Pharmaceutical composition comprising activated blood coagulation factor vii and blood coagulation factor x

Abstract

Provided is a pharmaceutical composition for treating a subject receiving treatment with a direct oral anticoagulant targeting activated blood coagulation factor X. This pharmaceutical composition contains activated blood coagulation factor VII and blood coagulation factor X and is for treating a subject receiving treatment with a direct oral anticoagulant targeting activated blood coagulation factor X.

Description

Pharmaceutical composition containing activated blood coagulation factor VII and blood coagulation factor X

[0001] RELATED APPLICATIONS This application claims the benefit of priority from Application No. 2024-041408, filed with the Japan Patent Office on March 15, 2024. The priority application is incorporated herein by reference in its entirety.

[0002] The present invention relates to a pharmaceutical composition comprising activated blood coagulation factor VII (FVIIa) and blood coagulation factor X (FX) for the treatment of subjects receiving treatment with a direct oral anticoagulant (FXa-DOAC) that targets activated blood coagulation factor X (FXa).

[0003] BACKGROUND OF THE INVENTION FXa-DOACs, such as rivaroxaban, apixaban, and edoxaban, are clinically used for the treatment and prevention of thrombotic events, but may cause acute major bleeding such as intracranial hemorrhage and cerebral hemorrhage.

[0004] FXa-DOAC antagonists are considered a treatment for acute severe bleeding episodes associated with FXa-DOAC. However, prior to their launch, recombinant activated blood coagulation factor VII (rFVIIa) or prothrombin complex (PCC) preparations were used off-label (Non-Patent Document 1). However, rFVIIa preparations have issues with durability and cost, while PCC preparations have issues with safety and dosage. Specifically, the half-life of rFVIIa is 2-3 hours, necessitating frequent administration, which places a significant physical burden on patients and healthcare professionals (Non-Patent Document 2). In addition to frequent administration, their high cost can potentially lead to financial pressure on healthcare (the cost per episode for an adult patient is approximately 2.4 million yen). PCC preparations contain various blood coagulation factors (prothrombin, blood coagulation factor VII, blood coagulation factor IX, and FX) as active ingredients, which poses a risk of hypercoagulation (Non-Patent Document 3). In addition, while the dosage regimen for acute bleeding episodes associated with FXa-DOAC has not been established, the volume of fluid administered with PCC preparations is thought to be large (62.5 to 125 mL per administration assuming a patient weighing 50 kg), placing a physical burden on patients. Subsequently, a recombinant FXa decoy preparation, andexanet alfa, became available as an FXa-DOAC antagonist. Andexanet alfa (recombinant FXa decoy) is a modified, recombinant, inactive form of factor Xa specifically designed to bind to and sequester direct oral anticoagulants that target activated blood coagulation factor X, thereby rapidly reducing anti-factor Xa activity, a measure of the anticoagulant effect of factor Xa inhibitors. In healthy subjects receiving either rivaroxaban, apixaban, or edoxaban, andexanet alfa rapidly reduced both the unbound fraction of factor Xa inhibitors and anti-factor Xa activity while minimizing side effects (Patent Document 1). However, andexanet alfa has problems in terms of administration time and drug cost. Specifically, unlike blood coagulation factor preparations, andexanet alfa is administered intravenously over a continuous period (2 hours), which places a heavy physical burden on patients and medical staff (Non-Patent Document 4).In addition, the cost of andexanet alfa per episode is high (approximately 1.7 million to 3 million yen), which could put strain on the medical economy.

[0005] Special Table 2022-519327

[0006] 2020 Revised Edition of Cardiac Arrhythmia Drug Treatment Guidelines (Published March 13, 2020) Package Inserts for NovoSeven® HI Intravenous Injection 1 mg Syringe / NovoSeven® HI Intravenous Injection 2 mg Syringe / NovoSeven® HI Intravenous Injection 5 mg Syringe (Revised April 2022) Sorensen B, et al. Critical Care. (2011) 15:201 (DOI: 10.1186 / cc9311.) Package Insert for Ondexa® Intravenous Injection 200 mg (Revised May 2023)

[0007] The present invention provides a pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X.

[0008] Accordingly, the present disclosure includes the following: [Item 1] A pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X. [Item 2] The pharmaceutical composition according to Item 1, which is administered at a dose of 15 μg / kg body weight or more as FVIIa. [Item 3] The pharmaceutical composition according to Item 1 or 2, which is administered at a dose of about 150 μg / kg body weight or more as FX. [Item 4] The pharmaceutical composition according to any one of Item 1-3, wherein the direct oral anticoagulant is a pharmaceutical composition comprising rivaroxaban, apixaban, or edoxaban. [Item 5] The pharmaceutical composition according to any one of Item 1-4, which comprises activated blood coagulation factor VII and blood coagulation factor X in a protein weight ratio of 1:10. [Item 6] The pharmaceutical composition according to any one of Item 1-5, which is administered at a dose of about 15 to 120 μg / kg body weight as FVIIa. [Item 7] The pharmaceutical composition of any one of Items 1 to 6, wherein the FVIIa amount is administered at about 15 to 60 μg / kg body weight. [Item 8] The pharmaceutical composition of any one of Items 1 to 7, wherein the FX amount is administered at about 150 to 1200 μg / kg body weight. [Item 9] The pharmaceutical composition of any one of Items 1 to 8, wherein the FX amount is administered at about 150 to 600 μg / kg body weight. [Item 10] The pharmaceutical composition of any one of Items 1 to 9, wherein the FVIIa amount is about 15 μg / kg body weight, about 30 μg / kg body weight, about 45 μg / kg body weight, about 60 μg / kg body weight, about 75 μg / kg body weight, about 90 μg / kg body weight, about 105 μg / kg body weight, about 120 μg / kg body weight or more, preferably about 15 to 120 μg / kg body weight, and more preferably about 15 to 60 μg / kg body weight. [Item 11] The pharmaceutical composition according to any one of Items 1 to 10, wherein the FX amount is about 150 μg / kg body weight, about 300 μg / kg body weight, about 450 μg / kg body weight, about 600 μg / kg body weight, about 750 μg / kg body weight, about 900 μg / kg body weight, about 1050 μg / kg body weight, about 1200 μg / kg body weight or more, preferably about 150 to 1200 μg / kg body weight, more preferably about 150 to 600 μg / kg body weight.[Item 12] The pharmaceutical composition according to any one of Items 1 to 11, comprising activated blood coagulation factor VII and blood coagulation factor X in a protein weight ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15, preferably about 1:10. [Item 13] A method for treating a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X, comprising the step of administering the pharmaceutical composition according to any one of Items 1 to 12 to the subject receiving treatment with the direct oral anticoagulant that targets activated blood coagulation factor X.

