Blood coagulation factor X activator and preparation thereof for treating hemorrhagic diseases

Abstract

The present application relates to blood coagulation factor X activator (FX activator, e.g., RVV-X) compositions (e.g., pharmaceutical compositions) comprising sucrose, histidine, polysorbate 20, and mannitol, and the use of FX activator (e.g., RVV-X) or compositions thereof (e.g., pharmaceutical compositions) for the treatment of hemorrhagic diseases (e.g., hemophilia) in subjects (e.g., humans).

Description

[0001] Submit sequence listing as ASCII TEXT file

[0002] The contents of the following submitted ASCII TEXT text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Text Name: EN_202206200179_SEQLIST, Record Date: 2022.06.20, Size: 9KB). Technical Field

[0003] The present application relates to compositions (e.g., pharmaceutical compositions) of coagulation factor X activators (FX activators), and the use of FX activators or compositions thereof (e.g., pharmaceutical compositions) for treating bleeding disorders (e.g., hemophilia) in individuals (e.g., humans). Background Art

[0004] Hemostasis is a complex physiological process that stops bleeding. Under normal circumstances, after tissue damage, platelets, plasma proteins, blood vessels and endothelial cells immediately participate in the hemostasis process and quickly form a blood clot. In the coagulation cascade reaction, certain plasma proteins (or coagulation factors) are "cascaded" activated in turn by another coagulation factor that has been activated in advance, resulting in the rapid generation of thrombin.

[0005] Coagulation factor X (FX) is a vitamin K-dependent serine protease. Its active form (FXa) is the only physiological activator of prothrombin in the body and plays a key role in the common coagulation pathway. Coagulation factor X activator (FX activator) can specifically activate FX, fully expose the active site, and generate FXa. FXa forms a prothrombin complex with activated platelets, FVa, and calcium ions at the site of injury, thereby increasing thrombin generation. Thrombin activates platelets and factors V and VIII at the site of injury, and forms a hemostatic plug by converting fibrinogen into fibrin, thereby achieving the purpose of stopping bleeding for bleeding patients. FX activators have great potential application value in surgical wound bleeding, internal bleeding diseases, and coagulation system diseases such as hemophilia.

[0006] The earliest discovered and most active FX activator was extracted from the venom of the round-spotted viper (Vipera russelli, Daboia russellii siamensis), referred to as RVV-X. RVV-X is a metalloproteinase with a relative molecular weight (MW) of 92880D, consisting of a heavy chain with a molecular weight of 57600D and two light chains with molecular weights of 16400D and 19400D, respectively, through disulfide bonds.

[0007] Since McFarlane first discovered the FX activator RVV-X in the venom of the round-spotted viper (RG MacFarlane, "The coagulant action of Russell's viper venom; the use of antivenomin defining its reaction with a serum factor," Br J Haematol. 1961; 7: 496-511), FX activators have also been found in many other snake venoms. FX activators are commonly found in viper and rattlesnake venoms. Substances that can activate FX have also been found in a few cobra venoms. In addition, FX activators can also be obtained through bioengineering technology, by expressing recombinant human FX activators in cells and isolating and purifying recombinant human FX activators from cell culture fluid.

[0008] The biological activity of FX activators depends on their secondary and tertiary structures. Therefore, it is particularly important to maintain their biological activity during preparation, purification, storage and administration.

[0009] Usually, thrombin and FX activator are prepared into a composition together and used as a hemostatic drug, see CN1520880A, CN1727002A, CN108785665A and CN1810258A. As a macromolecular protein, if FX activator is used as a single active ingredient to prepare a FX activator pharmaceutical composition, it is easy to denature and is not conducive to long-term storage. Human serum albumin (HSA) is a commonly used protective agent, widely used in biological products, as a stabilizer for protein. However, due to the easy allergic reaction and potential risk of viral contamination caused by HSA, it has been gradually eliminated.

[0010] Bleeding disorders include congenital bleeding disorders and acquired bleeding disorders, such as those caused by a lack of coagulation factors (eg, FX) or by the presence of acquired inhibitors of coagulation factors (eg, autoantibodies).

