Method for treating intracranial hemorrhage and method for evaluating the effect

Abstract

The present disclosure relates to compositions and methods useful for assessing hemostatic efficacy in patients who have suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor. The method includes administering to the patient a fXa derivative that has reduced catalytic activity compared to wild-type fXa protein, is capable of binding to the factor Xa inhibitor, and is unable to assemble into a prothrombinase complex; obtaining a blood sample from the patient after administration; and measuring anti-fXa activity in the sample, where the anti-fXa activity reflects hemostatic efficacy in the patient. Once the assessment is made, appropriate medical intervention can be implemented.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 802,645, filed February 7, 2019, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Factor Xa inhibitors, e.g., apixaban, rivaroxaban, edoxaban, betrixaban, and enoxaparin, have a positive benefit-risk profile for the treatment and prevention of thrombotic events but can cause or exacerbate acute major bleeding with significant morbidity and mortality. Acute major bleeding episodes associated with factor Xa inhibitors can be difficult to treat due to the lack of specific reversal agents.

[0003] Andexanet alfa (coagulation factor Xa [recombinant] inactivated-zhzo) is a modified recombinant inactive form of factor Xa specifically designed to bind and sequester factor Xa inhibitor molecules, thereby rapidly reducing anti-factor Xa activity, a measure of the anticoagulant effect of factor Xa inhibitors. In healthy subjects receiving either apixaban or rivaroxaban, andexanet rapidly reduced both the unbound fraction of factor Xa inhibitors and anti-factor Xa activity with minimal side effects.

[0004] Monitoring the hemostatic effect after treatment with andexanet may be important, especially in elderly patients, and may provide useful information for determining whether a patient is suitable for a particular medical procedure and whether additional andexanet is required. Summary of the Invention [Means for solving the problem]

[0005] The present invention, in one embodiment, provides a method useful for assessing the hemostatic efficacy in patients who have suffered an intracranial hemorrhage while undergoing anticoagulant treatment with a factor Xa (fXa) inhibitor.

[0006] This evaluation is based on the unexpected finding that, although there is no correlation between anti-factor Xa activity and hemostatic effect in the general patient population after treatment with andexanet alfa, a surprisingly good correlation was found in patients with intracranial hemorrhage.

[0007] Thus, in one embodiment, the present disclosure provides a method for evaluating hemostatic efficacy in a patient suffering from intracranial hemorrhage while undergoing anticoagulant treatment with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; Including, The method, wherein the anti-fXa activity is reflective of hemostatic efficacy in the patient.

[0008] In some embodiments, the method further comprises estimating the hemostatic effect based on the anti-fXa activity. In some embodiments, the method further comprises measuring the anti-fXa activity in a sample obtained from the patient prior to the administration of the fXa derivative. In some embodiments, the estimating takes as input the anti-fXa activity before and after the administration.

[0009] In some embodiments, the method comprises a bolus injection of said fXa derivative. In some embodiments, said administering further comprises an infusion of said fXa derivative after said bolus injection. In some embodiments, said blood sample is obtained 10 minutes to 4 hours after said administering. In some embodiments, said anti-fXa activity is measured using a chromogenic assay.

[0010] In some embodiments, the method further includes determining, based on the anti-fXa activity, (a) whether to administer the fXa derivative a second time to the patient, (b) the likelihood that the patient will suffer a thrombotic event, (c) the minimum time for monitoring or treatment, or (d) whether the patient is suitable for surgery.

[0011] In some embodiments, the method further comprises administering a second dose of the fXa derivative to the patient if the anti-fXa activity exceeds a predetermined anti-fXa activity threshold or if the hemostatic effect falls below a predetermined hemostatic effect threshold.

[0012] In one embodiment, there is provided a method for treating intracranial hemorrhage in a patient undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; administering a second dose of the fXa derivative to the patient if the anti-fXa activity exceeds a predetermined anti-fXa activity threshold. The above method is also provided, comprising:

[0013] In some embodiments, the predetermined anti-fXa activity threshold is 30 to 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25 to 0.5 IU / ml when the fXa inhibitor is enoxaparin.

[0014] Another embodiment is a method of performing surgery in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; The method further comprises performing surgery on the patient if the anti-fXa activity is below a predetermined anti-fXa activity threshold or if the estimated hemostatic effect based on the anti-fXa activity exceeds a predetermined hemostatic effect threshold.

[0015] In some embodiments, the predetermined anti-fXa activity threshold is 30 to 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25 to 0.5 IU / ml when the fXa inhibitor is enoxaparin.

[0016] Yet another embodiment is a method for preventing a thrombotic event in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; determining the likelihood that the patient will suffer from a thrombotic event based on the anti-fXa activity; administering anticoagulant therapy to the patient if the patient is determined to be at high risk of suffering from a thrombotic event; The method includes the steps of:

[0017] In some embodiments, the patient is determined to have a high likelihood of suffering from a thrombotic event if the anti-fXa activity is less than 30-50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or less than 0.25-0.5 IU / ml when the fXa inhibitor is enoxaparin.

[0018] Yet another embodiment is a method for monitoring recovery in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; determining the duration of monitoring for said patient based on risk; The method includes the steps of:

[0019] In some embodiments, the method further includes removing the patient from monitoring if the anti-fXa activity is between 30 and 50 ng / ml if the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or between 0.25 and 0.5 IU / ml if the fXa inhibitor is enoxaparin.

[0020] The fXa derivatives disclosed herein can be two-chain proteins comprising a light chain comprising the amino acid sequence of SEQ ID NO: 3 or a first peptide having at least 85% sequence identity to SEQ ID NO: 3, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 or a second peptide having at least 85% sequence identity to SEQ ID NO: 4. In some embodiments, the fXa derivative is andexanet alpha.

[0021] While drawings are provided as embodiments of the present disclosure, these drawings illustrate by way of example only and not by way of limitation. [Brief description of the drawings]

[0022] [Figure 1-1] FIG. 1 shows anti-factor Xa activity in patients who experienced major bleeding while undergoing anticoagulant therapy with the factor Xa inhibitor apixaban. [Figure 1-2] FIG. 1 shows anti-factor Xa activity in patients who experienced major bleeding while undergoing anticoagulant therapy with the factor Xa inhibitor rivaroxaban. [Figure 1-3] FIG. 1 shows anti-factor Xa activity in patients who experienced major bleeding while undergoing anticoagulant therapy with the factor Xa inhibitor enoxaparin. [Diagram 2] FIG. 1 shows the hemostatic effect measured in the above samples. [Diagram 3] FIG. 1 shows receiver operating characteristic curves for change in anti-factor Xa activity (ng / ml) and hemostatic effect (good / excellent vs. poor / none) for all patients (left panel) and for patients with intracranial hemorrhage (right panel). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] I. Definitions All numerical designations, including ranges, e.g., pH, temperature, time, concentration, and molecular weight, are approximations (+ or -) that vary by increments of 0.1. It is to be understood, even if not always explicitly stated, that all numerical designations are preceded by the term "about." It is also to be understood, even if not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such reagents are known in the art.

[0024] A "composition" is intended to mean a combination of an active agent with another compound or composition, inert (eg, a detectable agent or label) or active.