[0009] According to the present disclosure, there is provided a pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X.

[0010]

[0033] Figure 1 shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on the prothrombin time in plasma samples collected from patients taking direct oral anticoagulants (rivaroxaban, apixaban, or edoxaban) that target activated blood coagulation factor X. X-axis: activated blood coagulation factor VII and blood coagulation factor X concentrations [μg / ml]. Y-axis: prothrombin time [%]. (High) or (low) next to the measurement item indicates the high or low concentration of the administered DOAC.

[0034] Figure 1 shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on the activated partial thromboplastin time in plasma samples collected from patients taking direct oral anticoagulants (rivaroxaban, apixaban, or edoxaban) that target activated blood coagulation factor X. X-axis: activated blood coagulation factor VII and blood coagulation factor X concentrations [μg / ml]. Y-axis: clotting time [sec]. (High) or (low) next to the measurement item indicates the level of the administered DOAC concentration. The figure shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on the ratio of inhibited thrombin generation (RITG) in plasma samples collected from patients taking direct oral anticoagulants (rivaroxaban, apixaban, or edoxaban) that target activated blood coagulation factor X. X-axis: activated blood coagulation factor VII and blood coagulation factor X concentrations [μg / ml]. Y-axis: ratio calculated by dividing the difference in diluted prothrombin time between the test plasma and normal plasma by the diluted prothrombin time of normal plasma [dimensionless units]. The (high) or (low) next to the measurement item indicates the level of the administered DOAC concentration. This figure shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on prothrombin fragment 1+2 (F1+2) in plasma samples collected from patients taking direct oral anticoagulants (rivaroxaban, apixaban, or edoxaban) that target activated blood coagulation factor X. X-axis: activated blood coagulation factor VII and blood coagulation factor X concentrations [μg / ml]. Y-axis: F1+2 concentration [pmol / l]. The (high) or (low) symbol to the right of the measurement item indicates the high or low concentration of the administered DOAC.This figure shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on fibrin monomer complex (FMC) in plasma samples collected from patients taking direct oral anticoagulants (rivaroxaban, apixaban, or edoxaban) that target activated blood coagulation factor X. X-axis: activated blood coagulation factor VII and blood coagulation factor X concentrations [μg / ml]. Y-axis: FMC concentration [μg / ml]. "High" or "Low" next to the measurement item indicates the high or low concentration of the administered DOAC. This figure shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on D-dimer in plasma samples collected from patients taking direct oral anticoagulants (rivaroxaban, apixaban, or edoxaban) that target activated blood coagulation factor X. X-axis: activated blood coagulation factor VII and blood coagulation factor X concentrations [μg / ml]. Y-axis: D-dimer concentration [μg / ml]. (High) or (low) next to the measurement item indicates the level of the administered DOAC concentration. The upper panel shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on the prothrombin time, activated partial thromboplastin time, or ratio of inhibited thrombin generation (RITG) in plasma samples collected from patients taking rivaroxaban, while the lower panel shows the effect of adding a reversal agent for direct oral anticoagulants. For the upper panel, the x-axis indicates the concentrations of activated blood coagulation factor VII and blood coagulation factor X [μg / ml]. The y-axis of the upper panel, from the left figure, shows the prothrombin time [%], clotting time [sec], and the ratio [dimensionless units] calculated by dividing the difference in diluted prothrombin time between the test plasma and normal plasma by the diluted prothrombin time of normal plasma. In the figure, "high" or "low" indicates the concentration of the administered DOAC. The upper panel shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on the prothrombin time, activated partial thromboplastin time, or ratio of inhibited thrombin generation (RITG) in plasma samples collected from patients taking apixaban, while the lower panel shows the effect of adding a reversal agent for direct oral anticoagulants. For the upper panel, the x-axis indicates the concentration of activated blood coagulation factor VII and blood coagulation factor X [μg / ml].The y-axis in the upper panel shows the prothrombin time [%], clotting time [sec], and the ratio [dimensionless units] calculated by dividing the difference in the diluted prothrombin time between the test plasma and normal plasma by the diluted prothrombin time of normal plasma. High and low in the panel indicate the level of the administered DOAC concentration. The upper panel shows the effect of adding activated blood coagulation factor VII and blood coagulation factor X on the prothrombin time, activated partial thromboplastin time, or ratio of inhibited thrombin generation (RITG) in plasma samples collected from patients taking edoxaban, while the lower panel shows the effect of adding a reversal agent for direct oral anticoagulants. In the upper panel, the x-axis shows the concentration of activated blood coagulation factor VII and blood coagulation factor X [μg / ml]. The y-axis in the upper panel shows the prothrombin time (%), clotting time (sec), and the ratio (dimensionless units) calculated by dividing the difference in the diluted prothrombin time between the test plasma and normal plasma by the diluted prothrombin time of normal plasma. High and low in the figure indicate the concentration of the administered DOAC. The figure shows the effect of adding a coagulation factor preparation (activated blood coagulation factor VII and blood coagulation factor X, activated blood coagulation factor VII, blood coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated blood coagulation factor X on the prothrombin time in a plasma sample in which the blood coagulation activity of activated blood coagulation factor X was inhibited by rivaroxaban. The x-axis shows the concentration of the added substance (μg / ml or units / ml). The y-axis shows the prothrombin time (%). The figure shows the effect of adding coagulation factor concentrates (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated coagulation factor X on the prothrombin time in plasma samples in which the coagulation activity of activated coagulation factor X was inhibited by apixaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: prothrombin time [%].