[0011] Hemophilia is usually an inherited disease that impairs the body's ability to make blood clots to stop bleeding. Hemophilia A (HA) and hemophilia B (HB) are the two main types of hemophilia. Hemophilia A is caused by low levels of clotting factor VIII (FVIII), while hemophilia B is caused by low levels of clotting factor IX (FIX). Hemophilia C (HC) is another type of hemophilia caused by low levels of factor XI (FXI). Parahemophilia is caused by low levels of factor V (FV). Acquired hemophilia may be associated with cancer, autoimmune diseases, or pregnancy.

[0012] Factor replacement therapy is commonly used to treat bleeding episodes (BEs) in patients with HA and HB; however, 20%-30% of patients with HA and 5% of patients with HB develop exogenous inhibitors of FVIII or FIX. HA and HB with inhibitors (e.g., autoantibodies) are difficult to control. In these patients, replacement of the defective factor may not achieve hemostasis (depending on the titer of the inhibitor), so administration of bypassing agents may be necessary. Although bypassing therapy agents, such as activated thrombin complex concentrate (aPCC, e.g., ), emicizumab-kxwh (e.g., ) and activated recombinant human coagulation factor VII (rFVIIa, e.g., RT) have been widely used as hemostatic therapeutic agents, but they still have disadvantages and limitations. For example, aPCC is not available in China, and the trace amount of FVIII in aPCC may induce a recall response to FVIII; and due to the high price, rFVIIa and emicizumab are not widely used.

[0013] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0015] On the one hand, the present application provides a pharmaceutical composition comprising: i) a coagulation factor X activator (FX activator, e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL; ii) a stabilizer in an amount of 2 mg / ml to 100 mg / ml; iii) a buffer in an amount of 0.1 mg / ml to 50 mg / ml; iv) a surfactant in an amount of 0.001% (w / v) to 0.1% (w / v) and v) an excipient in an amount of 1 mg / ml to 100 mg / ml; wherein the pH of the pharmaceutical composition is 6.0 to 8.0.

[0016] In some embodiments, as described in any of the pharmaceutical compositions described above, the FX activator is RVV-X. In some embodiments, the RVV-X comprises: a) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2, or a sequence having at least 80% sequence identity with SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5, or a sequence having at least 80% sequence identity with SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; or c) a mixture of a) and b). In some embodiments, the RVV-X is isolated from the venom of Daboia russellii siamensis. In some embodiments, the purity of RVV-X in the pharmaceutical composition is at least 95% (e.g., at least 96%, 97%, 98%, 99% or 100%).

[0017] In some embodiments, as described in any of the pharmaceutical compositions described above, the content of the FX activator (eg, RVV-X) is 1 U / mL to 100 U / mL, such as 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL.

[0018] In some embodiments, as described above in any pharmaceutical composition, the stabilizer comprises one or both of sucrose and trehalose. In some embodiments, the stabilizer is sucrose. In some embodiments, the content of the stabilizer (e.g., sucrose) is 2 mg / ml to 60 mg / ml, such as 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml.

[0019] In some embodiments, as described above in any pharmaceutical composition, the buffer comprises one or both of histidine and arginine. In some embodiments, the buffer is histidine. In some embodiments, the content of the buffer (e.g., histidine) is 2 mg / ml to 20 mg / ml, such as 2 mg / ml to 15 mg / ml, 3 mg / ml to 5 mg / ml, or 3 mg / ml.

[0020] In some embodiments, as described above in any pharmaceutical composition, the surfactant comprises one or both of polysorbate and poloxamer. In some embodiments, the surfactant is selected from one or more of polysorbate 20, polysorbate 80 and poloxamer 188. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the content of the surfactant (e.g., polysorbate 20) is 0.005% (w / v) to 0.05% (w / v), such as 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v), or 0.02% (w / v).

[0021] In some embodiments, as described above in any pharmaceutical composition, the excipient is mannitol. In some embodiments, the content of the excipient is 10 mg / ml to 60 mg / ml, such as 30 mg / ml to 60 mg / ml, or 40 mg / ml.