[0025] "Factor Xa" or "fXa" or "fXa protein" is a serine protease in the blood coagulation pathway that is produced from inactive factor X (fX). The nucleotide sequence encoding human factor X (fX) is present in GenBank under the accession number "NM_000504". fX is activated to fXa (SEQ ID NO:1, Table 1) upon catalytic cleavage of the first 52 residues of the heavy chain. fXa comprises a light chain and a heavy chain (as shown in Table 1). The first 45 amino acid residues of the light chain (residues 1-45 of SEQ ID NO:1) contain 11 post-translationally modified gamma-carboxyglutamic acid residues (Gla), and are therefore referred to as the Gla domain. The Gla domain also contains a short (6 amino acid residue) aromatic stack sequence (residues 40-45 of SEQ ID NO:1). Selective removal of residues 1-44 by chymotrypsin digestion produces Gla-domainless fXa. The serine protease catalytic domain of fXa is located on the C-terminal heavy chain, which has high homology to other serine proteases such as thrombin, trypsin, and activated protein C.

[0026] "Native fXa" or "wild-type fXa" refers to fXa that occurs naturally in plasma or is isolated in its original, unmodified form, which has the biological activity of activating prothrombin, thus promoting the formation of blood clots. The term includes naturally occurring polypeptides isolated from tissue samples, as well as recombinantly produced fXa. "Active fXa" refers to fXa that has the biological activity of activating prothrombin. "Active fXa" may be native fXa or modified fXa that retains procoagulant activity.

[0027] As used herein, "fXa derivative" refers to a modified fXa protein having a modification or substantial deletion of the Gla domain (e.g., deletion of at least 50%, 60%, 70%, 80%, or 90% of amino acid residues 6-39 of the light chain) and a modification such that the fXa derivative has a reduced ability to assemble the prothrombinase complex and has reduced or no catalytic activity compared to the wild-type protein. Nevertheless, the fXa derivative is capable of binding to and / or substantially neutralizing fXa inhibitors, similar to the wild-type protein. Examples of fXa derivatives are provided in WO2009 / 042962 and WO2010 / 117729, and their biological equivalent publications.

[0028] The term "active site" refers to the portion of an enzyme or antibody where a chemical reaction occurs. A "modified active site" is an active site that has been structurally modified to increase or decrease the chemical reactivity or specificity of the active site. Examples of active sites include, but are not limited to, the catalytic domain of human factor X comprising 235-488 amino acid residues and the catalytic domain of human factor Xa comprising 195-448 amino acid residues. Examples of modified active sites include, but are not limited to, the catalytic domain of human factor Xa comprising 195-448 amino acid residues of SEQ ID NO:2 with at least one amino acid substitution at positions Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424.

[0029] Examples of fXa derivatives include "Gla-domainless fXa" or "de-Gla fXa," which refers to fXa or fXa derivatives that do not have a Gla domain and encompasses fXa derivatives having other modification(s) in addition to removal of the Gla domain. Examples of Gla-domainless fXa in the present invention include, but are not limited to, fXa derivatives in which all or part of amino acid residues 1-39 (or 6-39) of SEQ ID NO:1 have been deleted.

[0030] Another example of a fXa derivative is "Gla-deficient fXa," which refers to a fXa or fXa derivative with a reduced number of free side chain γ-carboxyl groups in its Gla domain. Gla-deficient fXa may also have other modifications, similar to Gla-domainless fXa. Gla-deficient fXa includes uncarboxylated fXa, undercarboxylated fXa, and decarboxylated fXa. "Uncarboxylated fXa" or "decarboxylated fXa" refers to a fXa derivative that does not have the γ-carboxy groups of the γ-carboxyglutamic acid residues of the Gla domain, such as fXa in which all of its Gla domain γ-carboxyglutamic acids have been replaced by different amino acids, or fXa in which all of its side chain γ-carboxyls have been removed or masked by means of amination, esterification, or the like. For recombinantly expressed proteins, uncarboxylated fXa may also be referred to as non-carboxylated fXa. "Undercarboxylated fXa" refers to a fXa derivative having a reduced number of γ-carboxy groups in the Gla domain compared to wild-type fXa protein, e.g., a fXa in which one or more, but not all, of its Gla domain γ-carboxyglutamates have been replaced by one or more different amino acids, or a fXa in which at least one, but not all, of its side chain γ-carboxyls have been removed or masked by means such as amination and esterification.

[0031] In some embodiments, the fXa derivative has a deletion of at least amino acid residues 6-39 of the light chain of wild-type fXa (see light chain of SEQ ID NO:1). In some embodiments, the fXa derivative also has a deletion of the EGF1 domain (amino acids 46-84) and / or the EGF2 domain (amino acids 85-128 of the light chain). In some embodiments, the light chain of the fXa derivative contains at least amino acids 129-139 of the wild-type light chain (or 95-105 of SEQ ID NO:3).

[0032] SEQ ID NO:2 (Table 2), also referred to as "andexanet alpha" or simply "andexanet", contains two mutations compared to wild-type fXa. The first mutation is a deletion of amino acid residues 6-39 in the Gla domain of fXa. The second mutation is a mutation of active site residue S379 to an Ala residue. This amino acid substitution corresponds to amino acid 296 in SEQ ID NO:1. Andexanet contains two chains, designated SEQ ID NOs:3 (light chain) and 4 (heavy chain), respectively.

[0033] fXa derivatives also include derivatives that contain mutations, post-translational modifications, and / or changes in the protein during the manufacturing process. For example, in another embodiment, the fXa derivatives may contain modifications to the His residues (to Ala) and / or Asp residues (to Ala / Asn) in the catalytic triad, with or without a Ser379 modification, and deletion or modification of the Gla domain. These modifications provide fXa derivatives that have reduced enzymatic activity but do not compete with fXa in the assembly of the prothrombinase complex.

[0034] In some embodiments, the fXa derivative has a heavy chain with one or two O-linked glycosylations. In some embodiments, the heavy chain may have a C-terminal truncation of one amino acid residue or up to 13, 14, or 15 amino acid residues. In some embodiments, the light chain may have an N-terminal truncation.

[0035] The present disclosure provides various biological equivalents of the disclosed sequences of the above fXa derivatives, or polypeptides having specific sequence identity to these fXa derivatives. In one embodiment, such biological equivalents retain the structural features of these fXa derivatives, i.e., modifications of the active site or heavy chain, and deletion or modification of the Gla domain. In another embodiment, such biological equivalents retain the functional features of these fXa derivatives, i.e., no competition with fXa in the assembly of the prothrombinase complex, and reduced catalytic activity. In another embodiment, such biological equivalents are capable of binding to and / or substantially neutralizing fXa inhibitors. Table 1. Polypeptide sequence of activated human factor X, fXa (SEQ ID NO:1) [Table 1-1] Table 2. Polypeptide sequence of andexanet (SEQ ID NO:2) [Table 2-1]

[0036] The terms "factor Xa inhibitors" or "inhibitors of factor Xa" refer to compounds capable of either directly or indirectly inhibiting the activity of coagulation factor Xa, which catalyzes the conversion of prothrombin to thrombin in vitro and / or in vivo.

