Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6432—Coagulation factor Xa (3.4.21.6)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21006—Coagulation factor Xa (3.4.21.6)

Definitions

• Oral direct FXa inhibitors are emerging anticoagulants that have the potential to simplify dosing schemes and hemostatic monitoring in patients with prothrombotic diseases when compared to standard treatments, such as warfarin. Although these drugs have many advantages over warfarin, no fully efficacious reversal agent is available for these novel anticoagulants.
• the disclosure provides methods for reducing or preventing bleeding in a subject being treated with a direct Factor Xa (FXa) inhibitor by administering a composition comprising a Factor Xa variant containing at least one modification including substitution for the wild-type amino acid at position 16 (using the chymotrypsin numbering system) with Thr, Leu, Phe, Asp or Gly, or substitution for the wild-type amino acid at position 17 (using the chymotrypsin numbering system) with Leu, Ala, or Gly.
• treatment with a composition comprising a FXa variant results in at least a 50% reduction in bleeding.
• direct Factor Xa inhibitors include rivaroxaban or apixaban.
• the plasma concentration of the direct FXa inhibitor is a typical therapeutic amount or a supratherapeutic amount.
• the plasma concentration of rivaroxaban can be about 500 nM, or greater, and the plasma concentration of apixaban can be about 250 nM, or greater.
• the FXa variant contains the substitution 116L.
• the FXa variant is capable of countering the effect of the direct Factor Xa inhibitor at a plasma concentration that is at least 100-fold lower than the plasma concentration of the Factor Xa inhibitor.
• the composition comprising the FXa variant is administered before a planned surgery, after an injury, or after an intentional or accidental overdose with a direct FXa inhibitor.
• hemostasis in the subject is monitored using a hemostasis assay after a first dose with a FXa variant and, if adequate
• hemostasis is not attained by a predetermined time, at least one second dose of FXa variant is administered to achieve sufficient hemostasis.
• the predetermined time is about 15 mins, 30 mins, 45 mins or 60 mins after the first dose of FXa variant is administered. Other times are also possible.
• at least a second procoagulant is administered in addition to FXa variant, including for example, a different FXa variant, factor IX, factor Xla, factor Xlla, factor VIII, factor Vila, FEIBA or prothrombin complex concentrate (PCC).
• the disclosure provides methods for increasing the amount of thrombin produced in response to activation of the extrinsic or intrinsic clotting pathway in a subject being treated with a direct Factor Xa (FXa) inhibitor by administering a composition comprising a Factor Xa variant containing at least one modification including substitution for the wild-type amino acid at position 16 (using the chymotrypsin numbering system) with Thr, Leu, Phe, Asp or Gly, or substitution for the wild-type amino acid at position 17 (using the chymotrypsin numbering system) with Leu, Ala, or Gly.
• direct Factor Xa inhibitors include rivaroxaban or apixaban.
• the plasma concentration of the direct FXa inhibitor is a typical therapeutic amount or a supratherapeutic amount.
• the plasma concentration of rivaroxaban can be about 500 nM, or greater, and the plasma concentration of apixaban can be about 250 nM, or greater.
• the FXa variant contains the
• the amount of thrombin produced increases by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more.
• the FXa variant is capable of countering the effect of the direct Factor Xa inhibitor at a plasma concentration that is at least 100-fold lower than the plasma concentration of the Factor Xa inhibitor.
• the composition comprising the FXa variant is administered before a planned surgery, after an injury, or after an intentional or accidental overdose with a direct FXa inhibitor. In some
• hemostasis in the subject is monitored using a hemostasis assay after a first dose with a FXa variant and, if adequate hemostasis is not attained by a predetermined time, at least one second dose of FXa variant is administered to achieve sufficient hemostasis.
• the hemostasis assay after a first dose with a FXa variant and, if adequate hemostasis is not attained by a predetermined time, at least one second dose of FXa variant is administered to achieve sufficient hemostasis.
• predetermined time is about 15 mins, 30 mins, 45 mins or 60 mins after the first dose of FXa variant is administered. Other times are also possible.
• at least a second procoagulant is administered in addition to FXa variant, including for example, a different FXa variant, factor IX, factor Xla, factor Xlla, factor VIII, factor Vila, FEIBA or prothrombin complex concentrate (PCC).
• the disclosure provides methods for decreasing clotting time (as measured, for example, using PT or INR, or some other assay) in a subject being treated with a direct Factor Xa (FXa) inhibitor by administering a composition comprising a Factor Xa variant containing at least one modification including substitution for the wild-type amino acid at position 16 (using the chymotrypsin numbering system) with Thr, Leu, Phe, Asp or Gly, or substitution for the wild-type amino acid at position 17 (using the chymotrypsin numbering system) with Leu, Ala, or Gly.
• direct Factor Xa inhibitors include rivaroxaban or apixaban.
• the plasma concentration of the direct FXa inhibitor is a typical therapeutic amount or a supratherapeutic amount.
• the plasma concentration of rivaroxaban can be about 500 nM, or greater
• the plasma concentration of apixaban can be about 250 nM, or greater.
• the FXa variant contains the substitution 116L.
• clotting time is reduced by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
• the FXa variant is capable of countering the effect of the direct Factor Xa inhibitor at a plasma concentration that is at least 100-fold lower than the plasma concentration of the Factor Xa inhibitor.
• the composition comprising the FXa variant is administered before a planned surgery, after an injury, or after an intentional or accidental overdose with a direct FXa inhibitor.
• hemostasis in the subject is monitored using a hemostasis assay after a first dose with a FXa variant and, if adequate hemostasis is not attained by a predetermined time, at least one second dose of FXa variant is administered to achieve sufficient hemostasis.
• the predetermined time is about 15 mins, 30 mins, 45 mins or 60 mins after the first dose of FXa variant is administered. Other times are also possible.
• At least a second procoagulant is administered in addition to FXa variant, including for example, a different FXa variant, factor IX, factor Xla, factor Xlla, factor VIII, factor Vila, FEIBA or prothrombin complex concentrate (PCC).
• FXa variant including for example, a different FXa variant, factor IX, factor Xla, factor Xlla, factor VIII, factor Vila, FEIBA or prothrombin complex concentrate (PCC).