[0022] In some embodiments, as described above in any pharmaceutical composition, the pH of the pharmaceutical composition is 6.3 to 7.3, such as 6.8 to 7.0, or 6.85.

[0023] In some embodiments, as any of the pharmaceutical compositions described above, the pharmaceutical composition further comprises an antioxidant, such as methionine. In some embodiments, the content of the antioxidant is 0.01 mg / ml to 1 mg / ml, such as 0.05 mg / ml to 1 mg / ml.

[0024] In some embodiments, as any of the pharmaceutical compositions described above, the pharmaceutical composition further comprises a calcium salt, such as calcium chloride. In some embodiments, the content of the calcium salt is 0.1 mg / ml to 10 mg / ml.

[0025] In some embodiments, any pharmaceutical composition as described above, the pharmaceutical composition comprises: i) RVV-X at a content of 10 U / mL; ii) sucrose at a content of 30 mg / ml; iii) histidine at a content of 3 mg / ml; iv) polysorbate 20 at a content of 0.02% (w / v); and v) mannitol at a content of 40 mg / ml; wherein the pH value of the pharmaceutical composition is 6.85.

[0026] In some embodiments, any of the pharmaceutical compositions described above is lyophilized. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the pharmaceutical composition is stable under the following conditions: i) after reconstitution, placed at 25°C for at least 4 hours; ii) placed at 2-8°C for at least 3 hours; and / or iii) placed at 25°C accelerated stability conditions for at least 3 hours. In some embodiments, after storage at 25°C accelerated stability conditions for about 6 months and then reconstituted, the pharmaceutical composition contains less than about 5% of insoluble particles with a diameter greater than 10 μm.

[0027] In another aspect, the present application provides a method of treating a bleeding disorder (eg, hemophilia, such as hemophilia A or hemophilia B) in an individual (eg, a human), comprising administering to the individual an effective amount of any one of the pharmaceutical compositions described above.

[0028] On the other hand, the present application provides a method for treating a bleeding disorder (e.g., hemophilia, such as hemophilia A or hemophilia B) in an individual (e.g., a human), comprising administering an effective amount of a FX activator (e.g., RVV-X) to the individual, wherein the FX activator is administered at a dose of 0.01U / kg to 0.48U / kg. In some embodiments, the FX activator is administered at a dose of 0.08U / kg to 0.48U / kg, such as 0.01U / kg to 0.16U / kg, 0.08U / kg to 0.16U / kg, or 0.16U / kg. In some embodiments, the FX activator is administered once. In some embodiments, the FX activator is administered up to 6 times. In some embodiments, the FX activator is administered 4 times. In some embodiments, the FX activator is administered once every 4 hours (q4h) to every 8 hours (q8h), such as q4h. In some embodiments, the FX activator is administered once at a dose of 0.01U / kg to 0.48U / kg. In some embodiments, the FX activator is administered at a dose of 0.16 U / kg q4h, for a maximum of 6 administrations. In some embodiments, the FX activator is administered at a dose of 0.16 U / kg q4h, for a maximum of 4 administrations.

[0029] In some embodiments, according to any of the methods described above, the FX activator is RVV-X. In some embodiments, the RVV-X comprises: a) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2, or a sequence having at least 80% sequence identity with SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5, or a sequence having at least 80% sequence identity with SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; or c) a mixture of a) and b). In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the purity of RVV-X in the pharmaceutical composition is at least 95% (e.g., at least 96%, 97%, 98%, 99% or 100%).