[0037] "Direct factor Xa inhibitors" or "direct fXa inhibitors" bind directly to fXa, and non-limiting examples include apixaban, rivaroxaban, edoxaban, betrixaban, NAP-5, rNAPc2, tissue factor pathway inhibitors, DX-DX-9065a (e.g., as described in Herbert, JM, et al, J Pharmacol Exp Ther. 1996 276(3):1030-8), YM-60828 (e.g., as described in Taniuchi, Y., et al, Thromb Haemost. 1998 79(3):543-8), YM-150 (e.g., as described in Eriksson, BI et. al, Blood 2005;106(11), Abstract 1865), TAK-442, PD-348292 (e.g., as described in Pipeline Insight: Antithrombotics - Reaching the Untreated Prophylaxis Market, 2007), otamixaban, LY517717 (e.g., as described in Agnelli, G., et al, J. Thromb. Haemost. 2007 5(4):746-53), GSK913893, razaxaban, or pharma- ceutically acceptable salts thereof, and combinations thereof.

[0038] An "indirect factor Xa inhibitor" or "indirect fXa inhibitor" inhibits fXa activity through one or more other factors. Non-limiting examples of indirect factor Xa inhibitors include enoxaparin, fondaparinux, idraparinux, biotinylated idraparinux, fragmin, tinzaparin, low molecular weight heparin ("LMWH"), and combinations thereof.

[0039] In one embodiment, the factor Xa inhibitor is selected from apixaban, rivaroxaban, edoxaban, betrixaban, enoxaparin, and combinations thereof.

[0040] II. Evaluation of hemostatic effect Anticoagulants serve a market need for the treatment or prevention of unwanted blood clots in patients prone to forming blood clots, such as, for example, patients with coagulation disorders, patients who have limited mobility for a period of time, or patients undergoing medical surgery. However, the main limitations of anticoagulant treatment are the risk of bleeding associated with the treatment and the inability to rapidly reverse anticoagulant activity in the event of an overdose or when emergency surgical procedures are required.

[0041] Andexanet alfa (andexanet) is a recombinant modified human factor Xa decoy protein that can reverse the inhibition of factor Xa and is therefore an effective antidote to factor Xa-based anticoagulant therapeutics.

[0042] In certain circumstances, such as when additional andexanet is needed or when anticoagulant overdose is a concern, it is important to monitor the effectiveness of andexanet, especially in elderly patients. However, monitoring hemostatic efficacy can be difficult. For example, measuring hemostatic efficacy may require serial CT or MRI scans of changes in hematoma volume and / or thickness over 12 hours or more.

[0043] In contrast to the observation that there was no correlation between anti-factor Xa activity and hemostatic effect in the overall population, a surprisingly good correlation was found in patients with intracranial hemorrhage.

[0044] Thus, according to one embodiment of the present disclosure, a method for evaluating hemostatic efficacy in a patient suffering from intracranial bleeding while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor is provided. In some embodiments, the method involves administering to the patient a fXa derivative of the present disclosure, obtaining a blood sample from the patient after the administration, and measuring anti-fXa activity in the sample. The anti-fXa activity can be used to predict hemostatic efficacy in the patient.

[0045] "Intracranial hemorrhage," or ICH, refers to bleeding within the skull or brain. Intracranial hemorrhage is a serious medical emergency because the accumulation of blood within the skull increases intracranial pressure, which can crush delicate brain tissue or restrict the blood supply to the brain tissue. Severe increases in intracranial pressure (ICP) can lead to brain herniation, in which part of the brain protrudes beyond the skull structures. Intracranial hemorrhage occurs when blood vessels within the skull rupture or leak. Intracranial hemorrhage can result from physical trauma or non-traumatic causes. Treatment with anticoagulants as well as disorders involving blood clotting can increase the risk of developing an intracranial hemorrhage.

[0046] In some embodiments, the patient is suffering from an intracranial hemorrhage, hi some embodiments, the patient is suffering from a subtype of intracranial hemorrhage, such as an intra-axial bleed or hemorrhage (cerebral hemorrhage), an extra-axial bleed (including epidural, subdural, and subarachnoid hemorrhage), an epidural hematoma, a subdural hematoma, or a subarachnoid hemorrhage.

[0047] "Intra-axial hemorrhage" is bleeding within the brain itself, i.e., cerebral hemorrhage. Subtypes of this include intraparenchymal hemorrhage, i.e., bleeding within the brain tissue, and intraventricular hemorrhage, bleeding within the brain's ventricles (especially in premature infants). Intra-axial hemorrhage is more dangerous and difficult to treat than extra-axial hemorrhage.

[0048] "Extra-axial hemorrhage" refers to bleeding that occurs within the skull but outside the brain tissue. Examples of extra-axial hemorrhage include epidural hemorrhage, subdural hemorrhage, and subarachnoid hemorrhage.

[0049] "Epidural hemorrhage" (extradural hemorrhage), occurring between the dura mater (the outermost layer of the meninges) and the skull, can result from trauma. Epidural hemorrhage can result from a laceration of an artery, most commonly the middle meningeal artery. Epidural hemorrhage is a dangerous type of injury because it is bleeding from a high-pressure system and can cause a rapid and potentially fatal increase in intracranial pressure. However, epidural hemorrhage is the least common type of meningeal hemorrhage, occurring in only 1% to 3% of cases of head trauma.

[0050] A "subdural hemorrhage" results from the rupture of bridging veins in the subdural space between the dura and arachnoid mater.

[0051] "Subarachnoid hemorrhage", occurring between the arachnoid and leptomeningeal layers, can result from either trauma or rupture of an aneurysm or arteriovenous malformation, as can intraparenchymal hemorrhage. Blood can be found layered throughout the brain along grooves and fissures, or filling the subarachnoid cisterns (most often the anterior cerebral artery of the circle of Willis and the chiasmus cistern due to the presence of the branching points of the above arteries within its space). The classic symptom of subarachnoid hemorrhage is the sudden onset of a severe headache (thunderclap headache). This can be a dangerous entity, requiring urgent neurosurgical evaluation and, sometimes, emergency intervention.

[0052] An "epidural hematoma" (EDH) is a hematoma that rapidly accumulates between the dura and the skull. These patients have a history of head trauma with loss of consciousness, followed by a period of lucidity, followed by loss of consciousness. Clinical onset occurs over a period ranging from minutes to hours. Many of these injuries are associated with laceration of the middle meningeal artery. A CT scan of the head will likely show a "lenticular" or convex lens-shaped extracerebral hemorrhage that does not cross sutures.

[0053] "Subdural hematomas" occur when there is a laceration of the bridging veins between the cerebral cortex and the draining sinuses. Sometimes they are caused by arterial lacerations on the surface of the brain. Acute subdural hematomas are usually also associated with damage to the cerebral cortex, so the prognosis is not as good as epidural hematomas. Clinical features vary with the site of injury and the severity of the injury. Patients may have a history of loss of consciousness, but recover and do not relapse. Clinical onset occurs over several hours. A CT scan of the head shows a crescent-shaped hemorrhage crossing the suture lines and compressing the brain. Significant pressure effects on the brain require craniotomy and surgical removal. Complications include focal neurological deficits depending on the site of the hematoma and brain injury, elevated intracranial pressure leading to brain herniation, and ischemia due to reduced blood supply and seizures.

