 <210> 35 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 35 Arg Gln Leu His Pro Phe His His Phe His Gly   1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 36 Arg Ser Trp Leu Arg Tyr Gly Tyr Gly His   1 5 10 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 37 Phe Asn Trp Asn Asn Val Asp Glu Tyr Tyr Asp Trp   1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 38 Asp Gln Trp Asp Trp Glu Asp Tyr Asp Glu Ala Trp   1 5 10 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 39 Tyr Asp Ile Tyr Asp Asp Tyr Glu Ile Trp Ala   1 5 10 <210> 40 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 40 Tyr Pro Lys His Ile Tyr Ala Asp Phe Pro Ser Thr Arg Leu   1 5 10 <210> 41 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 41 Tyr Pro Arg His Ile Tyr Pro Asp Phe Pro Thr Asp Thr Thr   1 5 10 <210> 42 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 42 Tyr Leu Lys His Ala Trp Pro Asp Phe Pro Lys Leu Gln Gln   1 5 10 <210> 43 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 43 Tyr Val Arg His Arg Phe Glu Asp Phe Pro Thr Ala Leu Pro   1 5 10 <210> 44 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 44 Phe Pro Trp His Lys Tyr Glu Asp Phe Pro Ser Pro Arg Thr   1 5 10 <210> 45 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 45 Gln Pro Ala His Arg Tyr Pro Asp Phe Pro Arg Asn Asn His   1 5 10 <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 46 Leu Pro Lys Thr Arg Phe Leu Asp Phe Pro His Val Ser Phe   1 5 10 <210> 47 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 47 Leu Pro Pro Ala Arg Tyr Pro Asp Phe Pro Ala Ala Lys Lys   1 5 10 <210> 48 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 48 Ile Pro Lys Asn Arg Phe Ser Asp Phe Pro Asp Ala Gln Gly   1 5 10 <210> 49 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 49 Leu Pro Ser Phe Arg Phe Pro Asp Phe Pro Ala Thr Lys Thr   1 5 10 <210> 50 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 50 Arg Val Leu Asn Arg Tyr Pro Asp Phe Pro Thr Thr Asn Gln   1 5 10 <210> 51 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 51 Phe Phe Lys Lys Thr Tyr Ala Asp Phe Pro Thr Ser Gln Thr   1 5 10 <210> 52 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 52 Ile Phe Lys Lys Thr Tyr Glu Asp Phe Pro Arg Phe Val Tyr   1 5 10 <210> 53 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 53 Val Leu His Asn Lys Tyr Asp Asp Phe Pro Arg Val Lys Lys   1 5 10 <210> 54 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Specific binding peptide <400> 54 Lys Val Lys His Arg Phe Asn Asp Phe Pro Val Trp Gly Asn   1 5 10

Claims (47)