[0033] Figure 1 shows the effect of adding coagulation factor concentrates (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated coagulation factor X on the prothrombin time in plasma samples in which the coagulation activity of activated coagulation factor X was inhibited by edoxaban. X-axis: Concentration of the added substance [μg / ml or units / ml]. Y-axis: Prothrombin time [%].

[0034] Figure 1 shows the effect of adding coagulation factor concentrates (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated coagulation factor X on the activated partial thromboplastin time in plasma samples in which the coagulation activity of activated coagulation factor X was inhibited by rivaroxaban. X-axis: Concentration of the added substance [μg / ml or units / ml]. Y-axis: Clotting time [sec].

[0033] Figure 1 shows the effect of adding coagulation factor concentrates (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated coagulation factor X on the activated partial thromboplastin time in plasma samples in which the coagulation activity of activated coagulation factor X was inhibited by apixaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: clotting time [sec].

[0034] Figure 1 shows the effect of adding coagulation factor concentrates (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated coagulation factor X on the activated partial thromboplastin time in plasma samples in which the coagulation activity of activated coagulation factor X was inhibited by edoxaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: clotting time [sec].This figure shows the effect of adding a coagulation factor preparation (activated blood coagulation factor VII and blood coagulation factor X, activated blood coagulation factor VII, blood coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated blood coagulation factor X on the ratio of inhibited thrombin generation (RITG) in a plasma sample in which the blood coagulation activity of activated blood coagulation factor X was inhibited by rivaroxaban. X-axis: concentration of the added substance [μg / ml or units / ml]. Y-axis: ratio calculated by dividing the difference in diluted prothrombin time between the test plasma and normal plasma by the diluted prothrombin time of normal plasma [dimensionless units]. This figure shows the effect of adding a coagulation factor preparation (activated blood coagulation factor VII and blood coagulation factor X, activated blood coagulation factor VII, blood coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated blood coagulation factor X on the ratio of inhibited thrombin generation (RITG) in a plasma sample in which the blood coagulation activity of activated blood coagulation factor X was inhibited by apixaban. X-axis: concentration of the added substance [μg / ml or units / ml]. Y-axis: ratio calculated by dividing the difference in diluted prothrombin time between the test plasma and normal plasma by the diluted prothrombin time of normal plasma [dimensionless units]. This figure shows the effect of adding a coagulation factor preparation (activated blood coagulation factor VII and blood coagulation factor X, activated blood coagulation factor VII, blood coagulation factor X, or prothrombin complex) or a reversal agent of a direct oral anticoagulant targeting activated blood coagulation factor X on the ratio of inhibited thrombin generation (RITG) in a plasma sample in which the blood coagulation activity of activated blood coagulation factor X was inhibited by edoxaban. The x-axis represents the concentration of the added substance (μg / ml or units / ml). The y-axis represents the ratio (dimensionless units) calculated by dividing the difference in diluted prothrombin time between the test plasma and normal plasma by the diluted prothrombin time of normal plasma.1 shows the effect of adding a coagulation factor preparation (activated blood coagulation factor VII and blood coagulation factor X, activated blood coagulation factor VII, blood coagulation factor X, or prothrombin complex) on prothrombin fragment 1+2 (F1+2) in a plasma sample in which the blood coagulation activity of activated blood coagulation factor X was inhibited by rivaroxaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: F1+2 concentration [pmol / l]. 1 shows the effect of adding a coagulation factor preparation (activated blood coagulation factor VII and blood coagulation factor X, activated blood coagulation factor VII, blood coagulation factor X, or prothrombin complex) on prothrombin fragment 1+2 (F1+2) in a plasma sample in which the blood coagulation activity of activated blood coagulation factor X was inhibited by apixaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: F1+2 concentration [pmol / l]. 1 shows the effect of adding a coagulation factor preparation (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) on prothrombin fragment 1+2 (F1+2) in a plasma sample in which the coagulation activity of activated coagulation factor X was inhibited by edoxaban. X-axis: concentration of the added substance [μg / ml or units / ml]. Y-axis: F1+2 concentration [pmol / l]. 1 shows the effect of adding a coagulation factor preparation (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) on fibrin monomer complex (FMC) in a plasma sample in which the coagulation activity of activated coagulation factor X was inhibited by rivaroxaban. X-axis: concentration of the added substance [μg / ml or units / ml]. Y-axis: FMC concentration [μg / ml]. This figure shows the effect of adding a coagulation factor preparation (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) to fibrin monomer complex (FMC) in a plasma sample in which the coagulation activity of activated coagulation factor X was inhibited by apixaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: FMC concentration [μg / ml].Figure 1 shows the effect of adding a coagulation factor preparation (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) on fibrin monomer complex (FMC) in a plasma sample in which the coagulation activity of activated coagulation factor X was inhibited by edoxaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: FMC concentration [μg / ml]. Figure 1 shows the effect of adding a coagulation factor preparation (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) on D-dimer in a plasma sample in which the coagulation activity of activated coagulation factor X was inhibited by rivaroxaban. X-axis: concentration of added substance [μg / ml or units / ml]. Y-axis: D-dimer concentration [μg / ml]. Figure 1 shows the effect of adding a coagulation factor preparation (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) on D-dimers in plasma samples in which the coagulation activity of activated coagulation factor X has been inhibited by apixaban. X-axis: Concentration of added substance [μg / ml or units / ml]. Y-axis: D-dimer concentration [μg / ml]. Figure 1 shows the effect of adding a coagulation factor preparation (activated coagulation factor VII and coagulation factor X, activated coagulation factor VII, coagulation factor X, or prothrombin complex) on D-dimers in plasma samples in which the coagulation activity of activated coagulation factor X has been inhibited by edoxaban. X-axis: Concentration of added substance [μg / ml or units / ml]. Y-axis: D-dimer concentration [μg / ml]. The figure shows the effect (blood loss) of administration to rabbits of activated blood coagulation factor VII and blood coagulation factor X, or a reversal agent of a direct oral anticoagulant that targets activated blood coagulation factor X. X-axis: administered substance and its dose [μg / kg or mg / kg]. Y-axis: blood loss [g] 35 minutes after the start of administration. Saline: physiological saline, Riv-: vehicle administration, Riv+: rivaroxaban administration, BYC: activated blood coagulation factor VII and blood coagulation factor X administration. AA: administration of a reversal agent of a direct oral anticoagulant that targets activated blood coagulation factor X.The figure shows the effect (prothrombin time) of administration to rabbits of a reversal agent for a direct oral anticoagulant that targets activated blood coagulation factor VII and blood coagulation factor X, or activated blood coagulation factor X. X-axis: administered substance and its dose [μg / kg or mg / kg]. Y-axis (left side): prothrombin time [sec]. Y-axis (right side): PT ratio [%] calculated by dividing the difference between the prothrombin time before and after drug administration by the prothrombin time before drug administration. Saline: physiological saline; Vehicle: vehicle administration; Riv+: rivaroxaban administration; BYC: activated blood coagulation factor VII and blood coagulation factor X administration. AA: administration of a reversal agent for a direct oral anticoagulant that targets activated blood coagulation factor X. The figure shows the effect (fibrinogen, D-dimer) of administration to rabbits of a reversal agent for a direct oral anticoagulant that targets activated blood coagulation factor VII and blood coagulation factor X, or activated blood coagulation factor X administration. X-axis: administered substance and its dose [μg / kg or mg / kg]. Y-axis: fibrinogen concentration [mg / dl], D-dimer concentration [μg / ml]. Saline: physiological saline, vehicle: vehicle administration, Riv+: rivaroxaban administration, BYC: activated blood coagulation factor VII and blood coagulation factor X administration. AA: administration of a reversal agent for a direct oral anticoagulant that targets activated blood coagulation factor X.