[0054] "Subarachnoid hemorrhage" is bleeding into the subarachnoid space, the area between the arachnoid membrane and the pia membrane that surrounds the brain. Subarachnoid hemorrhage (SAH) can occur spontaneously, usually from the rupture of a cerebral aneurysm, as well as from head trauma. Symptoms of SAH include a rapid onset of severe headache (thunderclap headache), vomiting, confusion or decreased level of consciousness, and occasionally seizures. Diagnosis is usually confirmed by a CT scan of the head or, rarely, a lumbar puncture. Treatment is by prompt neurosurgical or radiology-guided intervention, along with medications and other treatments that help prevent recurrence and complications of hemorrhage.

[0055] The fXa derivative may be administered intravenously either by bolus, or a combination of bolus and infusion, or subcutaneously. In certain embodiments, about 10 to about 20% of the prescribed amount is administered as a bolus, and the remaining prescribed amount is infused over a period of time until bleeding has substantially ceased. It is contemplated that the infusion may be administered for about 6 hours, or about 6 to about 12 hours, or about 12 to about 24 hours, or 48 hours.

[0056] In some embodiments, the fXa derivative is administered as an intravenous (IV) bolus at a target rate of about 30 mg / min, followed by a continuous infusion for up to 120 minutes. In some embodiments, the initial IV bolus comprises about 400 mg of the fXa derivative. In some embodiments, the initial IV bolus comprises about 800 mg of the fXa derivative. In some embodiments, the subsequent IV infusions are administered at about 4 mg / min for 120 minutes. In some embodiments, the subsequent IV infusions are administered at about 8 mg / min for 120 minutes.

[0057] In one embodiment, the fXa derivative is administered at least about 5 minutes after the onset of bleeding (blood loss). Alternatively, the fXa derivative is administered at least about 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes after the onset of bleeding.

[0058] Anti-fXa activity can be measured by methods known in the art, such as the chromogenic assay disclosed in Example 4 of WO2009 / 042962 and Siegal et al., N Engl J Med 2015;373:2413-24. For example, to measure the anti-fXa activity, purified active fXa, various concentrations of the fXa inhibitors (e.g., apixaban, rivaroxaban, edoxaban, betrixaban, and enoxaparin), and andexanet were incubated in 20 mM Tris, 150 mM NaCl, 5 mM Ca, 10 mM NaCl ... 2+ , and 0.1% bovine serum albumin (BSA). After 20 min of incubation at room temperature, 100 μM Spectrozyme-fXa (chromogenic substrate for factor Xa, Chromogenix) is added to the mixture and the rate of substrate cleavage is continuously monitored by a plate reader at 405 nanometers (nm) for 5 min.

[0059] As demonstrated in the present disclosure, the measured anti-fXa activity can be used to calculate / estimate the hemostatic effect based on the correlation between anti-fXa activity and hemostatic effect. In some embodiments, the estimation takes as input data points the anti-fXa activity both before and after administration of the fXa derivative.

[0060] Post-treatment measurements may be taken at various time points. For example, blood samples may be taken within 20-35 minutes after treatment begins and within 10 minutes after treatment ends. In some embodiments, blood samples may be taken up to 1, 2, 3, 4, 6, 12, 18, 24, 36, 48 hours or more after treatment.

[0061] III. Assessment-Based Medical Interventions The measured anti-fXa activity can also be used directly to aid in direct medical intervention without first estimating hemostatic efficacy. For example, a medical provider can determine, based on the anti-fXa activity, (a) whether to administer a second dose of the fXa derivative to the patient, (b) the likelihood that the patient will suffer a thrombotic event, (c) the minimum time for monitoring or treatment, and / or (d) whether the patient can undergo a surgical procedure.

[0062] In one embodiment, a method is provided for treating intracranial hemorrhage in a patient undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, the method optionally comprising administering to the patient a fXa derivative of the present disclosure, obtaining a blood sample from the patient after said administration, measuring anti-fXa activity in the sample, and administering a second dose of the fXa derivative to the patient if the anti-fXa activity exceeds a predetermined anti-fXa activity threshold.

[0063] The "predetermined anti-fXa activity threshold" can be determined for any particular patient population and / or medical intervention of interest. In some embodiments, the predetermined anti-fXa activity threshold can be a value of 30-50 ng / ml for a direct fXa inhibitor (e.g., apixaban, rivaroxaban, edoxaban, or betrixaban), or 0.25-0.5 international units (IU) / ml when the fXa inhibitor is an indirect inhibitor (e.g., enoxaparin). Anti-fXa activity in these ranges can be considered to indicate a favorable therapeutic outcome in some embodiments.

[0064] Thus, if the measured anti-fXa activity is greater than 30, 40, 50, 60, 70, 80, 90, or 100 ng / ml for a direct fXa inhibitor, further administration of the fXa derivative may be justified and is administered.Similarly, if the measured anti-fXa activity is greater than 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 IU / ml for an indirect fXa inhibitor, further administration of the fXa derivative may be justified and is administered.

[0065] In another embodiment, a method is provided for performing surgery on a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor. In some embodiments, the method involves administering to the patient a fXa derivative of the present disclosure, obtaining a blood sample from the patient after said administration, measuring anti-fXa activity in the sample, and performing surgery on the patient if the anti-fXa activity is below a predetermined anti-fXa activity threshold.

[0066] Here, the "predetermined anti-fXa activity threshold" can also be empirically determined. Nevertheless, in some embodiments, the predetermined anti-fXa activity threshold can be a value of 30-50 ng / ml for direct fXa inhibitors (e.g., apixaban, rivaroxaban, edoxaban, or betrixaban), or 0.25-0.5 IU / ml when the fXa inhibitor is an indirect inhibitor (e.g., enoxaparin). Anti-fXa activity in these ranges can be considered to indicate favorable therapeutic results with fXa derivatives in some embodiments.

[0067] Thus, in some embodiments, the patient may be suitable for surgery if the measured anti-fXa activity is less than 50, 40, 30, 25, 20, 15, 10, or 5 ng / ml for direct fXa inhibitors. If the measured anti-fXa activity is too high, the patient may be at high risk of bleeding during the required surgery. Similarly, the patient may be suitable for surgery if the measured anti-fXa activity is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.15 IU / ml for indirect fXa inhibitors.

[0068] In another embodiment, a method for preventing a thrombotic event in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant treatment with a factor Xa (fXa) inhibitor is also provided. In some embodiments, the method includes administering to the patient a fXa derivative of the present disclosure, obtaining a blood sample from the patient after the administration, measuring anti-fXa activity in the sample, determining the likelihood that the patient will suffer from a thrombotic event based on the anti-fXa activity, and administering anticoagulant therapy to the patient if the patient is determined to be likely to suffer from a thrombotic event.

[0069] In some embodiments, the predetermined anti-fXa activity threshold may be a value of 30-50 ng / ml for a direct fXa inhibitor (e.g., apixaban, rivaroxaban, betrixaban, or edoxaban), or 0.25-0.5 IU / ml if the fXa inhibitor is an indirect inhibitor (e.g., enoxaparin). Anti-fXa activity in these ranges may, in some embodiments, be considered to indicate that the patient is relatively safe from a thrombotic event. If the anti-fXa activity is too low, the patient may be at risk of developing a thrombotic event, and appropriate precautions are therefore recommended.