• Figures 1A-B show inhibition of free wt-FXa or FXa l16L by rivaroxaban.
• the initial velocity of peptidyl substrate (SpecXa; 200 uM) hydrolysis by A) wt-FXa (2 nM) or B) FXa l16L (6 nM) was determined at increasing concentrations of rivaroxaban.
• the Ki value is given on each graph.
• Figures 2A-B show rivaroxaban inhibition of wt-FXa or FXa l16L assembled in prothrombinase.
• the initial velocity of peptidyl substrate (SpecXa; 200 uM) hydrolysis by A) wt-FXa (2 nM) or B) FXa l16L (6 nM) in the presence of PCPS (20 uM) and FVa (40 nM) was determined at increasing concentrations of rivaroxaban.
• Figure 3 shows the effect of different concentrations of FXa l16L on reversing the effects on thrombin generation of rivaroxaban.
• Figures 4A-D show the effect of FXa l16L on reversing the effects of rivaroxaban.
• Normal human plasma was incubated with 500 nM rivaroxaban and in the absence or presence of increasing concentrations of FXa l16L .
• peak thrombin (A and C) and total thrombin generated (ETP; B and D) were plotted.
• rivaroxaban Normal human plasma was incubated with 7.5 uM rivaroxaban and in the absence or presence of increasing concentrations of FXa l16L . Following data analysis, peak thrombin (A) and total thrombin generated (ETP; B) were plotted.
• FIGS 6A-B show FXa l16L or FXa l16T reverses the effects of 250 nM apixaban.
• Normal human plasma was incubated with 250 nM apixaban and in the absence or presence of increasing concentrations of FXa l16L or FXa l16T .
• peak thrombin (A) and total thrombin generated (ETP; B) were plotted.
• FIGS 7A-B show FXa l16L or FXa l16T reverses the effects of high dose apixaban.
• Normal human plasma was incubated with 2.0 uM Apixaban and in the absence or presence of increasing concentrations of FXa l16L or FXa l16T .
• peak thrombin (A) and total thrombin generated (ETP; B) were plotted.
• Figures 8A-B show FXa corrects whole blood clotting in the presence of rivaroxaban. Whole blood thromboelastography was used to assess the ability of FXa l16L to reverse the effects of rivaroxaban at a typical (A) and a high (B) dose.
• FIGS 9A-B show FXa l16L corrects whole blood clotting in the presence of apixaban.
• Whole blood thromboelastography was used to assess the ability of FXa l16L to reverse the effects of apixaban at a typical (A) and a high (B) dose.
• Figures 10A-B show that FXa l16L counteracts rivaroxaban in a thrombin generation assay.
• Figure 10A shows a dose response of rivaroxaban and
• Figure 10B shows a dose response of FXa l16L in the presence of rivaroxaban.
• Figure 1 1 shows that FXa l16L counteracts rivaroxaban in a mouse tail clip bleeding model.
• Figure 12 demonstrates that rivaroxaban administered to mice delays clotting time of whole blood measured using ROTEM and that administration with FXa l16L dose-responsively counteracts the effect of rivaroxaban.
• Figure 13 shows that rivaroxaban administered to a mouse prevents clot formation at a site of vascular injury in the cremaster muscle caused by laser and that administration with FXa l16L counteracts the effect of rivaroxaban. Clot formation was visualized using intravital microscopy and fluorescently labeled antibodies against fibrin and platelets.
• Figure 13A shows clot formation in an untreated mouse.
• Figure 13B shows that rivaroxaban delayed and reduced platelet accumulation and prevented fibrin deposition.
• Figure 13C shows that in a mouse administered rivaroxaban and FXa l16L clot formation occurred at the site of injury.
• Figure 14 is the amino acid sequence of wild-type human Factor X preprotein (SEQ ID NO: 1 ).
• the signal peptide corresponds to amino acids 1 -23.
• the propeptide corresponds to amino acids 24-40.
• the mature light chain of activated Factor X (FXa) corresponds to amino acids 41 -179.
• the mature heavy chain of activated FXa (after removal of the activation peptide) corresponds to amino acids 235-488.
• the activation peptide (AP) corresponds to amino acids 183-234.
• Figure 15 is the nucleotide sequence of the cDNA encoding wild-type human Factor X preprotein (SEQ ID NO:2). The coding sequence corresponds to nucleotides 58 to 1524.
• the disclosure provides compositions and methods for counteracting the anti-coagulant effect of a direct FXa inhibitor in a subject in need thereof.
• Applicants have discovered that certain FXa variants rapidly and completely counteract the effect of a direct FXa inhibitor in a dose dependent manner. More specifically, applicants have discovered that a relatively small amount of an FXa variant restores normal coagulation activity in vivo in the presence of FXa inhibitor at therapeutic concentrations and even at supratherapeutic concentrations.
• Applicants' disclosure therefore contributes to fulfilling the promise of these advantageous anti-coagulants.
• Coagulation factor X is a zymogen which, upon activation by the intrinsic factor IX/factor VIII or extrinsic pathway (tissue factor/factor Vila), becomes FXa, which is the protease moiety of prothrombinase. Following proteolytic cleavage of the Arg-lle scissile bond, releasing an activation peptide (AP), a series of well defined structural changes in the zymogen drives the activation process to the mature active serine protease (See Toso et al., (2008) J. Biol. Chem.
• the mature FXa has a light chain and a heavy chain that contains the catalytic domain.
• the mature FXa becomes an active serine protease upon formation of the prothrombinase complex, which includes binding of an activated cofactor, Factor Va (FVa).
• FVa Factor Va
• FXa variants have been developed that upon activation cleavage yield a zymogen-like FXa variant. That is, once cleaved, the resulting FXa variant has poor active site function and is more resistant to inactivation by circulating inhibitors (i.e. antithrombin III and TFPI).
• the FXa variants thus, have longer half-lives in plasma than wild-type FXa.
• the FXa variant binds FVa, lipid membrane and calcium to form a fully active prothrombinase complex that efficiently activates prothrombin.
• the enzymes of coagulation are trypsin-like enzymes that belong to the S1 peptidase family of proteases that bear a chymotrypsin-like fold.