[0011] DETAILED DESCRIPTION OF THE INVENTION Hereinafter, embodiments for carrying out the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0012] In the present disclosure, the direct oral anticoagulant targeting activated blood coagulation factor X is not particularly limited, and can be appropriately selected by a person skilled in the art, as long as it specifically interacts with activated blood coagulation factor X and inhibits the function of activated blood coagulation factor X. For example, examples of direct oral anticoagulants targeting activated blood coagulation factor X include pharmaceutical compositions containing rivaroxaban, apixaban, or edoxaban.

[0013] As used herein, "blood clotting factor," "clotting factor," or "(blood) clotting factor" or the abbreviated form simply "F" before the respective blood clotting factor number (e.g., FVII, FX, etc.) are used synonymously and refer to the respective human blood clotting factor of the human clotting system. Activated clotting factors are abbreviated, for example, as Factor VIIa, Factor Xa. Unactivated clotting factors are abbreviated, for example, as Factor VII, Factor X, etc.

[0014] Byclot is a preparation containing activated blood coagulation factor VII (FVIIa) and blood coagulation factor X (FX) derived from domestically donated plasma, and is indicated for suppressing bleeding tendency in hemophilia patients with inhibitors to FVIII or FIX. Byclot contains FVIIa and FX in a weight ratio of 1:10, specifically, 0.6 mg / ml FVIIa and 6.0 mg / ml FX. By producing FXa in the presence of FVIII or FIX inhibitors, Byclot increases thrombin and fibrin production, enabling hemostatic management in target patients.

[0015] PCC is a preparation containing plasma-derived FII, FVII, FIX, and FX as active ingredients, and is indicated for suppressing bleeding tendency in patients with hemophilia B or in patients receiving vitamin K antagonists. By producing FXa in a state of FIX deficiency or in the presence of a vitamin K antagonist, PCC increases thrombin and fibrin production, enabling hemostatic management in target patients.

[0016] NovoSeven is a recombinant FVIIa preparation indicated for use in suppressing bleeding tendency in hemophilia patients with inhibitors to FVIII or FIX. By producing FXa in the presence of FVIII or FIX inhibitors, it increases thrombin and fibrin production, enabling hemostatic management in target patients. There is no significant difference in specific activity or in vivo half-life between recombinant FVIIa and plasma-derived FVIIa.

[0017] Factor VII is one of the enzymes that make up the blood coagulation cascade. Factor VII is typically activated to factor VIIa by tissue factor-FVIIa complex, FXa, FIXa, or thrombin. Factor VIIa typically acts by cleaving factor X or factor IX at one or two sites (factor X: arg-ile bond, factor IX: arg-ala bond, and then arg-val bond) to generate factor Xa or factor IXa.

[0018] Factor X is one of the enzymes that make up the blood coagulation cascade. Factor X is typically activated to factor Xa by intrinsic or extrinsic tenases. Factor Xa typically acts by cleaving prothrombin at two sites (the arg-thr bond and then the arg-ile bond) to generate activated thrombin.

[0019] In one embodiment, a pharmaceutical composition is provided comprising activated blood coagulation factor VII and blood coagulation factor X for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X. In another embodiment, a treatment method is provided using a combination of activated blood coagulation factor VII and blood coagulation factor X for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X. In another embodiment, a use of activated blood coagulation factor VII and blood coagulation factor X for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X is provided. In yet another embodiment, a use of activated blood coagulation factor VII and / or blood coagulation factor X in the manufacture of a medicament for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X, wherein the medicament comprises activated blood coagulation factor VII and blood coagulation factor X.

[0020] In the present disclosure, a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X refers to a subject in a state affected by the direct oral anticoagulant that targets activated blood coagulation factor X, such as a subject who has received or is receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X, or a subject who has previously received a direct oral anticoagulant that targets activated blood coagulation factor X and who subsequently requires hemostasis, such as due to elective or emergency surgery.

[0021] In this disclosure, the term "treatment" is meant to encompass alleviating, suppressing, or preventing a disorder, disease, or condition, or one or more symptoms associated with that disorder, disease, or condition; or reducing or eradicating the cause of the disorder, disease, or condition itself. For example, as used herein, treatment also includes improving bleeding conditions in patients who are unable, slow to, or have difficulty in achieving hemostasis.