[0030] In some embodiments, any of the methods described above, wherein the FX activator is included in a pharmaceutical composition. In some embodiments, the concentration of the FX activator in the pharmaceutical composition is 1U / mL to 200U / mL, such as 1U / mL to 100U / mL, 5U / mL to 100U / mL, 5U / mL to 50U / mL or 10U / mL. In some embodiments, the pharmaceutical composition further comprises one or more stabilizers, buffers, surfactants and excipients. In some embodiments, the stabilizer comprises one or both of sucrose and trehalose. In some embodiments, the stabilizer is sucrose. In some embodiments, the content of the stabilizer (e.g., sucrose) is 2mg / ml to 100mg / ml, such as 2mg / ml to 60mg / ml, 15mg / ml to 60mg / ml, 30mg / ml to 50mg / ml or 30mg / ml. In some embodiments, the buffer comprises one or both of histidine and arginine. In some embodiments, the buffer is histidine. In some embodiments, the content of the buffer (e.g., histidine) is 0.1 mg / ml to 50 mg / ml, such as 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 5 mg / ml or 3 mg / ml. In some embodiments, the surfactant comprises one or both of polysorbate and poloxamer. In some embodiments, the surfactant is one or more selected from polysorbate 20, polysorbate 80 and poloxamer 188. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the content of the surfactant (e.g., polysorbate 20) is 0.001% (w / v) to 0.1% (w / v), such as 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v) or 0.02% (w / v). In some embodiments, the excipient is mannitol. In some embodiments, the content of the excipient is 1mg / ml to 100mg / ml, such as 10mg / ml to 60mg / ml, 30mg / ml to 60mg / ml or 40mg / ml. In some embodiments, the pH value of the pharmaceutical composition is 6.0 to 8.0, such as 6.3 to 7.3, 6.8 to 7.0 or 6.85. In some embodiments, the pharmaceutical composition further comprises an antioxidant, and the antioxidant is methionine. In some embodiments, the content of the antioxidant is 0.01mg / ml to 1mg / ml, such as 0.05mg / ml to 1mg / ml. In some embodiments, the pharmaceutical composition further comprises a calcium salt. In some embodiments, the calcium salt is calcium chloride. In some embodiments, the content of the calcium salt is 0.1mg / ml to 10mg / ml.In some embodiments, the pharmaceutical composition comprises: i) RVV-X at 10 U / mL; ii) sucrose at 30 mg / ml; iii) histidine at 3 mg / ml; iv) polysorbate 20 at 0.02% (w / v); and v) mannitol at 40 mg / ml; wherein the pH value of the drug combination is 6.85. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the pharmaceutical composition is stable under the following conditions: i) after reconstitution, placed at 25°C for at least 4 hours; ii) placed at 2-8°C for at least 3 hours; and / or iii) placed at 25°C accelerated stability conditions for at least 3 hours. In some embodiments, after storage at 25°C accelerated stability conditions for about 6 months and then reconstituted, the pharmaceutical composition contains less than about 5% of insoluble particles with a diameter greater than 10 μm.

[0031] In some embodiments, as in any of the methods described above, the FX activator (eg, RVV-X) is administered by intravenous injection.

[0032] In some embodiments, as described above in any method, the bleeding disease is a congenital bleeding disease or an acquired bleeding disease. In some embodiments, the bleeding disease is selected from the group consisting of a disease caused by lack of coagulation factors, a disease caused by the presence of acquired coagulation factor inhibitors, a blood disease, a bleeding disease, von Willebrands' disease, a disease caused by anticoagulation therapy with vitamin K antagonists, a hereditary platelet disease, vitamin K epoxide reductase C1 deficiency, gamma carboxylase deficiency, bleeding associated with trauma or injury or surgery, thrombosis, thrombocytopenia, stroke, coagulation disorders, disseminated intravascular coagulation (DIC), Bernard-Soulier syndrome, Glanzman thrombosis and storage pool deficiency. In some embodiments, the bleeding disease is due to a lack of coagulation factors. In some embodiments, the bleeding disease is hemophilia, such as hemophilia A or hemophilia B. In some embodiments, the hemophilia is hemophilia with inhibitors.

[0033] In some embodiments, as in any of the methods described above, the individual is a human.