[0070] Thus, in some embodiments, if the measured anti-fXa activity is less than 50, 40, 30, 25, 20, 15, 10, or 5 ng / ml for direct fXa inhibitors, then a suitable prescription to prevent possible thrombotic events is recommended for the patient. Similarly, if the measured anti-fXa activity is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.15 IU / ml for indirect fXa inhibitors, then the patient should receive a drug therapy to prevent thrombotic events. In some embodiments, the drug therapy is an anticoagulant therapy, such as one using a factor Xa inhibitor.

[0071] In some embodiments, the present disclosure provides a method for monitoring recovery of a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor. In some embodiments, the method involves administering to the patient a fXa derivative of the present disclosure, obtaining a blood sample from the patient after said administration, measuring anti-fXa activity in the sample, and determining a period of time to monitor the patient based on risk.

[0072] In the intracranial hemorrhage population, one third of patients are at risk for hematoma expansion and 5-15% for thrombotic events. The risk is significantly reduced after 2-3 days, so patients are usually discharged at this time. If the anti-fXa activity level is within a safe window after treatment with a fXa derivative, e.g., 30-50 ng / ml for direct fXa inhibitors or 0.25-0.5 IU / ml for indirect fXa inhibitors, patients can be discharged less than 24, 36, or 48 hours after treatment. If the measured anti-fXa activity level is significantly higher or lower than this, further monitoring may be required. As disclosed above, if the anti-fXa activity is too high, perhaps more of the fXa derivative may be administered. If the anti-fXa activity is too low, perhaps more fXa inhibitor may be administered to prevent thrombotic events.

[0073] IV. Factor Xa derivative composition In various embodiments, a factor Xa derivative is administered to a patient.Therefore, a composition comprising the factor Xa derivative is also provided.In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier.In some embodiments, the fXa derivative is conjugated with a moiety capable of extending the circulatory half-life of the derivative.

[0074] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the composition of the present disclosure. Pharmaceutically acceptable carriers include physiological saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salts or electrolytes such as zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylic acid esters, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. Suitable pharmaceutical carriers are preferably selected for the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and are consistent with conventional pharmaceutical practice.

[0075] The formulations of the present disclosure can be manufactured by methods well known in the art, such as conventional granulation, mixing, dissolution, encapsulation, lyophilization, or emulsification processes, among others. The compositions can be manufactured in a variety of forms, including granules, precipitates, or particles, powders, including lyophilized, rotary dried, or spray dried powders, amorphous powders, injection solutions, emulsions, elixirs, suspensions, or solutions. The formulations may optionally include stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0076] In one embodiment, the fXa derivative is lyophilized. Methods for lyophilization of polypeptides are well known in the art. In some embodiments, the fXa derivative is provided in single-use vials containing 100, 200, or 400 mg of lyophilized fXa derivative formulated with the inactive ingredients tromethamine (Tris), L-arginine hydrochloride, sucrose (2% w / v), mannitol (5% w / v), and polysorbate 80 (0.01% w / v) at pH 7.8. It can be reconstituted with sterile water for injection for intravenous (IV) administration.

[0077] Pharmaceutical preparations can also be formulated as suspensions or solutions using sterile liquids such as oils, water, alcohols, and combinations thereof. For oral or parenteral administration, pharma- ceutically suitable surfactants, suspending agents, or emulsifying agents may be added. Suspensions may include oils such as peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. Suspension formulations may also include esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. Suspension formulations may include alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. Ethers such as poly(ethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum, and water may also be used in suspension formulations.

[0078] The formulations are for administration to a mammal, preferably a human. Such formulations of the present disclosure can be administered by various methods, preferably parenteral administration.

[0079] As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. However, if the plasma half-life of the fXa inhibitor being neutralized is long, a continuous infusion or sustained release formulation may be required to bind the fXa derivative to the fXa inhibitor, and such an infusion or formulation will release active fXa before the fXa inhibitor is cleared from the body. Thus, in one embodiment, the formulation is administered to the subject as a bolus. In another embodiment, the formulation is administered by infusion. In another embodiment, the formulation is administered by a combination of bolus and infusion.

[0080] The sterile injectable liquid forms of the compositions of the present disclosure may be aqueous or oleaginous suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable liquid preparations may also be sterile injectable liquids or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any sterile, fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the formulation of injectable liquids, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, particularly in their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharma- ceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers, which are commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compositions may be formulated for parenteral administration by injection, such as a bolus injection, or by continuous infusion. Unit dosage forms for injection may be in ampoules or multi-dose containers.

[0081] In addition to the above dosage forms, pharma- ceutically acceptable excipients and carriers and dosage forms are generally known to those skilled in the art and are included in the present disclosure. It should be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular fXa derivative used; the age, weight, general health, sex and diet, renal and hepatic function of the patient; as well as the time of administration, rate of excretion, drug combinations, the judgment of the treating physician or veterinarian, and the severity of the particular disease being treated. EXAMPLES

[0082] The present disclosure will be further understood by reference to the following examples, which are intended to be purely exemplary of the present disclosure. The present disclosure is not limited in scope by the illustrated embodiments, which are intended to be merely illustrative of a single aspect of the present disclosure. Any method that is functionally equivalent is within the scope of the present disclosure. From the above description and the accompanying figures, various modifications of the present disclosure will become apparent to those skilled in the art in addition to those described herein. Such modifications are within the scope of the appended claims.

[0083] Example 1 Treatment of Factor Xa Inhibitor-Associated Bleeding with Andexanet Alfa In this example, the ability of andexanet alfa to reduce anti-factor Xa activity and restore hemostatic efficacy in patients with acute major bleeding associated with factor Xa inhibitor-based anticoagulant therapy was examined.

[0084] The trial evaluated 352 patients who experienced acute major bleeding within 18 hours of receiving a factor Xa inhibitor. They received a bolus of andexanet followed by a 2-hour infusion. The two primary endpoints were the percent change in antifactor Xa activity after treatment with andexanet and the proportion of patients with excellent or good hemostatic efficacy 12 hours after the end of the infusion, which was determined based on prespecified criteria. Efficacy was evaluated in the subgroup of patients with confirmed major bleeding and baseline antifactor Xa activity of at least 75 ng per milliliter (or ≥ 0.25 IU per milliliter for patients receiving enoxaparin).

[0085] The mean age of the patients was 77 years, and most patients had substantial cardiovascular disease. Bleeding was primarily intracranial (227 patients [64%]) or gastrointestinal (90 patients [26%]). In patients receiving apixaban, median antifactor Xa activity decreased from 149.7 ng per milliliter at baseline to 11.1 ng per milliliter after a bolus of andexanet (a 92% decrease; 95% confidence interval [CI], 91 to 93); in patients receiving rivaroxaban, the median decreased from 211.8 ng per milliliter to 14.2 ng per milliliter (a 92% decrease; 95% CI, 88 to 94). Excellent or good hemostasis occurred in 204 of 249 evaluable patients (82%). Within 30 days, 49 patients (14%) died and 34 (10%) had a thrombotic event. Decreased antifactor Xa activity was not predictive of hemostatic outcome overall, but was somewhat predictive in patients with intracranial hemorrhage.