[0022] The administration interval and number of administrations can be determined appropriately by those skilled in the art. The pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X of the present disclosure contains a high dose of FX, and therefore exhibits a longer pharmacological effect than rFVIIa, allowing for a longer administration interval. Therefore, the administration interval of the composition of the present disclosure can be longer than in treatment with rFVIIa, making it possible to reduce the physical burden on patients and medical professionals.

[0023] The FVIIa or FX of the present invention can be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, e.g., intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are included, including, but not limited to, single administration or repeated administration over various time periods, bolus administration, and pulse infusion. Preferably, bolus intravenous administration or continuous intravenous administration is desired.

[0024] A pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X is administered, for example, at a single dose of about 15 μg / kg body weight, about 30 μg / kg body weight, about 45 μg / kg body weight, about 60 μg / kg body weight, about 75 μg / kg body weight, about 90 μg / kg body weight, about 105 μg / kg body weight, about 120 μg / kg body weight, or more, as FVIIa. Preferably, a pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X is administered at a single dose of about 15 to 120 μg / kg body weight, more preferably about 15 to 60 μg / kg body weight, as FVIIa.

[0025] A pharmaceutical composition containing activated blood coagulation factor VII and blood coagulation factor X preferably contains more blood coagulation factor X than activated blood coagulation factor VII from the viewpoint of enzymatic chemical reaction theory (in the blood coagulation reaction between activated blood coagulation factor VII (enzyme) and blood coagulation factor X (substrate), the reaction rate depends on the concentration of the substrate, but since the physiological plasma concentration of blood coagulation factor X is approximately the same as its Michaelis-Menten constant, the blood coagulation reaction can only reach approximately half of the theoretical maximum reaction rate, i.e., the substrate concentration can be said to be insufficient). The pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X contains activated blood coagulation factor VII and blood coagulation factor X in a protein weight ratio of about 1:1 or more, for example, about 1:1 to about 1:15, for example, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, or about 1:15. Preferably, the pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X contains activated blood coagulation factor VII and blood coagulation factor X in a protein weight ratio of about 1:10.

[0026] A pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X is administered, for example, at an FX amount of about 150 μg / kg body weight, about 300 μg / kg body weight, about 450 μg / kg body weight, about 600 μg / kg body weight, about 750 μg / kg body weight, about 900 μg / kg body weight, about 1050 μg / kg body weight, about 1200 μg / kg body weight, or more per dose. Preferably, a pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X is administered at an FX amount of about 150 to 1200 μg / kg body weight, more preferably about 150 to 600 μg / kg body weight per dose.

[0027] The blood coagulation factors used in the present invention may be natural proteins derived from living organisms or recombinant proteins. When derived from living organisms, they may be derived from plasma, which is preferably human plasma.

[0028] As used herein, a "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. A "pharmaceutical composition" as used herein may contain ingredients in the same container, may contain ingredients in separate containers that are mixed at the time of use, or may be in a form in which the ingredients are administered separately.

[0029] In one embodiment of the present invention, the combination of FVIIa and FX improves blood coagulation activity compared to treatment with FVIIa alone, FX alone, PCC, or an FXa-DOAC reversal agent. In one embodiment, an FXa-DOAC reversal agent is a drug that binds to an anticoagulant (FXa-DOAC) that targets factor Xa (FXa) and neutralizes its anticoagulant activity, thereby suppressing excessive bleeding, such as Ondexa (andexanet alfa). In another embodiment of the present invention, the blood coagulation activity of the treatment is improved in a dose-dependent manner with FVIIa and FX. For example, blood coagulation activity can be assessed by measuring prothrombin time (PT), activated partial thromboplastin time (APTT), ratio of inhibited thrombin generation (RITG), or prothrombin fragment 1 + 2 (F1 + 2). For example, blood coagulation activity is improved when the blood clotting time or RITG is shortened or F1+2 is increased. For example, in this specification, blood coagulation activity is improved when the PT is 70% or more, the APTT is 38.0 seconds or less, the RITG is 13.7 or less, or F1+2 is 69 pmol / L or more. Preferably, blood coagulation activity is improved when the PT is 70% to 100%, the APTT is 26.0 to 38.0 seconds, the RITG is -18.2 to 13.7, or F1+2 is 69 to 229 pmol / L.

[0030] In one embodiment of the present invention, the combination of FVIIa and FX may increase thrombin generation in patients. Thrombin is a key enzyme in coagulation, and enhancing thrombin production is known to have various effects on the coagulation system. Thrombin generally converts fibrinogen to fibrin, which then activates platelets. Increased thrombin production can also lead to increased fibrin formation and platelet activation. Furthermore, regular thrombin production can also lead to a decrease in fibrinolytic activity. For example, thrombin production in biological plasma can be measured using enzyme-linked immunosorbent assay (ELISA) to measure prothrombin fragment 1+2 (F1+2). F1+2 is a peptide released from prothrombin during its activation to thrombin, and therefore can be considered indirect evidence of thrombin production.

[0031] In one embodiment of the present invention, the pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X is intended for rapid intravenous administration, and is therefore expected to impose a lower physical burden on patients and medical staff than an FXa-DOAC reversal agent, which requires continuous intravenous administration (over two hours). While rFVIIa requires administration every two to three hours, the pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X allows for longer administration intervals, for example, an additional administration after an eight-hour interval followed by administration every 48 hours or every 36 hours, thereby reducing the physical burden on patients and medical staff. Furthermore, for example, in the case of a patient weighing 50 kg, the required amount of FVIIa per episode will be 0.75 to 6 mg and FX will be 7.5 to 60 mg, and the drug cost is expected to be approximately 268,000 to 1,072,000 yen, which is expected to be cheaper than the drug cost of an FXa-DOAC reversal agent (approximately 1.7 million to 3,000,000 yen), which requires 880 mg or 1,760 mg, or the drug cost of rFVIIa (approximately 2.4 million yen).

[0032] The combination of the present invention is used in advance when bleeding occurs or when bleeding is expected, although the period of use is not particularly limited. For example, the combination of the present invention can be used when bleeding occurs after an injury or during or before surgery.