[0034] Also provided are compositions (e.g., pharmaceutical compositions), kits, and manufactured articles comprising any of the pharmaceutical compositions described above. Also provided are methods for treating individual (human) bleeding disorders (e.g., bleeding disorders such as hemophilia A or hemophilia B) using any of the methods described herein or their pharmaceutical compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figures 1A-1D The PK characteristics of single and multiple injections of RVV-X were described. Patients received a single intravenous injection of RVV-X at three doses, 0.16 U / kg, 0.32 U / kg, and 0.48 U / kg. Figure 1A Shown is the concentration-time curve of RVV-X in plasma after a single injection. Figure 1B Shown is the semi-logarithmic drug concentration-time curve of RVV-X in plasma after a single injection. Figure 1C Shown is a single injection of C max Analysis of plasma RVV-X concentrations between doses. Figure 1D Shown is the concentration-time curve of RVV-X in plasma after multiple injections.

[0036] Figure 2A-2B The pharmacodynamic (PD) characteristics of a single injection of RVV-X were described. Patients received a single intravenous injection of RVV-X at 6 doses of 0.01U / kg, 0.04U / kg, 0.08U / kg, 0.16U / kg, 0.32U / kg, and 0.48U / kg. Figure 2A Shown are changes in peak height of thrombin generation (TG). Figure 2B Shown are changes in intrinsic thrombin generation potential (ETP). Change values ​​were obtained by subtracting the baseline value from the measured value at each tested time point after RVV-X administration.

[0037] Figure 3A-3B To describe the plasma concentration of FX (FX:C) after a single injection of RVV-X. Patients received a single intravenous injection of RVV-X at 6 doses of 0.01 U / kg, 0.04 U / kg, 0.08 U / kg, 0.16 U / kg, 0.32 U / kg, and 0.48 U / kg. Figure 3A Shown is the variation of FX:C. Figure 3B The values ​​of FX:C are shown. The results are expressed as mean ± standard deviation.

[0038] Figure 4 The relationship between RVV-X concentration and APTT in plasma samples is described.

[0039] Figure 5 Shown are the numbers of insoluble particles in each lyophilized formulation at 0 month.

[0040] Figure 6 Shown are the numbers of insoluble particles in each lyophilized formulation after 6 months of acceleration. DETAILED DESCRIPTION OF THE INVENTION

[0042] Thrombin and FX activator are usually prepared into a composition together and used as a hemostatic drug. As a macromolecular protein, if FX activator is used as a single active ingredient to prepare a FX activator pharmaceutical composition, FX activator is prone to denaturation, which is not conducive to long-term storage. In addition, the biological activity of FX activator depends on its secondary and tertiary structures. Therefore, it is particularly important to maintain its biological activity during preparation, purification, storage and administration.

[0043] Bleeding episodes in hemophilia A (HA) and hemophilia B (HB) patients with inhibitors (e.g., autoantibodies) are difficult to control. Current bypassing therapeutics have disadvantages and limitations. For example, activated thrombin complex concentrates (aPCC, e.g., ) is not widely used. In addition, the trace amount of FVIII in aPCC may induce a recall response to FVIII. PCC may induce a memory immune response to FIX. In addition, PCC and aPCC are derived from blood products, so there is a potential risk of viral infection and allergy. Emicizumab-kxwh is a bispecific humanized monoclonal antibody that restores the function of inactivated FVIII by bridging activated FIX and FX, thereby promoting effective hemostasis. The FDA has approved it for the treatment of HA and HB patients with FVIII or FIX inhibitors, respectively. However, due to its high price, it is not widely used. Activated recombinant factor VII (rFVIIa, e.g., The same price problem exists for RT. rFVIIa works by forming a complex with tissue factor, which activates FX to FXa via the extrinsic pathway and then converts prothrombin to thrombin to achieve hemostasis.

[0044] On the one hand, the present invention provides a composition (e.g., a pharmaceutical composition) of a FX activator (e.g., RVV-X), such as a lead formulation of RVV-X, which is stable and biologically active under long-term storage, accelerated or stressed conditions, and / or after reconstitution. On the other hand, the present invention provides a FX activator or a composition thereof, such as any FX activator pharmaceutical composition described herein, for treating a bleeding disorder (e.g., hemophilia, such as HA or HB with inhibitors) in an individual (e.g., human), wherein the FX activator or a composition thereof exhibits excellent therapeutic effects (e.g., high specificity, high activity, low dose, rapid onset), PK / PD curves, and safety in vivo. This property is consistent with the FDA-approved rFVIIa Equivalent or better.