• the coagulation proteases contain catalytic domains that are highly homologous to each other and to the ancestral serine proteases of digestion. The structural homology/identity is so great (>70%) that residues in the catalytic domains of the coagulation enzymes (including Factor Xa) are numbered according to the corresponding residues in chymotrypsinogen. (Chymotrypsin numbering system; see Bajaj and Birktoft, Methods Enzymol.
• an FXa variant may be an FXa protein comprising an amino acid substitution that makes the variant more zymogen-like compared to a wild-type FXa protein in vivo or in vitro.
• FXa variants of the disclosure substantially regain wild-type FXa activity upon formation of
• FXa variants that are useful in methods of the disclosure are variants comprising a modification selected from the group consisting of: a) lie at position 16 is Thr, Leu, Phe, Asp or Gly and b) Val at position 17 is Leu, Ala, or Gly, according to the chymotrypsin numbering system. Amino acids 16 and 17 in the chymotrypsin numbering system occur at amino acids 235 and 236, respectively, of SEQ ID NO: 1 (human Factor X preproprotein).
• FXa variants are FXa l16L and FXa l16T (the nomenclature used herein for the FXa variants recites the original amino acid at the numbered position according to the chymotrypsin numbering system followed by the substituted amino acid).
• the FXa variants can be variants of any mammalian FXa. Of particular interest, however, are FXa variants of human FXa.
• the FX variant that is activated into a variant FXa of the disclosure may be further modified by inserting a non-native
• intracellular proteolytic cleavage site can be inserted between the Arg at position 234 of SEQ ID NO: 1 (position 15 in the chymotrypsin numbering system) and the amino acid at the position corresponding to position 235 of SEQ ID NO: 1 (position 16 in the chymotrypsin numbering system) in the variant FX zymogen.
• the non-native intracellular protease cleavage site is Arg-Lys-Arg or Arg-Lys-Arg-Arg-Lys-Arg (SEQ ID NO:3). These cleavage sites are efficiently recognized by proteases (PACE/furin-like enzymes) within the cell and are removed.
• This cleavage may result in a processed variant FXa in which the mature heavy chain of the molecule now begins at the amino acid at the position corresponding to position 235 of SEQ ID NO: 1 (position 16 in the chymotrypsin numbering system). Introduction of this cleavage site at said position allows for the intracellular conversion of FX to FXa.
• the entire amino acid sequence of the FX variant activation peptide (AP) i.e., amino acids 183-234 of SEQ ID NO: 1
• the non-native intracellular protease cleavage site is Arg- Lys-Arg or Arg-Lys-Arg-Arg-Lys-Arg (SEQ ID NO:3).
• this modification allows for intracellular cleavage of the FX variant expressed by cells.
• the intracellular cleavage converts FX variant to activated zymogen-like FXa variant which is then secreted by cells for subsequent purification. This approach obviates the need for extracellular cleavage that would otherwise be required to activate the variant clotting factor, for example, after isolating the protein or just before blood clotting.
• FXa variants of the disclosure are derived from FX variant preproteins comprising native wild-type human signal sequence and/or propeptide sequence.
• FX signal sequences and/or propeptide from non-human species can be used in place of the corresponding native amino acid sequences.
• signal sequence and/or propeptide sequence from other human or non-human secreted proteins can be used in place of the corresponding native amino acid sequences.
• a FXa variant comprises amino acids 41 - 179 and amino acids 235-488 of SEQ ID NO: 1 , wherein the amino acid at position 235 (isoleucine in the wild-type sequence) is substituted with a different amino acid selected from the group consisting of threonine (Thr), leucine (Leu), phenylalanine (Phe), aspartic acid (Asp), or glycine (Gly).
• Thr threonine
• Leu leu
• Phe phenylalanine
• Asp aspartic acid
• Gly glycine
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO:1 , wherein the amino acid at position 235 is substituted with Thr (i.e., I235T or I16T).
• a FXa variant comprises amino acids 41 -179 and amino acids 235- 488 of SEQ ID NO: 1 , wherein the amino acid at position 235 is substituted with Leu (i.e., I235L or I16L).
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO: 1 , wherein the amino acid at position 235 is substituted with Phe (i.e., I235F or I16F).
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO: 1 , wherein the amino acid at position 235 is substituted with Asp (i.e., I235D or 116D).
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO: 1 , wherein the amino acid at position 235 is substituted with Gly (i.e., I235G or I16G).
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO:1 , wherein the amino acid at position 236 (valine in the wild-type sequence) is substituted with a different amino acid selected from the group consisting of leucine (Leu), alanine (Ala), or glycine (Gly).
• Leu leucine
• Al alanine
• Gly glycine
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO: 1 , wherein the amino acid at position 236 is substituted with Leu (i.e., V236L or V17L).
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO: 1 , wherein the amino acid at position 236 is substituted with Ala (i.e., V236A or V17A).
• a FXa variant comprises amino acids 41 -179 and amino acids 235-488 of SEQ ID NO: 1 , wherein the amino acid at position 236 is substituted with Gly (i.e., V236G or V17G).
• FXa variants of the disclosure can include various isoforms of the light and/or mature heavy chain of the protein.
• Non-limiting exemplary isoforms of the FXa variant mature heavy chain include the alpha and beta versions of the mature heavy chain. Jesty et al., J Biol Chem. 1975 Jun 25;250(12):4497-504, incorporated by reference herein.
• Compositions of the disclosure can include FXa variant proteins in which one or the other or both alpha and beta mature heavy chain isoforms are represented.
• isoforms of FXa variant proteins can include isoforms in which a variable number of amino acids (for example, 1 , 2, 3, 4, 5, 6, or more amino acids) are either missing from or added to the carboxy terminus of the light chain and/or mature heavy chains of the protein.
• a variable number of amino acids for example, 1 , 2, 3, 4, 5, 6, or more amino acids
• FXa variants of the disclosure include proteins with a certain minimal degree of homology or sequence identity compared to the amino acid sequence of wild-type FXa in SEQ ID NO: 1 .