[0033] In one embodiment of the present invention, the half-life of factor X (40 hours) is longer than that of activated factor VII (2.5 hours), which can reduce the burden of frequent injections by maintaining longer administration intervals compared to activated factor VII alone.

[0034] In one embodiment of the present invention, a pharmaceutical composition containing activated blood coagulation factor VII and blood coagulation factor X can reduce the risk of thrombosis compared to PCC preparations containing prothrombin, which have a relatively high risk of hypercoagulation. Furthermore, because the efficacy of the pharmaceutical composition is exerted in the presence of tissue factor and phospholipids specific to the bleeding site, it is considered unlikely that nonspecific thrombus formation will occur.

[0035] In one embodiment of the present invention, the subject to be treated is preferably, but not limited to, a human. In another embodiment, the subject to be treated is receiving FXa-DOAC. The combination of the present invention can reduce or eliminate the administration of an FXa-DOAC reversal agent.

[0036] As used herein, "about" means within a range of ±10%, preferably ±5%, and more preferably ±2.5%.

[0037] The present invention will be specifically and in detail explained below by showing examples. However, the examples are used to illustrate the present invention and are not intended to limit the present invention.

[0038] Experimental Procedures: Example 1 Coagulation Activity (PT, APTT, RITG, F1+2, FMC, D-Dimer) in Plasma of Patients Taking FXa-DOAC Plasma samples collected from patients taking FXa-DOAC (rivaroxaban, apixaban or edoxaban) were used as an anticoagulation model by FXa-DOAC.

[0039] FVIIa / FX was added to plasma samples at concentrations of 0.375, 0.75, or 1.5 μg / ml. Vicrot (containing 0.6 mg / ml of FVIIa and 6.0 mg / ml of FX; FVIIa / FX may be referred to as vicrot herein) was used as FVIIa / FX. An FXa-DOAC reversal agent (andexanet alfa; AstraZeneca) was added to plasma samples at concentrations of 60 or 120 μg / ml.

[0040] Tables 1 and 2: Tested combinations of additives FVIIa / FX: Activated blood clotting factor VII and blood clotting factor X (byclot) FVIIa / FX: Activated blood coagulation factor VII and blood coagulation factor X (byclot) FXa-DOAC: Direct oral anticoagulant targeting activated blood coagulation factor X

[0041] PT was determined by measuring the time until clotting after activating the extrinsic blood coagulation reaction with a sufficient amount of tissue factor (the device was from Sekisui Medical Co., Ltd., and the reagents were from Sysmex Corporation).

[0042] PT measurement method: PT in biological plasma was measured by the one-stage coagulation assay. Briefly, the extrinsic blood coagulation reaction was activated by adding tissue factor and calcium ions to the plasma sample, and the time (seconds) until fibrin precipitation was measured using a fully automated blood coagulation analyzer. A dilution series of standard plasma (100%, 50%, 25%) was prepared, and the dilution standard curve was calculated and applied to the measured values ​​of the test plasma to express the PT in percentage.

[0043] PT reference value: 70-100%

[0044] The APTT was measured by measuring the time until clotting after activating the intrinsic coagulation reaction using a sufficient amount of phospholipid and an activator (the device was from Sekisui Medical Co., Ltd., and the reagents were from Sysmex Corporation).

[0045] APTT measurement method: APTT in biological plasma was measured by a one-stage coagulation assay. Briefly, the intrinsic blood coagulation reaction was activated by adding phospholipids, an activator, and calcium ions to the plasma sample, and the time (seconds) until fibrin precipitation was measured using an automated blood coagulation analyzer.

[0046] APTT reference value: 26.0 to 38.0 seconds

[0047] RITG was calculated using diluted prothrombin time (dPT) (the device was from Fujirebio Inc., and the reagents were from IL Japan Co., Ltd.).

[0048] RITG measurement method: RITG in biological plasma was measured by a one-stage coagulation assay. Briefly, the intrinsic coagulation reaction was activated by adding a 500-fold diluted PT reagent to the plasma sample, and the time (seconds) until fibrin deposition was measured using an automated blood coagulation analyzer. RITG was calculated using the following formula: RITG = {(dPT of test plasma - dPTc of control) / dPTc of control} x 100

[0049] Reference values ​​for RITG: <-18.2 (hypercoagulable), >13.7 (anticoagulable)

[0050] F1+2 was measured using enzyme-linked immunosorbent assay (ELISA) (reagents provided by Siemens Healthcare Diagnostics).

[0051] F1+2 measurement method: F1+2 in biological plasma was measured by sandwich ELISA using a plate coated with anti-human F1+2 mouse monoclonal antibody. Briefly, F1+2 in the plasma sample was sandwiched between two antibodies (anti-human F1+2 mouse monoclonal antibody and peroxidase-labeled anti-human F1+2 mouse monoclonal antibody) to form a complex. After washing away excess peroxidase-labeled anti-human F1+2 mouse monoclonal antibody, the amount of F1+2 in the sample (pmol / L) was measured by optically measuring the color intensity of the bound labeled enzyme using an absorbance measurement device.

[0052] Standard value for F1+2: 69-229 pmol / l

[0053] FMC was measured using the latex agglutination method (equipment from Fujirebio Inc., reagents from Nissui Pharmaceutical Co., Ltd.).

[0054] FMC measurement method: FMC in biological plasma was measured by agglutination method using anti-fibrin monomer antibody-conjugated latex. Briefly, FMC in the plasma sample was agglutinated by antigen-antibody reaction with anti-fibrin monomer antibody-conjugated latex, and the amount of FMC in the sample (μg / ml) was measured by optically measuring the turbidity caused by agglutination using an absorbance measuring device.

[0055] FMC reference value: <6.1 μg / ml

[0056] D-Dimer was measured using the latex agglutination method (the device was from Sekisui Medical Co., Ltd., and the reagents were from Sekisui Medical Co., Ltd.).

[0057] D-Dimer measurement method: D-Dimer in biological plasma was measured by agglutination method using anti-human D-Dimer mouse monoclonal antibody-sensitized latex. Briefly, FMC in the plasma sample was agglutinated by antigen-antibody reaction with anti-human D-Dimer mouse monoclonal antibody-sensitized latex, and the amount of D-Dimer in the sample (μg / ml) was measured by optically measuring the turbidity caused by agglutination using a spectrophotometer.