[0045] The FX activator described herein (e.g., RVV-X, such as isolated and purified from the venom of Vipera rotundifolia) and its pharmaceutical composition (e.g., RVV-X lead agent), as well as its use in treating bleeding disorders as described herein (e.g., with or without inhibitors HA or HB), has one or more excellent therapeutic effects: 1) it exhibits good biological activity in promoting hemostasis in patients with hemophilia A and hemophilia B with inhibitors (FVIII inhibitors or FIX inhibitors, respectively), who are known to be difficult to treat; 2) it can effectively promote hemostasis in various bleeding events, such as during surgery (see Examples 1-4); 3) it has comparable or superior therapeutic effects compared to FDA-approved expensive or limited-use hemostatic agents, for example, the therapeutic effect of RVV-X in a human Phase II clinical trial (100% effective hemostasis) was significantly better than that of thrombin complex concentrate (PCC; effective hemostasis rate 50%), and comparable to injectable recombinant human coagulation factor VIIa (e.g., RT; effective hemostasis rate of about 90%), in KOFVIII hemophilia mice, a single low-dose injection of RVV-X can effectively promote hemostasis and reduce mortality, and show similar RT and other similar or even better therapeutic effects; 4) it has excellent safety and is well tolerated within the dose range demonstrated herein, without causing any thromboembolic events, disseminated intravascular coagulation (DIC), serious adverse events (SAE), adverse events (AE) leading to discontinuation of the drug during clinical trials, or suspected unexpected serious adverse reactions (SUSAR) (see Examples 1 and 4); 5) it does not induce inhibitors (binding antibodies) or anti-RVV-X neutralizing antibodies (undesirable immune responses) in vivo; 6) it has a wide range of hemostatic applications from mammals (e.g., mice) to human subjects; 7) it exhibits excellent pharmacokinetic (PK) and pharmacodynamic (PD) curves, such as rapid onset and long in vivo half-life, for example, T 1 / 2 Up to 7-9 hours, C begins to appear 5-10 minutes after administration max and therapeutic indicators (e.g., increased thrombin generation (TG), increased intrinsic thrombin generating potential (ETP), shortened activated partial thromboplastin time (APTT)) for at least 24 hours, while FDA-approved RT 1 / 2Only 1.7-3.11 hours (U.S. FDA NovoSeven package insert); 8) The FX activator and its pharmaceutical composition as described herein (e.g., RVV-X lead agent) have a long half-life and high biological activity, and therefore can be administered at a low frequency and low dose (e.g., once every 4 hours, a total of 4 times, each dose of 0.16U / kg; or a single dose, a dose of 0.16U / kg-0.48U / kg) to achieve effective hemostasis, and can be administered by intravenous injection, which provides convenience for patients and reduces costs; and 9) The FX activator and its pharmaceutical composition as described herein (e.g., RVV-X lead agent) and formulation scheme (e.g., powdered lyophilized injection) provide excellent stability and safety (e.g., compared with a prescription containing human serum albumin as a protein stabilizer), such as under long-term storage conditions (2-8°C), accelerated conditions (e.g., 25°C, RH 65±5%), stress conditions (e.g., high temperature (40±2°C), high humidity or light) or after reconstitution, it has stable appearance, moisture, pH value and bioactivity stability, and the aggregates and / or insoluble particles are significantly reduced, thereby effectively maintaining the drug properties, while also avoiding the risks of allergic reactions and / or viral contamination that are common when HSA is used as a protein stabilizer.

[0046] Also provided are methods of preparing (eg, formulation regimens) any of the FX activator pharmaceutical compositions described herein, as well as articles of manufacture comprising a FX activator (eg, RVV-X) or a pharmaceutical composition thereof.