• FXa variants include proteins that contain a light and mature heavy chain that are at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99%
• FXa variants also include a substitution at the amino acid position corresponding to position 235 of SEQ ID NO: 1 with Thr, Leu, Phe, Asp, or Gly, or a substitution at the amino acid position corresponding to position 236 of SEQ ID NO: 1 with Leu, Ala, or Gly, and further wherein such FXa variants are zymogenic until incorporated into prothrombinase complex.
• the wild-type FXa light chain sequence corresponds to amino acids 41 to 179 and the wild-type FXa mature heavy chain sequence corresponds to amino acids 235 to 488.
• Percentage amino acid sequence homology or identity can readily be determined using software such as Protein BLAST available at the website of the National Center for Biotechnology Information (h ⁇ tp.//blas .ncbi nirn mh qoy/8iast.cqi).
• FXa variants of the disclosure can also include FXa variants containing one or more post-translational modifications including, without limitation, one or more O-linked or N-linked carbohydrate groups or a variable number of gamma-carboxyglutamic acid (Gla) residues.
• FXa variants of the disclosure can further include chemically modified FXa variant proteins.
• Other FXa variants useful in the methods of the disclosure are also possible.
• FXa l16x refers to a variant of activated Factor X wherein the amino acid corresponding to position 235 in SEQ ID NO: 1
• amino acid "x" can be threonine (Thr or T), leucine (Leu or L), phenylalanine (Phe or F), aspartic acid (Asp or D), or glycine (Gly or G).
• FXa v17y refers to a variant of activated Factor X wherein the amino acid corresponding to position 236 in SEQ ID NO: 1
• amino acid "y" can be leucine (Leu or L), alanine (Ala or A), or glycine (Gly or G).
• FXa l16x and FXa v17y are not limited by the protein sequence set forth in SEQ ID NO: 1 . Rather these terms additionally include the variety of isoforms and homologous proteins described herein with the specified substitution mutations at positions 16 or 17 in the chymotrypsin numbering system that behave as zymogens until incorporated into prothrombinase complex.
• An FXa variant of the disclosure may be produced by any technique for expressing a protein.
• isolated protein is a protein, polypeptide or variant that by virtue of its origin or source of derivation (1 ) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
• a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
• a protein may also be rendered substantially free of naturally-associated components by isolation, using protein purification techniques well known in the art.
• a protein or polypeptide is “substantially pure,” “substantially
• polypeptide or protein may be monomeric or multimeric.
• a substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and may be over 99% pure. Protein purity or
• homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.
• a direct FXa inhibitor is an inhibitor that binds directly to FXa and selectively binds FXa over other proteases.
• Direct FXa inhibitors are
• noncompetitive inhibitors of FXa with respect to prothrombin They bind the substrate binding cleft and inhibit FXa competitively with respect to small peptide substrates that also bind this region. They inhibit FXa with high picomolar affinity and are highly protein bound in plasma.
• Examples of direct FXa inhibitors are rivaroxaban, apixaban, betrixaban, darexaban, edoxaban and otamixaban. In certain embodiments, direct FXa inhibitors are selected from rivaroxaban and apixaban.
• an FXa variant can be used to counteract a direct FXa inhibitor that binds FXa or that binds FXa that has formed
• the direct FXa inhibitors may or may not require cofactors of FXa for inhibition.
• an FXa variant such as FXa l16L and FXa l16T , are administered to a subject whose blood contains a direct FXa inhibitor.
• the disclosure encompasses the use of a FXa variant to counteract direct FXa inhibitors, including but not limited to synthetic inhibitors, small molecule inhibitors, orally available inhibitors, or reversible inhibitors.
• the FXa inhibitor may be any combination of these features, such as an orally available, synthetic, reversible, small molecule inhibitor.
• the direct FXa inhibitors may be selected from rivaroxaban, apixaban, betrixaban, darexaban, edoxaban and otamixaban (see Perzborn et al., Nat Rev Drug Discov. 201 1 Jan; 10(1 ):61 -75; Turpie, Arterioscler Thromb Vase Biol. 2007
• direct FXa inhibitors are selected from rivaroxaban or apixaban.
• a FXa variant of the disclosure can be any FXa variant of the disclosure.
• a FXa variant of the disclosure can be administered to a subject to reverse the effects of a direct FXa inhibitor where such inhibitor occurs at supratherapeutic
• a supratherapeutic concentration is one that is higher than that ordinarily considered required to safely achieve anti-coagulation in a particular subject or class of subjects.
• Supratherapeutic concentrations of a direct FXa inhibitor can result from accidental or intentional overdose.
• Supratherapeutic concentrations of a direct FXa inhibitor can also result from unexpected effects in particular subjects, such as an unexpectedly high sensitivity to these drugs, or unexpectedly slow rate of clearance, due for example to drug interactions or other factors. Determination of what would be a therapeutic concentration or supratherapeutic concentration of direct FXa inhibitor in a particular subject or class of subjects is within the knowledge of those ordinarily skilled in the art.
• an FXa variant is used to counteract a direct FXa inhibitor or inhibitors that selectively bind FXa over other trypsin-like proteases by at least 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least, 500-fold, at least 1 ,000-fold, at least 5,000-fold or at least 10,000-fold.
• the direct FXa inhibitor may bind an FXa variant with a Kj of about 2 x 10 "7 M or less.
• Kj refers to the inhibitor constant of a particular inhibitor-target interaction, which is the concentration required to produce half maximum
• Kj by using methods known in the art. The disclosure contemplates, thus, counteracting a direct FXa inhibitor that binds an FXa variant free of the prothrombinase complex with a Kj of about 2 x 10 "8 M or less, about 1 x 10 "8 M or less, about 9 x 10 "9 M or less, about 8 x 10 "9 M or less, about 7 x 10 "9 M or less, about 6 x 10 "9 M or less, about 5 x 10 "9 M or less, about 4 x 10 "9 M or less, about 3 x 10 "9 M or less, about 2 x 10 "9 M or less , about 1 x 10 "9 M or less, about 9 x 10 "10 M or less, about 8 x 10 "10 M or less, about 7 x 10 "10 M or less, about 6 x 10 "10 M or less, about 5 x 10 "10 M or less, about 4 x 10 "10 M or less, about 3 x 10 "10 "10
• the direct FXa inhibitor to be counteracted by an FXa variant according to the methods of the disclosure may bind a wild-type FXa with a Kj at least 1 .5 fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 50-fold less than it binds the FXa variant.