[0086] In patients with acute major bleeding associated with the use of factor Xa inhibitors, treatment with andexanet significantly reduced anti-factor Xa activity, with 82% of patients achieving excellent or good hemostatic response at 12 hours, as assessed by prespecified criteria.

[0087] method Clinical Trial Design and Monitoring This was a multicenter, prospective, open-label, single-arm trial. The protocol, consent form, and supporting materials were approved by the Institutional Review Boards at each institution.

[0088] Clinical trial population Patients were enrolled at 63 centers in North America and Europe. Patients were eligible if they were at least 18 years of age, had an acute major hemorrhage, and received one of the following within 18 hours: any dose of apixaban, rivaroxaban, or edoxaban, or enoxaparin at a dose of at least 1 mg per kilogram of body weight per day. Acute major hemorrhage was defined by one or more of the following characteristics: potentially life-threatening bleeding with signs or symptoms of hemodynamic compromise (e.g., severe hypotension, poor skin perfusion, confusion, or otherwise unexplained low cardiac output); bleeding associated with a decline in hemoglobin level of at least 2 g per deciliter (or, if baseline hemoglobin levels were unavailable, a hemoglobin level of ≦8 g per deciliter); or bleeding in a critical area or organ (e.g., retroperitoneal, intra-articular, pericardial, epidural, or intracranial hemorrhage or intramuscular hemorrhage with compartment syndrome). Written informed consent was obtained from all patients either directly, by proxy consent from a legally authorized representative, or by emergency consent.

[0089] The main exclusion criteria were elective surgery (excluding minimally invasive surgery or procedures) within 12 hours of treatment with andexanet; intracranial hemorrhage in patients with a Glasgow Coma Scale score less than 7 (scores range from 15 [normal] to 3 [deep coma]) or an estimated hematoma volume greater than 60 cc; expected survival less than 1 month; occurrence of a thrombotic event within 2 weeks prior to enrollment; or use of any of the following medications within the past 7 days: vitamin K antagonists, dabigatran, prothrombin complex concentrates, recombinant factor VIIa, whole blood, or plasma.

[0090] Study Procedures and Data Collection Eligible and consenting patients received a bolus of andexanet over 15 to 30 minutes, followed by a 2-hour infusion of the same drug. The original protocol used the following doses: for all patients who received apixaban and for patients who received rivaroxaban more than 7 hours before the bolus, the bolus dose was 400 mg over 15 minutes and the infusion dose was 480 mg. For patients who received enoxaparin, edoxaban, or a single dose of rivaroxaban within 7 hours or at an unknown time before the bolus, the bolus dose was 800 mg over 30 minutes and the infusion dose was 960 mg. Protocol amendment 4 resulted in minor changes to this dosing schedule.

[0091] Blood samples were taken before and during treatment with andexanet, and 4, 8, and 12 hours after the end of treatment to measure plasma levels of anti-factor Xa activity and the unbound fraction of factor Xa inhibitor.

[0092] For patients with intracranial hemorrhage, a head CT or MRI was scheduled to be performed 2 hours before andexanet treatment, and 1 and 12 hours after the end of andexanet treatment.

[0093] Clinical Trial Evaluation Items The trial had two primary efficacy endpoints: percent change from baseline in anti-factor Xa activity after andexanet treatment and the proportion of patients with excellent or good hemostatic efficacy 12 hours after andexanet infusion, which was assessed by an independent adjudication committee based on prespecified criteria. Primary safety endpoints were death, thrombotic events, and occurrence of antibodies to andexanet or native factors X and Xa. All analyses were censored at 30 days, although some patients completed their final safety visit by 45 days after andexanet treatment.

[0094] statistical analysis The safety analysis included all patients who received andexanet. The efficacy analysis population included only patients who retrospectively met two criteria: baseline anti-factor Xa activity of at least 75 ng per milliliter (or ≥ 0.25 IU per milliliter for patients receiving enoxaparin) and major bleeding as assessed by the adjudication committee and confirmed at the time of submission. Initially, a sample of 250 patients was planned, which would provide 80% power to demonstrate a greater than 50% proportion of patients with "excellent" or "good" hemostatic efficacy. In a protocol amendment, the sample was adjusted to 350 patients to meet new regulatory requirements for sufficient numbers of patients for each factor Xa inhibitor and to include at least 120 patients with intracranial bleeding in the efficacy analysis population.

[0095] Continuous variables are summarized as mean and standard deviation or median and interquartile range, and categorical variables are presented as frequencies. Percentage changes from baseline in anti-factor Xa activity were calculated using two-sided nonparametric confidence intervals around the median. The proportion of patients with effective hemostasis is presented with 95% confidence intervals calculated by binomial test. Associations between hemostatic efficacy and change in anti-factor Xa activity were examined using receiver operating characteristic (ROC) curves. Analyses were performed using SAS software, version 9.4 (SAS Institute).

[0096] result: All 352 patients received andexanet and were followed for at least 30 days or until death. The mean age of the patients was 77 years, and baseline medical history included myocardial infarction in 48 patients (14%), stroke in 69 (20%), and deep vein thrombosis in 67 (19%) (Table 1). Atrial fibrillation was present in 280 patients (80%) and was the primary indication for anticoagulation therapy. 128 patients (36%) received rivaroxaban (median daily dose, 20 mg), 194 patients (55%) received apixaban (median daily dose, 10 mg), 10 patients (3%) received edoxaban (daily dose, 30 mg [five patients] or 60 mg [five patients]), and 20 patients (6%) received enoxaparin. The primary site of bleeding was intracranial in 227 patients (64%) and gastrointestinal in 90 patients (26%). 254 patients (72%) met the criteria for the efficacy analysis population (those judged to meet criteria for bleeding severity and baseline antifactor Xa activity ≥ 75 ng per milliliter or ≥ 0.25 IU per milliliter for patients receiving enoxaparin). Table 1. Baseline patient characteristics * [Table 1-2] [Table 1-3]

[0097] Anti-factor Xa activity In the efficacy population, 134 patients who received apixaban experienced a decrease in median anti-factor Xa activity from 149.7 ng per milliliter at baseline to 11.1 ng per milliliter at the end of the bolus (a 92% decrease; 95% confidence interval [CI], 91 to 93) (Figure 1). In 100 patients who received rivaroxaban, median anti-factor Xa activity decreased from 211.8 ng per milliliter at baseline to 14.2 ng per milliliter at the end of the bolus (a 92% decrease; 95% CI, 88 to 94). In 16 patients who received enoxaparin, median anti-factor Xa activity decreased from 0.48 IU per milliliter at baseline to 0.15 IU per milliliter at the end of the bolus (a 75% decrease; 95% CI, 66 to 79). At 4, 8, and 12 hours after andexanet infusion, median anti-factor Xa activity decreased from baseline by 32%, 34%, and 38%, respectively, for apixaban and 42%, 48%, and 62%, respectively, for rivaroxaban.