[0047] I. Definitions

[0048] Unless the context clearly indicates otherwise, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described in detail below for illustration. Such techniques are fully explained in the literature. See Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubelet al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: APractical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0049] As described herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result, including a clinical result. In view of the purposes of the present invention, the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, alleviating the disease (partially or completely), reducing the dosage of one or more other drugs required for treating the disease, delaying disease progression, improving or improving the quality of life, increasing body weight, and / or prolonging survival. At the same time, "treatment" also includes reducing the pathological results of the disease. The method of the present application takes into account any one or more aspects of these treatments. For example, if one or more symptoms associated with the disease are alleviated or eliminated, including but not limited to reducing the symptoms caused by the disease, improving the quality of life of the patient with the disease, reducing the dosage of other drugs required for treating the disease, and / or prolonging the survival of the individual, the patient is considered to be successfully "treated".

[0050] The term "prevent" and similar words, such as "prevented", "preventing", etc., refer to methods of preventing, inhibiting, or reducing the likelihood of recurrence of a disease or condition. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of a disease or condition. As used herein, "preventing" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the disease or condition recurs.

[0051] As used herein, "delaying" the development of a disease means postponing, hindering, slowing down, slowing down, stabilizing and / or postponing the development of a disease. The time of delay may be different depending on the history of the disease and / or the individual being treated. A method of "delaying" the development of a disease refers to a method that reduces the likelihood of disease development and / or reduces the extent of the disease within a given time frame compared to not using the method. This comparison is usually based on clinical studies using statistically significant numbers of individuals.

[0052] As used herein, the term "effective amount" refers to a dose of a drug or a dose of a pharmaceutical composition sufficient to treat a particular disorder, condition or disease, such as improving, alleviating, weakening and / or delaying one or more symptoms. In some embodiments, the effective amount is an amount sufficient to delay the development of the disease. In some embodiments, the effective amount is an amount sufficient to prevent or delay the recurrence of the disease. In some embodiments, the effective amount is an amount sufficient to alleviate one or more symptoms associated with the disease to a certain extent. The effective amount can be administered in one or more administrations. In some embodiments, the effective amount of a drug or composition can: (i) reduce bleeding volume and / or time; (ii) increase coagulant activity; (iii) activate FX; (iv) increase thrombin generation (TG) and / or endogenous thrombin generation potential (ETP); (v) shorten the activated partial thromboplastin time (APTT), prothrombin time (PT) and / or thrombin time (TT); (vi) promote hemostasis, such as reducing bleeding time and / or bleeding volume; (vii) reduce mortality and / or (viii) promote wound healing, etc.

[0053] As used herein, "coagulation pathway" or "coagulation cascade" refers to a series of activation events that lead to the formation of an insoluble fibrin clot. In the coagulation cascade or pathway, a serine protease (also known as a zymogen) is converted into an active protease by cleaving one or more peptide bonds in the inactive protein, which then serves as the activating protease for the next zymogen molecule in the cascade. In the last proteolytic step of the cascade, thrombin hydrolyzes fibrinogen into fibrin, which then cross-links at the site of injury to form a clot.

[0054] As used herein, "hemostasis" refers to stopping bleeding or stopping the flow of blood in an organ or body part. The term "hemostasis" may include the entire blood coagulation process that prevents blood loss after blood vessel injury until the blood clot dissolves after tissue repair.

[0055] As used herein, "clotting" or "coagulation" refers to the formation of an insoluble fibrin clot, or the process by which coagulation factors in the blood interact in the coagulation cascade, ultimately leading to the formation of an insoluble fibrin clot.

[0056] As used herein, "coagulant" refers to any substance that promotes blood clotting.

[0057] As used herein, "anticoagulant" refers to any substance that inhibits blood clotting.

[0058] As used herein, the term "osmotic pressure" refers to a measure of solute concentration, defined as the number of millimoles of solute (non-ionic and ionic forms) per kilogram of solution. The desired osmotic pressure level can be achieved by adding one or more stabilizers, such as sugars or sugar alcohols, including but not limited to mannitol, glucose, trehalose and / or sucrose. References detail other stabilizers suitable for providing osmotic pressure, such as "Handbook of Pharmaceutical Excipients" (4th edition, Royal Pharmaceutical Society, Science and Practice Press) or "Remington: The Science and Practice of Pharmacy" (19th edition, Mack Publishing Company).