[0058] Reference value for D-Dimer: <1.0 μg / ml

[0059] Negative control measurements: Plasma from patients receiving FXa-DOAC (rivaroxaban, apixaban, or edoxaban) without added medication Analysis of plasma from patients receiving FXa-DOAC without added medication revealed, as expected, a state of coagulation inhibition (referred to as the value on the Y-axis of each graph), indicating the physiological basis for the anticoagulated state in patients receiving FXa-DOAC.

[0060] Addition of FVIIa / FX: A dose-dependent increase in PT (%) was observed, regardless of the type or concentration of FXa-DOAC added (Figure 1). Similarly, a dose-dependent decrease in APTT was observed, regardless of the type or concentration of FXa-DOAC added (Figure 2). Similarly, a dose-dependent decrease in RITG was observed, regardless of the type or concentration of FXa-DOAC added (Figure 3). Similarly, except for edoxaban, a dose-dependent increase in F1+2 was observed, regardless of the type or concentration of FXa-DOAC added (Figure 4). No dose-dependent increase in FMC was observed, regardless of the type or concentration of FXa-DOAC added (Figure 5). Similarly, no tendency for D-Dimer to increase in a dose-dependent manner with added FVIIa / FX was observed, regardless of the type or concentration of FXa-DOAC (FIG. 6).

[0061] Comparison with negative control Regardless of the type or concentration of FXa-DOAC, compared with the negative control, the FVIIa / FX-added samples tended to show shorter APTT and RITG and increased PT (%) and F1+2 (Figures 1, 2, 3, 4).

[0062] Comparison with Control Substances: FVIIa / FX significantly improved PT (%) compared to FXa-DOAC reversal agents at high concentrations, regardless of the type of FXa-DOAC. Furthermore, FVIIa / FX significantly improved APTT compared to FXa-DOAC reversal agents at high concentrations of FXa-DOAC (apixaban, edoxaban). Furthermore, FVIIa / FX significantly improved RITG compared to FXa-DOAC reversal agents at high concentrations of FXa-DOAC (apixaban) (Figures 7-9). Meanwhile, FVIIa / FX significantly improved various measurement parameters (PT, PT-INR, APTT) compared to FXa-DOAC reversal agents at low concentrations, regardless of the type of FXa-DOAC (Figures 7-9).

[0063] Example 2 Coagulation Activity (PT, APTT, RITG, F1+2, FMC, D-Dimer) in Plasma Containing FXa-DOAC Commercially available plasma samples containing FXa-DOAC (rivaroxaban, apixaban, or edoxaban) (HYPHEN BioMed) were used as an anticoagulation model by FXa-DOAC.

[0064] FVIIa / FX was spiked into plasma samples at concentrations of 0.75, 1.5, or 3.0 μg / ml. FVIIa was spiked into plasma samples at concentrations of 0.75, 1.5, or 3.0 μg / ml. FX was spiked into plasma samples at concentrations of 7.5, 15, or 30 μg / ml. PCC (CSL Behring) was spiked into plasma samples at a concentration of 0.875 units / ml. FXa-DOAC reversal agent (AstraZeneca) was spiked into plasma samples at a concentration of 60 or 120 μg / ml.

[0065] Table 3: Tested dosage combinations FVIIa / FX: Activated blood coagulation factor VII and blood coagulation factor X (byclot) PCC: Prothrombin complex concentrate FXa-DOAC: Oral anticoagulant targeting activated blood coagulation factor X

[0066] Negative Control Measurement: Plasma Containing FXa-DOAC (Rivaroxaban, Apixaban, or Edoxaban) Without Added Drug Analysis of plasma containing FXa-DOAC without added drug revealed, as expected, a state of coagulation inhibition, indicating the physiological basis for the anticoagulant state in patients taking FXa-DOAC.

[0067] A dose-dependent increase in PT (%) was observed with added FVIIa / FX, regardless of the type or concentration of FXa-DOAC (Figures 10-12). Similarly, a dose-dependent decrease in APTT was observed with added FVIIa / FX, regardless of the type or concentration of FXa-DOAC (Figures 13-15). Similarly, a dose-dependent decrease in RITG was observed with added FVIIa / FX, regardless of the type or concentration of FXa-DOAC (Figures 16-18). Similarly, a dose-dependent increase in F1+2 was observed with added FVIIa / FX, regardless of the type or concentration of FXa-DOAC (Figures 19-21). No dose-dependent increase in FMC was observed with added FVIIa / FX, regardless of the type or concentration of FXa-DOAC (Figures 22-24). Similarly, no tendency for D-Dimer to increase in a dose-dependent manner with added FVIIa / FX was observed, regardless of the type or concentration of FXa-DOAC (FIGS. 25-27).

[0068] Comparison with negative control Regardless of the type or concentration of FXa-DOAC, compared with the negative control, the FVIIa / FX-added samples tended to show shorter APTT and RITG and increased PT (%) and F1+2 (Figures 10-21).

[0069] Comparison with Control Substances: FVIIa / FX significantly improved various measurement parameters (PT, APTT, RITG, F1+2) compared with FVIIa alone at high FXa-DOAC concentrations. The FVIIa measurement parameters (PT, APTT, RITG, F1+2) showed similar behavior to FVIIa / FX, but the improvement was weaker at high FXa-DOAC concentrations than FVIIa / FX. The changes observed with FVIIa were even more gradual with FX. The reason for the weak improvement with FX is considered to be due to the assumed mechanism of action against FXa-DOAC: Ondexa is a recombinant FXa decoy that binds to FXa-DOAC in a 1:1 ratio, neutralizing its efficacy. For Ondexa to exert its efficacy, a large amount of decoy protein must be administered. When FX is used as an FXa-DOAC antagonist, its mechanism of action is neutralization, similar to Ondexa, and therefore the dose of FX has a significant impact on efficacy. Since the FX concentration of FVIIa / FX shown in the examples is less than approximately 25% of the clinical Ondexa concentration, it was thought that FX alone hardly exerts any FXa-DOAC neutralizing effect. Compared to FVIIa / FX, PCC showed higher values ​​only in F1+2. Meanwhile, the other measurement items (PT, APTT, RITG) did not show the same improvement effect as FVIIa / FX, and the changes were also minimal. While FXa-DOAC neutralizers showed improvement effects in all measurement items, the improvement effects were weaker than those of FVIIa / FX.