[0098] In Figures 1A-1C, the median levels of anti-factor Xa activity at each time point are shown as horizontal lines within boxes. The top and bottom of the boxes indicate the 75th and 25th percentiles, respectively, and the whiskers indicate the 90th and 10th percentiles. Outliers are displayed as points. The andexanet bolus was delivered over a period of 15-30 minutes, and the infusion of the drug lasted for 2 hours. Subsequent time points were measured from the end of the infusion. Plots for 134 patients who received apixaban, 100 who received rivaroxaban, and 16 who received enoxaparin are shown in Figures 1A, 1B, and 1C, respectively (four patients in the efficacy analysis population who received edoxaban are not shown). Values ​​below the graphs indicate the median, the percentage change in median from baseline, and the 95% confidence interval (CI) of this change.

[0099] Hemostatic effect Of 254 patients in the efficacy analysis population, 249 were evaluable for hemostatic efficacy, and 204 (82%) were judged to have excellent or good hemostatic efficacy at 12 hours (95% CI, 77 to 87) (Figure 2). Of these patients, 171 were judged to have excellent hemostatic efficacy and 33 were judged to have good hemostatic efficacy. The proportion of patients with excellent or good efficacy was 85% (95% CI, 76 to 94) for gastrointestinal bleeding and 80% (95% CI, 74 to 86) for intracranial bleeding.

[0100] Figure 2 shows the proportion of patients in the efficacy analysis population who achieved excellent or good hemostatic outcomes at 12 hours as assessed by an independent adjudication committee based on prespecified criteria. The size of the red box is proportional to the number of patients in the analysis subgroups. The study hypothesis was that the proportion of patients with excellent or good hemostatic outcomes would exceed 50% (represented by the vertical dashed line). There were five patients in the efficacy analysis population for whom hemostatic outcomes could not be determined due to administrative reasons. The four patients in the efficacy analysis population who received edoxaban are not shown for drug subgroups.

[0101] Safety evaluation items Thirty-four patients (10%) had thrombotic events during the 30-day follow-up period (Table 2). Of these patients, 11 had events within 5 days of andexanet treatment, 11 had events within 6 to 14 days, and 12 had events within 15 to 30 days. Myocardial infarction occurred in 7 patients, ischemic stroke in 14 patients, deep venous thrombosis in 13 patients, and pulmonary embolism in 5 patients. Two patients had infusion reactions, none of which were severe. No patients developed antibodies to factor X or factor Xa after treatment with andexanet, and no neutralizing antibodies to andexanet occurred. Forty-nine patients (14%) died within 30 days of enrollment; 35 from cardiovascular causes, 12 from noncardiovascular causes, and 2 from unknown causes.

[0102] Resumption of anticoagulation therapy and thrombotic events All patients were immediately discontinued from factor Xa inhibitor therapy at enrollment. Thirty days after andexanet treatment, 220 (62%) of these patients restarted anticoagulation therapy, and 8 (2%) had a thrombotic event after restarting anticoagulation therapy. Of these 220 patients, 100 (28%) restarted oral anticoagulation therapy during follow-up. No thrombotic events occurred after restarting oral anticoagulation therapy (Table 2). Table 2. Thrombotic events and timing of resumption of anticoagulant therapy [Table 2-2] [Table 2-3]

[0103] Biomarker-Efficacy Correlations The relationship between the change in antifactor Xa activity during treatment with andexanet and the assessment of hemostatic efficacy was assessed using a receiver operating characteristic curve. Overall, there was no significant relationship between hemostatic efficacy and the decrease in antifactor Xa activity during treatment with andexanet (Figure 3). For patients with intracranial hemorrhage, the magnitude of the decrease in antifactor Xa activity from baseline to the nadir during treatment was predictive of hemostatic efficacy, with an area under the receiver operating characteristic curve of 0.64 (95% CI, 0.54 to 0.74).

[0104] However, for patients with intracranial hemorrhage, the magnitude of the decline in antifactor Xa activity from baseline to treatment nadir was predictive of hemostatic response, with an area under the receiver operating characteristic curve of 0.64 (95% CI, 0.54 to 0.74) (Figure 3).

[0105] FIG. 3 shows receiver operating characteristic curves for the association between anti-factor Xa activity (measured in nanograms per milliliter) and hemostatic outcome (excellent or good versus poor or absent) in all patients receiving oral factor Xa inhibitors (part A) and in patients with intracranial hemorrhage receiving oral factor Xa inhibitors (part B). Patients were included in the analysis if an assessment of hemostatic outcome was available and if levels of anti-factor Xa activity were available at baseline and during treatment with andexanet (at the end of either the bolus or infusion dose). The dashed line is the reference line showing chance prediction. AUC represents the area under the curve.

[0106] Although reversal of anticoagulation is only one of several clinical factors that determine hemostatic efficacy, it was hypothesized that a decrease in antifactor Xa activity might be a predictor of clinical response. However, this was not true for the overall population, likely due to confounding by variations in bleeding source (venous, arterial), platelet function, type of factor Xa inhibitor, and other patient characteristics. Correlation of antifactor Xa activity with hemostatic efficacy and clinical response may be useful in monitoring clinical response.

[0107] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this specification, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0108] Applicants reserve the right to physically incorporate into this application any and all materials and information from such articles, patents, patent applications, or other physical and electronic documents.