• the direct FXa inhibitor may bind a wild-type FXa with a Kj of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less than the Kj with an FXa variant free of the prothrombinase complex.
• the direct FXa inhibitor may bind a prothrombinase complex comprising a wild-type FXa with about the same Kj as it binds a prothrombinase complex comprising an FXa variant.
• the disclosure provides methods for counteracting the effects of a direct FXa inhibitor in a subject who is bleeding (internally or externally) or is at risk of bleeding (e.g., in the course of a planned surgery) by administering a FXa variant.
• the direct FXa inhibitor may be present in the subject at a therapeutic concentration or a higher concentrations (i.e., a supratherapeutic concentration).
• the therapeutic concentration may be an overdose in sensitive individuals.
• the methods of the disclosure thus, are useful for providing an antidote to an overdose of a direct FXa inhibitor.
• the subject of treatment may be a human or a veterinary subject.
• Direct inhibitor overdose can be detected based on existence of symptoms or signs of excessively reduced clotting ability.
• Non-limiting examples include evidence of gastrointestinal bleeding, including dark tarry stools, bloody stools, and vomiting of blood.
• Other examples include nosebleeds, and increased tendency to, or severity of, bruising or bleeding from minor cuts and scrapes.
• direct inhibitor overdose can be detected directly or by measuring the ability of subject blood to clot and detecting deviations from the expected degree of anti-coagulation.
• Blood clotting potential can be measured in ways familiar to those ordinarily skilled in the art.
• overdose may be suspected when a subject's prothrombin time is excessively prolonged.
• overdose is confirmed when the prothrombin time expressed as an International Normalized Ratio (INR) is measured to be greater than about 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 12, 14, 16, 18, 20, or greater.
• ILR International Normalized Ratio
• the FXa variant may be administered whenever it is desired to counteract the effects of the direct FXa inhibitor, including but not limited to before a planned surgery, after an injury resulting in external or internal bleeding or after a direct FXa inhibitor overdose.
• the FXa variant may be administered at least about 12 hours, at least about 6 hours, at least about 3 hours, at least about 2 hours, at least about 1 hour, at least about 30 minutes, at least about 10 minutes, or at least about 5 minutes of when the desired
• the disclosure provides a method of administering a FXa variant to effect the urgent reversal of acquired coagulopathy due to FXa inhibition therapy in a subject with acute major bleeding.
• subjects are adult human patients. In other embodiments, subjects are pediatric human patients.
• acute major bleeding is caused by trauma.
• acute major bleeding occurs during surgery or other type of interventional procedure.
• exemplary non-limiting interventional procedures include incisions, drainage, vascular surgery, appendectomy, herniotomy or hernioplasty, abdominal surgery, cholecystectomy, trephination (burr hole), lumbar puncture, cardiac pacemaker insertion, hip fracture surgery, and others.
• acute major bleeding can be spontaneous bleeding with no apparent cause.
• sites of acute major bleeding include gastrointestinal bleeding, subcutaneous or intramuscular bleeding, bladder bleeding,
• hemarthrosis subdural hematoma, nasal bleeding, peritoneal bleeding, uterine bleeding, and other sites of bleeding.
• Effective treatment with FXa variants of the disclosure can reverse the effects of a direct FXa inhibitor.
• Successful reversal of such effects by a FXa variant can be determined in a variety of ways and be measured or monitored using different assays, methods, or endpoints.
• treatment with a FXa variant to reverse the effects of a direct FXa inhibitor is monitored using tests or assays performed on blood or plasma from a subject treated with FXa variant.
• a blood sample can be taken from a subject at a predetermined time after treatment with FXa variant.
• the blood, or plasma prepared from it, is then subjected to one or more tests to determine if certain hemostatic pharmacodynamic parameters have been normalized despite the presence of direct FXa inhibitor. If normalization is found then the subject need not be further treated with FXa variant. If normalization is not found, however, then further treatment with FXa variant in accordance with the methods of the disclosure may be required to reverse the effect of a direct FXa inhibitor.
• Tests for monitoring the effectiveness of treatment with a FXa variant include tests that directly or indirectly measure the ability to clot or that measure the activity of a direct FXa inhibitor.
• Non-limiting exemplary tests include prothrombin time or the related International Normalized Ratio, the
• thromboelastography chromogenic anti-FXa assay, thrombin generation assay, level of prothrombin fragment 1 + 2, level of thrombin-antithrombin III complex, activated partial thromboplastin time, and partial thromboplastin time.
• Other tests are also possible within the knowledge of those of ordinary skill in the art.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant reduces bleeding in the subject.
• treatment with FXa variant reduces bleeding in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the presence of a direct FXa inhibitor compared to absence of treatment with FXa variant.
• treatment with FXa variant reduces bleeding in a subject about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%- 35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%- 70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant reduces the activity of a direct FXa inhibitor in the subject.
• treatment with FXa variant reduces activity of the direct FXa inhibitor in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the presence of a direct FXa inhibitor compared to absence of treatment with FXa variant.
• treatment with FXa variant reduces the activity of a direct FXa inhibitor in a subject about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.
• Activity of a direct FXa inhibitor can be monitored using a chromogenic anti-FXa assay, such as that described in Asmis, et al., Thromb Res., 129:492- 498 (2012), or Barrett, et al., Thromb Haemost. 104: 1263-71 (2010), each of which are incorporated by reference herein.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant increases the amount of thrombin produced in the blood or plasma of the subject.
• treatment with FXa variant increases thrombin production in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in the presence of a direct FXa inhibitor compared to the absence of an FXa variant.
• Thrombin production in the blood or plasma of a subject can be determined using the thrombin generation assay (TGA) or other technique familiar to those of ordinary skill in the art.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant increases clotting in the subject.
• treatment with FXa variant increases clotting in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 .5 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in the presence of a direct FXa inhibitor compared to the absence of an FXa variant.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant reduces clotting time in the subject.
• treatment with FXa variant reduces clotting time in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the presence of a direct FXa inhibitor compared to absence of treatment with FXa variant.