[0070] Example 3 Blood loss, PT, D-Dimer, and fibrinogen concentration in a rivaroxaban-administered rabbit hepatic lobar hemorrhage model. Rivaroxaban-administered rabbits were used as an animal model of anticoagulation with FXa-DOAC.

[0071] Rivaroxaban was administered to rabbits (Japanese white, female) at a dose (0.5 mg / kg) that exerts an anticoagulant effect.

[0072] Table 4: Tested dosage combinations Riv: Rivaroxaban FVIIa / FX: Activated blood coagulation factor VII and blood coagulation factor X (byclot) FXa-DOAC: Direct oral anticoagulant targeting activated blood coagulation factor X

[0073] Blood loss measurement method: Thirty minutes after administration of rivaroxaban or vehicle to rabbits, liver lobes (right lobe, medial left lobe) were excised, and five wounds, 1 cm long and 2 mm deep, were created in each lobe using a scalpel. Three minutes later, FVIIa / FX, FXa-DOAC neutralizer, or saline was continuously administered for 5 minutes using an infusion pump (36 mL / kg / h), and blood was absorbed into tared gauze from the start of administration. Blood was absorbed for a total of 35 minutes from the start of administration, and the weight of the blood was recorded as the blood loss.

[0074] PT was determined by measuring the time until clotting after activating the extrinsic blood coagulation reaction with a sufficient amount of tissue factor (the apparatus was from Sysmex Corporation, and the reagents were from IL Japan Co., Ltd.).

[0075] PT measurement method: PT in biological plasma was measured by a one-stage coagulation method. Briefly, the extrinsic blood coagulation reaction was activated by adding tissue factor and calcium ions to the plasma sample, and the time (seconds) until fibrin precipitation was measured using a fully automated blood coagulation measuring device. Only the FVIIa / FX sample was diluted with rivaroxaban-containing rabbit plasma before measurement. The PT ratio was calculated using the following formula: PT ratio = (PT before drug administration - PT after drug administration) / PT before drug administration x 100

[0076] PT reference value: 9 to 12 seconds

[0077] Fibrinogen was measured using the thrombin time method (apparatus and reagents from Sysmex Corporation).

[0078] Fibrinogen measurement method: Fibrinogen in biological plasma was measured by the thrombin time method based on the Clauss method. Briefly, a coagulant (thrombin reagent) was added to the plasma sample, and the time it took for the plasma to clot was measured. A calibration curve was created using fibrinogen standard plasma, and the fibrinogen content (mg / dl) of the plasma sample was calculated from the measured clotting time.

[0079] Fibrinogen reference value: 170-310 mg / dl

[0080] D-Dimer was measured using an antigen-antibody reaction (analysis software from Fujifilm Wako Pure Chemical Industries, Ltd., reagents from LifeSpan BioSciences, Inc.).

[0081] D-Dimer measurement method: D-Dimer in biological plasma was measured by sandwich ELISA. Briefly, D-Dimer in a plasma sample was captured with a target-specific antibody immobilized on a solid phase, and then bound to a detection antibody. Next, the D-Dimer was bound to an enzyme-labeled antibody that binds to the bound antibody, and then reacted with a chromogenic substrate. The intensity of the color development was optically measured using a spectrophotometer to determine the amount of D-Dimer (ng / ml) in the sample.

[0082] Reference value for D-Dimer: 70.5 to 617.3 ng / ml

[0083] Positive control: Rabbits administered with vehicle containing no rivaroxaban or saline

[0084] Negative control measurements: rivaroxaban and saline treated rabbits Analysis of rivaroxaban treated rabbit plasma revealed, as expected, an inhibited state of coagulation, indicating the physiological basis for the anticoagulated state in patients taking FXa-DOAC.

[0085] A dose-dependent decrease in bleeding volume was observed with FVIIa / FX (Figure 28). Similarly, a dose-dependent decrease in PT was observed with FVIIa / FX (Figure 29). No significant differences were observed in fibrinogen and D-Dimer levels between FVIIa / FX groups (Figure 30).

[0086] Comparison with the negative control: Compared with the negative control, the high-dose FVIIa / FX (1200 μg / kg) group showed lower bleeding volume (Figure 28). Furthermore, compared with the negative control, the PT of FVIIa / FX was lower at all doses (Figure 29). Furthermore, compared with the negative control, the fibrinogen and D-Dimer levels of FVIIa / FX were similar (Figure 30).

[0087] Comparison with the Control: Compared to the control, the high dose FVIIa / FX (1200 μg / kg) group showed a lower amount of bleeding (FIG. 28).

[0088] A pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X for the treatment of a subject receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X is provided.

Claims

1. A pharmaceutical composition comprising activated blood coagulation factor VII and blood coagulation factor X for the treatment of subjects receiving treatment with a direct oral anticoagulant that targets activated blood coagulation factor X.

2. The pharmaceutical composition according to claim 1, wherein the amount of FVIIa administered is about 15 μg / kg body weight or more.

3. The pharmaceutical composition according to claim 1 or 2, wherein the FX amount is administered at about 150 μg / kg body weight or more.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the direct oral anticoagulant is a pharmaceutical composition containing rivaroxaban, apixaban, or edoxaban.

5. A pharmaceutical composition according to any one of claims 1 to 4, comprising activated blood coagulation factor VII and blood coagulation factor X in a protein weight ratio of 1:

10.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the amount of FVIIa administered is about 15 to 120 μg / kg body weight.

7. The pharmaceutical composition according to any one of claims 1 to 6, which is administered in an amount of FVIIa of about 15 to 60 μg / kg body weight.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the FX amount is administered at about 150 to 1200 μg / kg body weight.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the FX amount is administered at about 150 to 600 μg / kg body weight.

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