A method of treating a bleeding event comprising administering an agent comprising a Factor XI (FXI) or FXI-cognate polypeptide in a therapeutically effective amount to a patient in need thereof. The method of claim 1, wherein said administration results in reduced clotting time in said patient. The method of claim 1, wherein said administration results in enhanced hemostasis in said patient. The method of claim 1, wherein said administering results in an increase in the time for coagulation lysis in said patient. The method of claim 1, wherein said administration results in an increase in coagulation intensity in said patient. The method of claim 1, wherein said administration results in an increase in overall coagulation quality (OCQ) in said patient. The method of claim 1, wherein after the administration, the patient exhibits an effective FXI plasma concentration of at least about 5 nM. 8. The method of claim 7, wherein the effective FXI plasma concentration is at least about 10 nM. The method of claim 8, wherein the effective FXI plasma concentration is at least about 30 nM. The method of claim 1, wherein the FXI or FXI-cognate polypeptide comprises a sequence of SEQ ID NO: 1, or a fragment thereof that retains at least one FXI-related biological activity. The method of claim 1, wherein the FXI or FXI-cognate polypeptide comprises the sequence of SEQ ID NO: 2, or a fragment thereof that retains at least one FXI-related biological activity. The method of claim 1, wherein the patient does not suffer from congenital FXI defects. The method of claim 1, wherein the bleeding event is accompanied by a condition selected from the group consisting of surgery, dental procedures, trauma, or hemodilution. The method of claim 1, further comprising: (a) obtaining a blood sample from the patient prior to the administration; (b) measuring at least one of FXI concentration, FXIa: FXI ratio, or amount of exogenous FXI required to restore coagulation; And (c) determining said amount of FXI effective for treatment based on the results of step (b). administering to said patient an agent comprising (i) an agent comprising a first amount of FXI polypeptide and (ii) a second amount of non-factor VII / factor VIIa coagulant, wherein said first amount and agent are combined 2 A method of treating a bleeding event, characterized in that the amount is effective for treatment. The method of claim 15, wherein the non-factor VII / factor VIIa coagulant is Factor XIII; Tissue factor pathway inhibitor (TFPI) inhibitors; Factor IX; Thrombin activated fibrinolytic inhibitor (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitors; Protein S inhibitors; And a tissue plasminogen activator (tPA) inhibitor. The method of claim 1, wherein the method does not comprise administration of Factor VII / Factor VIIa coagulant. A pharmaceutical formulation comprising (i) an isolated recombinant FXI polypeptide and (ii) a pharmaceutically acceptable carrier or excipient. Use of FXI Polypeptides for the Treatment of Bleeding Events. 20. The use of claim 19, wherein the bleeding event is accompanied by a condition selected from the group consisting of surgery, dental procedures, trauma, or hemodilution. Use according to claim 19 or 20, wherein the bleeding event is not treated with Factor VII / Factor VIIa coagulant. Use of FXI polypeptides to promote hemostasis in patients in need. Use of FXI polypeptide to increase coagulation lysis time in patients in need. Use of FXI polypeptides to increase coagulation intensity in patients in need. Use of FXI polypeptides to increase overall coagulation quality (OCQ) in patients in need. Use of FXI polypeptides to reduce clotting time in patients in need. The use according to any one of claims 19 to 25, wherein the effective FXI plasma concentration in the patient is increased to at least about 5 nM. 27. The use of claim 26, wherein the effective FXI plasma concentration is increased to at least about 10 nM. The use of claim 27, wherein the effective FXI plasma concentration is increased to at least about 30 nM. The use according to any of claims 19 to 28, wherein the patient to be treated is not treated with Factor VII / Factor VIIa coagulant. Use of FXI polypeptides in the manufacture of pharmaceutical formulations to treat bleeding events. 32. Use according to claim 31, wherein the bleeding event is accompanied by a condition selected from the group consisting of surgery, dental procedures, trauma, or hemodilution. 33. Use according to claim 31 or 32, wherein the bleeding event is not treated with Factor VII / Factor VIIa coagulant. Use of FXI polypeptides in the manufacture of pharmaceutical formulations for the promotion of hemostasis in patients in need. Use of FXI polypeptides in the manufacture of a pharmaceutical formulation to increase coagulation lysis time in patients in need. Use of FXI polypeptides in the manufacture of pharmaceutical formulations for increasing coagulation intensity in patients in need. Use of FXI polypeptides in the manufacture of pharmaceutical formulations to increase overall coagulation quality (OCQ) in patients in need. Use of FXI polypeptides in the manufacture of pharmaceutical formulations to reduce clotting time in patients in need. 39. The use according to any one of claims 31 to 38, wherein the effective FXI plasma concentration in the patient is increased to at least about 5 nM. 40. The use of claim 39, wherein the effective FXI plasma concentration is increased by at least about 10 nM. 41. The use of claim 40, wherein the effective FXI plasma concentration is increased by at least about 30 nM. 42. The use according to any one of claims 31 to 41, wherein the patient to be treated is not treated with Factor VII / Factor VIIa coagulants. 43. The use according to any one of claims 19 to 42, wherein the patient to be treated does not suffer from congenital FXI defects. 44. The use according to any one of claims 19 to 43, wherein said FXI polypeptide comprises a sequence of SEQ ID NO: 1, or a fragment thereof that retains at least one FXI-related biological activity. The use according to any of claims 19 to 43, wherein said FXI polypeptide comprises a sequence of SEQ ID NO: 2, or a fragment thereof that retains at least one FXI-related biological activity. 46. The use according to any one of claims 19 to 45, wherein said FXI polypeptide can be administered with a non-factor VII / factor VIIa coagulant. 47. The method of claim 46, wherein said non-factor VII / factor VIIa coagulant is Factor XIII; Tissue factor pathway inhibitor (TFPI) inhibitors; Factor IX; Thrombin activated fibrinolytic inhibitor (TAFI); Plasminogen activator inhibitor-1 (PAI-1); Factor V; Protein C inhibitors; Protein S inhibitors; And a tissue plasminogen activator (tPA) inhibitor.

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