• treatment with FXa variant reduces clotting time in a subject about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%- 30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%- 65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%- 100%.
• clotting time is determined by measuring the subject's prothrombin time (PT) which decreases as hemostasis is restored.
• PT is the amount of time it takes for serum to clot after addition of tissue factor. PT therefore measures the capability of the extrinsic clotting system to support clotting.
• PT can vary depending on the particular reagents a lab uses to run the test, but a normal PT is about 1 1 to 13 seconds.
• Clotting time can also be expressed using the International Normalized Ratio (INR), which eliminates lab to lab variability in clotting time measurements. Using the INR, a ratio of 0.8 to 1 .1 indicates normal clotting.
• INR International Normalized Ratio
• a ratio of 0.8 to 1 .1 indicates normal clotting.
• PT or INR can be determined at a predetermined time after a FXa variant is administered to a subject in need of reversal of the effects of a direct FXa inhibitor.
• treatment with a FXa variant to reverse the effects of a direct FXa inhibitor reduces the PT of a subject to about 25 seconds, 24 seconds, 23 seconds, 22 seconds, 21 seconds, 20 seconds, 19 seconds, 18 seconds, 17 seconds, 16 seconds, 15 seconds, 14 seconds, 13 seconds, 12 seconds, 1 1 seconds, 10 seconds, or less.
• treatment with a FXa variant reduces the INR or a subject to about 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1 , 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1 , 2.0, 1 .9, 1 .8, 1 .7, 1 .6, 1 .5, 1 .4, 1 .3, 1 .2, 1 .1 , 1 .0, 0.9, 0.8, 0.7, or less.
• treatment with FXa variant reduces PT or INR in a subject about 5%-10%, 10%- 15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%- 50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%- 85%, 85%-90%, 90%-95%, or 95%-100%.
• Prothrombin time can be measured at a predetermined after
• PT is measured 15 mins, 20 mins, 30 mins, 40 mins, 45 mins, 50 mins, 60 mins or more after administration of FXa. Other times are also possible according to the knowledge of those of ordinary skill in the art.
• Clotting time can also be measured using the one-step prothrombinase- induced clotting time (PiCT) assay as described in Graff, et al., Monitoring effects of direct FXa-inhibitors with a new one-step prothrombinase-induced clotting time (PiCT) assay: comparative in vitro investigation with heparin, enoxaparin, fondaparinux and DX 9065a, Int J Clin Pharmacol Then, 45:237-43 (2007) and Harder, et al., Monitoring direct FXa-inhibitors and fondaparinux by
• Prothrombinase-induced Clotting Time (PiCT): relation to FXa-activity and influence of assay modifications, Thromb Res., 123:396-403 (2008), each of which are incorporated by reference.
• the methods of thromboelastometry or thromboelastography may be used to analyze clot formation or clotting time.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant increases the level of prothrombin fragment 1 + 2 (PF1 + 2) in the blood or plasma of the subject.
• treatment with FXa variant increases PF1 + 2 in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 .5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in the presence of a direct FXa inhibitor compared to the absence of an FXa variant.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant increases the level of thrombin-antithrombin III complex (TAT) in the blood or plasma of the subject.
• treatment with FXa variant increases TAT in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 1 .5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, at least 50-fold, or more in the presence of a direct FXa inhibitor compared to the absence of an FXa variant.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant reduces activated partial thromboplastin time (aPTT) in the subject.
• treatment with FXa variant reduces activated partial thromboplastin time (aPTT) in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the presence of a direct FXa inhibitor compared to absence of treatment with FXa variant.
• treatment with FXa variant reduces aPTT in a subject about 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.
• reversing the effects of a direct FXa inhibitor in a subject by administering a FXa variant reduces partial thromboplastin time (PTT) in the subject.
• treatment with FXa variant reduces partial thromboplastin time (PTT) in a subject at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the presence of a direct FXa inhibitor compared to absence of treatment with FXa variant.
• treatment with FXa variant reduces PTT in a subject about 5%- 10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%- 45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%- 80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.
• clinical endpoints can be relied upon to determine if hemostasis has been adequately restored in a subject treated with a FXa variant to reverse the effects of a direct FXa inhibitor. For example, where a subject presents with acute bleeding, clinical hemostatic efficacy can be scored "very good” where prompt cessation of existing bleeding occurs after treatment with FXa variant; "satisfactory” where there is a 1 -2 hr delay in bleeding cessation; “questionable” where there is a >2 hr delay in bleeding cessation; and “none” where an effect on bleeding is absent. Where treatment with FXa variant is determined to be less than satisfactory, then an additional dose of FXa variant can be administered to effect adequate hemostasis.
• clinical hemostatic efficacy can be scored "very good” where normal hemostasis is attained during the procedure; "satisfactory” where intraprocedural hemostasis is mildly abnormal as judged by quantity or quality of blood loss (e.g., slight oozing); “questionable” where intraprocedural hemostasis is moderately abnormal as judged by quantity or quality of blood loss (e.g., controllable bleeding); and "none" where
• intraprocedural hemostasis is severely abnormal as judged by quantity or quality of blood loss (e.g., severe refractory hemorrhage).
• a therapeutically effective dose of a direct FXa inhibitor depends upon numerous factors that are well known to a medical practitioner of skill in the art.
• a typical therapeutic plasma concentration of rivaroxaban is about 500 nM.
• an FXa variant can be administered to counteract lower or higher concentrations of inhibitor.
• the plasma concentration of rivaroxaban in a subject to be treated with an FXa variant may be lower or higher than the typical therapeutic concentration, for example about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM or about 1 ,000 nM.
• a typical therapeutic plasma concentration of apixaban is about 250 nM.
• the FXa variant is administered to a subject with a plasma concentration of apixaban of about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM or about 1 ,000 nM.
• an FXa variant can be used to counteract a direct FXa inhibitor in cases of overdose, such as when the plasma concentration of the inhibitor is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 1 .5 fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 50-fold higher than the typical therapeutic plasma concentration.
• the FXa variants are surprisingly effective in counteracting a direct FXa inhibitor at a plasma concentration that is lower than the plasma concentration of the direct FXa inhibitor.