[0109] The disclosure has been described broadly and generally herein. Each of the narrower species and subgeneric groupings falling within the general disclosure also form part of the disclosure. This includes a general description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, whether or not the removed portion is specifically described herein. Other embodiments are within the scope of the following claims. Furthermore, when features or aspects of the disclosure are described in Markush groups, those skilled in the art will recognize that the disclosure is therefore also described in terms of any individual members or subgroups of members of the Markush group. The present invention provides, for example, the following items. (Item 1) 1. A method for evaluating hemostatic efficacy in a patient suffering from intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to a wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in said sample; Including, The method, wherein the anti-fXa activity reflects the hemostatic effect in the patient. (Item 2) 2. The method of claim 1, further comprising estimating the hemostatic effect based on the anti-fXa activity. (Item 3) 3. The method of claim 2, further comprising measuring the anti-fXa activity in a sample obtained from the patient prior to the administration of the fXa derivative. (Item 4) 4. The method of claim 3, wherein said estimating takes as input said anti-fXa activity before and after said administering. (Item 5) 4. The method according to any one of items 1 to 3, wherein said administering comprises a bolus injection of said fXa derivative. (Item 6) 6. The method of claim 5, wherein the administration further comprises an infusion of the fXa derivative after the bolus injection. (Item 7) 7. The method of claim 6, wherein the blood sample is obtained 10 minutes to 4 hours after the administration. (Item 8) 8. The method according to any one of items 1 to 7, wherein the anti-fXa activity is measured using a chromogenic assay. (Item 9) 9. The method of any one of items 1 to 8, further comprising determining based on the anti-fXa activity (a) whether to administer a second dose of the fXa derivative to the patient, (b) the likelihood of the patient suffering from a thrombotic event, (c) the minimum time for monitoring or treatment, or (d) whether the patient can undergo a surgical procedure. (Item 10) 10. The method according to any one of items 1 to 9, further comprising administering to the patient a second dose of the fXa derivative if the anti-fXa activity exceeds a predetermined anti-fXa activity threshold or if the hemostatic effect falls below a predetermined hemostatic effect threshold. (Item 11) 11. The method according to item 10, wherein the predetermined anti-fXa activity threshold is 30 to 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25 to 0.5 IU / ml when the fXa inhibitor is enoxaparin. (Item 12) 1. A method for treating intracranial hemorrhage in a patient undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to a wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; administering a second dose of the fXa derivative to the patient if the anti-fXa activity exceeds a predetermined anti-fXa activity threshold. The method comprising: (Item 13) Item 13. The method according to item 12, wherein the predetermined anti-fXa activity threshold is 30 to 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25 to 0.5 IU / ml when the fXa inhibitor is enoxaparin. (Item 14) 1. A method of performing surgery in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to a wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; The method further comprises performing the surgery in the patient if the anti-fXa activity is below a predetermined anti-fXa activity threshold or if the estimated hemostatic effect based on the anti-fXa activity exceeds a predetermined hemostatic effect threshold. (Item 15) Item 15. The method according to item 14, wherein the predetermined anti-fXa activity threshold is 30 to 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25 to 0.5 IU / ml when the fXa inhibitor is enoxaparin. (Item 16) 1. A method for preventing a thrombotic event in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to a wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; determining the likelihood that the patient will suffer a thrombotic event based on the anti-fXa activity; administering anticoagulant therapy to the patient if the patient is determined to be at high risk of suffering from a thrombotic event; The method comprising: (Item 17) Item 17. The method according to item 16, wherein the patient is determined to have a high likelihood of suffering from a thrombotic event when the anti-fXa activity is less than 30 to 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or less than 0.25 to 0.5 IU / ml when the fXa inhibitor is enoxaparin. (Item 18) 1. A method for monitoring recovery in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: administering to said patient a fXa derivative having at least 50% of amino acid residues 6-39 of the light chain deleted and a modified active site on the heavy chain, said derivative having reduced catalytic activity compared to a wild-type fXa protein and the ability to bind to said factor Xa inhibitor; obtaining a blood sample from said patient after said administration; Measuring anti-fXa activity in the sample; determining a period of time for monitoring said patient based on risk; The method comprising: (Item 19) 19. The method of claim 18, further comprising removing the patient from monitoring if the anti-fXa activity is between 30 and 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or between 0.25 and 0.5 IU / ml when the fXa inhibitor is enoxaparin. (Item 20) 20. The method according to any one of items 1 to 19, wherein the fXa derivative is a two-chain protein comprising a light chain comprising the amino acid sequence of SEQ ID NO: 3 or a first peptide having at least 85% sequence identity with SEQ ID NO: 3, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 or a second peptide having at least 85% sequence identity with SEQ ID NO: 4. (Item 21) 21. The method of claim 20, wherein the fXa derivative is andexanet alpha.

Claims

1. 1. A method for providing an index for evaluating hemostatic efficacy in a patient suffering from intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, comprising: the patient has been administered a fXa derivative comprising a two-chain protein comprising a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4; the method comprising measuring anti-fXa activity in a blood sample obtained from the patient; The method, wherein the anti-fXa activity is reflective of the hemostatic effect in the patient.

2. The method of claim 1 , wherein the anti-fXa activity is used to assess the hemostatic effect.

3. The method of claim 2, wherein the method further comprises measuring the anti-fXa activity in a sample obtained from the patient prior to the administration of the fXa derivative.

4. The method of claim 3 , wherein said estimating takes as input said anti-fXa activity before and after said administering.

5. The method of any one of claims 1 to 3, wherein said administering comprises a bolus injection of said fXa derivative.

6. The method of claim 5 , wherein the administration further comprises an infusion of the fXa derivative after the bolus injection.

7. The method of claim 6, wherein the blood sample is obtained 10 minutes to 4 hours after the administration.

8. The method of any one of claims 1 to 7, wherein the anti-fXa activity is measured using a chromogenic assay.

9. The method according to any one of claims 1 to 8, wherein the anti-fXa activity can be used as an indicator for determining (a) whether to administer the fXa derivative a second time to the patient, (b) whether the patient is likely to suffer from a thrombotic event, (c) whether to determine the minimum time for monitoring or treatment, or (d) whether the patient can undergo a surgical procedure.

10. The method of any one of claims 1 to 9, wherein the patient is a candidate for receiving a second administration of the fXa derivative if the anti-fXa activity exceeds a predetermined anti-fXa activity threshold or if the hemostatic effect falls below a predetermined hemostatic effect threshold.

11. 11. The method of claim 10, wherein the predetermined anti-fXa activity threshold is 30-50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25-0.5 IU / ml when the fXa inhibitor is enoxaparin.

12. 1. A method for providing an indication of whether a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant treatment with a factor Xa (fXa) inhibitor is a candidate for receiving a second administration of a fXa derivative, the patient having received an administration of the fXa derivative comprising a two-chain protein comprising a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4, the method comprising measuring anti-fXa activity in a blood sample obtained from the patient; The method, wherein said anti-fXa activity above a predetermined anti-fXa activity threshold is an indication that said patient is a candidate to receive a second dose of said fXa derivative.

13. 13. The method of claim 12, wherein the predetermined anti-fXa activity threshold is 30-50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25-0.5 IU / ml when the fXa inhibitor is enoxaparin.

14. 1. A method for providing an indication of whether a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant treatment with a factor Xa (fXa) inhibitor is a candidate for surgery, wherein the patient has been administered a fXa derivative comprising a two-chain protein comprising a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4, the method comprising measuring anti-fXa activity in a blood sample obtained from the patient; The method, wherein said anti-fXa activity below a predetermined anti-fXa activity threshold or an estimated hemostatic effect based on said anti-fXa activity above a predetermined hemostatic effect threshold is an indication that said patient is a candidate for undergoing said surgery.

15. 15. The method of claim 14, wherein the predetermined anti-fXa activity threshold is 30-50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or 0.25-0.5 IU / ml when the fXa inhibitor is enoxaparin.

16. 1. A method for providing an indication of whether a thrombotic event can be prevented in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant treatment with a factor Xa (fXa) inhibitor, said patient having been administered a fXa derivative comprising a two-chain protein comprising a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4, said method comprising measuring anti-fXa activity in a blood sample obtained from said patient; wherein the patient is determined to have a high likelihood of suffering from a thrombotic event when the anti-fXa activity is less than 30 to 50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or when the anti-fXa activity is less than 0.25 to 0.5 IU / ml when the fXa inhibitor is enoxaparin.

17. 1. A method for providing an indication of whether to discontinue monitoring for recovery in a patient who has suffered an intracranial hemorrhage while undergoing anticoagulant therapy with a factor Xa (fXa) inhibitor, said patient having been administered a fXa derivative comprising a two-chain protein comprising a light chain comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain comprising the amino acid sequence of SEQ ID NO:4, said method comprising measuring anti-fXa activity in a blood sample obtained from said patient; wherein the anti-fXa activity is 30-50 ng / ml when the fXa inhibitor is apixaban, rivaroxaban, betrixaban, or edoxaban, or the anti-fXa activity is 0.25-0.5 IU / ml when the fXa inhibitor is enoxaparin, is an indication that the patient should be taken off monitoring.

18. The method according to any one of claims 1 to 17, wherein the fXa derivative is andexanet alpha.

Images