• the FXa variant counters the effect of a direct FXa inhibitor at a plasma concentration ratio of variant to inhibitor of about 1 to 10, about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 250, about 1 to 500, about 1 to 1 ,000, about 1 to 2,500, about 1 to 5,000 or about 1 to 10,000.
• the FXa variant counters the effect of a direct FXa inhibitor at a plasma concentration of at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1 , 000-fold, at least 2,500-fold, at least 5,000-fold, or at least 10,000-fold lower than the plasma concentration of the direct FXa inhibitor.
• the plasma concentration of an FXa variant sufficient to reverse the effect of a direct FXa inhibitor is calculated by multiplying the plasma concentration of the direct inhibitor by a conversion factor ranging from about 0.1 x 10 "4 to about 1000 x 10 "4 , about 4 x 10 "4 to about 40 x 10 "4 , about 20 x 10 "4 to about 200 x 10 "4 , or other ranges.
• the conversion factor ranging from about 0.1 x 10 "4 to about 1000 x 10 "4 , about 4 x 10 "4 to about 40 x 10 "4 , about 20 x 10 "4 to about 200 x 10 "4 , or other ranges.
• conversion factor can be about 0.1 x 10 "4 , 0.5 x 10 “4 , 1 x 10 “4 , 2 x 10 “4 , 3 x 10 “4 , 4 x 10 “4 , 5 x 10 "4 , 6 x 10 “4 7 x 10 "4 , 8 x 10 “4 , 9 x 10 “4 , 10 x 10 “4 1 1 x 10 “4 , 12 x 10 "4 13 x 10 "4 , 14 x 10 "4 15 x 10 "4 , 16 x 10 “4 17 x 10 "4 , 18 x 10 “4 , 19 x 10 "4 20 x 10 "4 21 x 10 "4 , 22 x 10 “4 , 23 x 10 "4 , 24 x 10 “4 , 25 x 10 "4 , 26 x 10 “4 , 27 x 10 “4 , 28 x 10 “4 , 29 x 10 “4 , 30 x 10 "4 31 x 10 "4
• Achieving a target plasma concentration of FXa variant sufficient to reverse overdose of a direct FXa inhibitor is within the knowledge of those ordinarily skilled in the art.
• estimates of relevant pharmacokinetic parameters such as subject plasma volume or other parameters, can be made based on upon subject sex, height and weight, or other factors, and used to calculate how much FXa variant needs be administered to achieve the target concentration.
• plasma concentrations can be monitored according to the knowledge of those ordinarily skilled in the art and this information used to maintain the concentration in any desired range.
• compositions and methods of the disclosure include a
• a “therapeutically effective amount” or a “prophylactically effective amount” of an FXa variant refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutically effective amount of the FXa variant may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the FXa variant to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the FXa variant are outweighed by the therapeutically beneficial effects.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. For example, a dose may be given prior to a planned surgery.
• Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required
• a therapeutically or prophylactically-effective amount of an FXa variant administered is about 0.0001 to 50 mg/kg, about 0.001 to 50 mg/kg, about 0.001 to 5 mg/kg, about 0.001 to 0.5 mg/kg, about 0.001 to 0.05 mg/kg, about 0.01 to 5 mg/kg or about 0.01 to 0.5 mg/kg.
• a therapeutically or prophylactically-effective serum concentration of an FXa variant of the disclosure is about 0.0003 to 300 nM, about 0.003 to 300 nM, about 0.03 to 300 nM, about 0.003 to 30 nM, about 0.03 to 30 nM or about 0.3 to 3 nM.
• concentration of the FXa variant for example in blood or plasma, may be measured by any method known in the art.
• dosage values may vary with FXa inhibitor concentration. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
• kits comprising an FXa variant or a composition comprising such an FXa variant.
• a kit may include, in addition to the FXa variant or composition, diagnostic or additional therapeutic agents.
• a kit can also include instructions for use in a therapeutic method, as well as packaging material such as, but not limited to, ice, dry ice, styrofoam, foam, plastic, cellophane, shrink wrap, bubble wrap, cardboard and starch peanuts.
• the kit includes the FXa variant or a composition comprising it and one or more therapeutic agents that can be used in a method described herein.
• the FXa variant may be administered, for example in a composition comprising it, once or multiple times to a subject until adequate hemostasis is restored or the direct FXa inhibitor or inhibitors are no longer effective. Where multiple administrations are used they may administered hourly, daily, or at any other appropriate interval, including for example multiple daily doses. Multiple doses may be administered on a schedule such as every 10 minutes, every 15 minutes, every 20 minutes, every 30 minutes, every hour, every two hours, every three hours, every four hours, three times daily, twice daily, once daily, once every two days, once every three days, and once weekly.
• the FXa variant may also be administered continuously, e.g. via a minipump.
• the FXa variant may be administered, for example, via a parenteral route (e.g., intravenously,
• the FXa variant will generally be administered as part of a pharmaceutical composition as described below.
• the FXa variant may be co-administered with another procoagulant including another FXa variant, Factor IX, Factor Xla, Factor Xlla, Factor VIII, Factor Vila, FEIBA and prothrombin complex concentrate (PCC).
• another procoagulant including another FXa variant, Factor IX, Factor Xla, Factor Xlla, Factor VIII, Factor Vila, FEIBA and prothrombin complex concentrate (PCC).
• Co-administration of an FXa variant of the disclosure with an additional therapeutic agent encompasses administering a
• Co-administration or combination therapy further includes administering the FXa variant and additional therapeutic agent(s) simultaneously or sequentially, or both.
• the FXa variant may be administered once every three days, while the additional therapeutic agent is administered once daily at the same as the FXa variant, or at a different time.
• An FXa variant may be administered prior to or subsequent to treatment with the additional therapeutic agent.
• administration of an FXa variant of the disclosure may be part of a treatment regimen that includes other treatment modalities including surgery.
• the combination therapy may be administered to prevent recurrence of the condition.
• the combination therapy may be
• the administrations may be on a schedule such as every 10 minutes, every 15 minutes, every 20 minutes, every 30 minutes, every hour, every two hours, every three hours, every four hours, three times daily, twice daily, once daily, once every two days, once every three days, once weekly, or may be administered continuously, e.g. via a minipump.
• the combination therapy may be administered, for example, via a parenteral route (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly).
• the disclosure provides a composition comprising an FXa variant for use in counteracting a direct FXa inhibitor in a subject.
• the composition may comprise a pharmaceutically acceptable carrier, vehicle or other ingredients that are physiologically compatible.
• suitable carriers, vehicles and other ingredients include solvents (e.g., water, ethanol, saline, phosphate buffered saline), detergents, surfactants, dispersion media, coatings, antibacterial or antifungal agents, isotonifying agents, absorption delaying agents, sugars (e.g., sucrose, dextrose, lactose), polyalcohols (e.g., glycerol, mannitol, sorbitol), salts (e.g., sodium chloride, potassium chloride), wetting agents, emulsifying agents, preservatives, buffers, and agents capable of enhancing the stability or effectiveness of the FXa variant.
• solvents e.g., water, ethanol, saline, phosphate
• compositions for use according to the disclosure may be in any suitable form for administration to a subject, such as liquid solutions (e.g., injectable and infusible solutions).
• Compositions can be provided in a pre-mixed format ready for administration to a subject, for example, in a vial or pre-filled syringe. Such formats do not require reconstitution with diluent before
• compositions can be provided in lyophilized form requiring reconstitution with diluent (e.g., sterile water or saline) before diluent (e.g., sterile water or saline) before diluent (e.g., sterile water or saline) before diluent (e.g., sterile water or saline) before diluent (e.g., sterile water or saline) before
• compositions can be formulated for storage under refrigeration or at room temperature.
• the form of the composition depends, at least in part, on the intended mode of administration. In certain embodiments, the mode of
• administration is parenteral, including for example intravenous, subcutaneous, intraperitoneal, or intramuscular administration.
• compositions typically must be sterile and stable under the conditions of manufacture and storage.
• the composition can be formulated as a solution, microemulsion, dispersion, in liposomes, or other ordered structure suitable to high drug concentration.
• Sterile injectable solutions can be prepared by incorporating the FXa variant in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
• compositions herein may be administered to a subject being treated with a direct FXa inhibitor.
• the thrombin generation assay was used to assess whether the zymogen-like FXa variants can reverse the effects of direct FXa inhibitors in a more physiologic environment.
• the TGA measures thrombin production in plasma over time following the initiation of coagulation and was performed as previously described (See Bunce et al. , (201 1 ) Blood 117, 290-298, incorporated by reference herein in their entirety).
• Thrombin generation in normal human plasma was measured for 90 min at 37°C in the presence or absence of 500 nM rivaroxaban.
• increasing amounts of FXa l16L was added to plasma containing 500 nM
• Thrombin generation was initiated with 2.0 pM tissue factor/4 uM phospholipid as well as CaC and a thrombin fluorogenic substrate.
• FXa zymogen-like variants could reverse the effects of another direct FXa inhibitor, apixaban.
• FXa l16L was also compared.
• FXa l16T is similar to FXa l16L , however it has intrinsically less activity, has a longer plasma half-life, and has ⁇ 3-5-fold reduced activity compared to FXa l16L when assembled in the prothrombinase complex.
• FXa l16L could restore peak thrombin (Figure 6A) and total thrombin (Figure 6B) generated in the presence of 250 nM apixaban (a typical therapeutic plasma concentration) in a dose-dependent manner, which appears to reach a maximum between 1 -3 nM of FXa l16L .
• FXa l16T was also effective at reversing the effects of apixaban; however, it appears that higher concentrations of this variant are needed to fully restore thrombin generation ( Figure 6). Both variants were still effective even in the presence of a higher concentration of apixaban (2 ⁇ ). However, under these conditions it appears that higher concentrations of both variants are needed to fully restore thrombin generation ( Figure 7A and B).
• ROTEM thromboelastometry
• thrombography (CAT) system Thrombinoscope BV, Maastricht, The
• Normal human plasma was obtained from George King Biomedical (Overland Park, KS).
• 20 ⁇ _ of PPP-Reagent LOW containing 4 ⁇ phospholipids and 1 pM tissue factor was added to 70 ⁇ _ of pooled citrated normal human plasma (treated with 250 nM rivaroxaban, within the therapeutic plasma concentration range) in an Immulon 2HB round bottom 96 well plate with reactions duplicated.
• 10 ⁇ _ of vehicle or FXa l16L was added to plasma at final concentrations ranging from 0.03125 nM to 0.5 nM FXa l16L , given a 120 ⁇ _ total reaction volume.
• Reactions were initiated by addition of 20 ⁇ _ FluCa buffer containing calcium chloride and fluorogenic substrate. Fluorescence of plasma reactions was read at 37°C at 20 second intervals on a Fluoroskan Ascent fluorometer and compared to those of reference thrombin calibrator reactions to determine thrombin concentrations. The intensity of the fluorescence signal (FU) was continuously monitored at 37°C using the CAT. Thrombin generation curves (nM thrombin vs. time) were analyzed to extract lag time, peak height, time to peak, and the area under the curve representing the endogenous thrombin potential (ETP) using the Thromboscope software
• mice Male C57BI/6 mice (The Jackson Laboratory, Bar Harbor, ME) received a single intravenous injection of rivaroxaban at a dose of 10, 25 or 50 mg/kg. Thirty minutes later, mice were anesthetized with isoflurane and placed on a heated platform, and the body temperature of the mice was maintained at 37°C prior to the tail cut. The tails were immersed in 50 mL pre-warmed phosphate buffered saline (PBS) at 37°C for 2 minutes. A 3 mm tail cut was made and blood was collected into PBS for a 10 minute period.
• PBS pre-warmed phosphate buffered saline
• a quantitative assessment of the amount of bleeding was determined by hemoglobin content of the blood collected into PBS. Tubes were centrifuged to collect erythrocytes, resuspended in 5 mL lysis buffer (8.3 g/L NH 4 CI, 1 .0 g/L KHC0 3 , and 0.037 g/L EDTA), and the absorbance of the sample was measured at 575 nm. The absorbance values were converted to total blood loss ( ⁇ ) using a standard curve. The administration of rivaroxaban resulted in a dose dependent increase in blood loss following a tail cut ( Figure 11 ).

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