[0059] As used herein, the terms "isosmotic" and "isotonic" are used interchangeably with the term "substantially isotonic," which refers to a formulation having an osmotic pressure that is the same as, or at least substantially equivalent to, that of another solution, and a total concentration of solutes (including osmotic and non-osmotic solutes) in the formulation that is the same as, or at least substantially equivalent to, the total number of solutes in the other solution. Thus, although those skilled in the art understand that "isosmotic" and "isotonic" formulations for in vivo administration generally have an osmotic pressure range of about 270 mmol / kg to 310 mmol / kg, in the context of the high concentration, low viscosity formulations described in the embodiments herein, the terms "isosmotic", "isotonic", "substantially isosmotic" and "substantially isotonic" are used interchangeably to refer to formulations having an osmotic pressure range of about 240 mOsm / kg to 400 mOsm / kg, or 270 mOsm / kg to 370 mOsm / kg, or 300 mOsm / kg to 330 mOsm / kg.

[0060] As used herein, "subject" or "subject" refers to an animal (e.g., a mammal), including but not limited to humans, cows, horses, cats, dogs, rodents, birds, or primates. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse, rat, or monkey.

[0061] As used herein, the terms "specifically binds," "specifically recognizes," or "specific for" refer to a measurable and reproducible interaction, such as the binding of a ligand to a receptor, that can be determined to be present in a heterogeneous population of molecules, including biomolecules. For example, a ligand that specifically recognizes a receptor means that the ligand binds to the receptor with greater affinity, avidity, more readily, and / or more persistently than it binds to other receptors. In some embodiments, the binding affinity of the ligand to an unrelated receptor is less than 10% of its binding affinity to the target receptor as measured, for example, by radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (K) of a ligand that specifically binds to a target receptor is 100%. d )≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 M. In some embodiments, the ligand can specifically bind to a receptor that is conserved between different species. In some embodiments, specific binding includes but does not require exclusive binding. The binding specificity of the ligand can be experimentally determined by methods known in the art. These methods include, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore testing and peptide scanning.

[0062] As used herein, "covalent bond" refers to a stable bond formed between two atoms by sharing one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, "peptide bond" refers to a covalent bond formed between the carboxyl group of an amino acid and the amine group of an adjacent amino acid. As used herein, "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments are bound by one or more disulfide bonds. One or more disulfide bonds between two fragments may be formed by connecting thiol groups in the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines of two Fc fragments. Oxidation of two thiol groups can form a disulfide bond. In some embodiments, the covalent bond is formed by direct connection through a covalent bond. In some embodiments, the covalent bond is directly connected through a peptide bond or a disulfide bond.

[0063] The "percentage (%) of amino acid sequence identity" or "homology" of a peptide or polypeptide sequence is defined as the percentage of identical amino acid residues in a candidate sequence to a particular polypeptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to maximize the percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. The percentage of amino acid sequence identity can be determined by a variety of alignments within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0064] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide, the amine group of which forms a peptide bond with the carboxyl group of its adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid of the polypeptide, the carboxyl group of which forms a peptide bond with the amine group of its adjacent amino acid residue.

[0065] An "isolated" polypeptide is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from all other components of its production environment. Contaminating components of the production environment, such as those produced by recombinant transfected cells, often interfere with the study, diagnosis, or treatment of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide will be purified to: (1) greater than 95% polypeptide by weight as determined by the Lowry method, and in some embodiments, greater than 99% polypeptide by weight; (2) to a degree sufficient to obtain sequence of at least 15 N-terminal residues or internal amino acids using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated polypeptide includes the polypeptide in situ within recombinant cells, since at least one element of the polypeptide's natural environment is absent. However, generally, at least one purification step is required to produce an isolated polypeptide.

[0066] An "isolated" nucleic acid molecule encoding a construct is a nucleic acid molecule identified and separated from a nucleic acid molecule containing at least one impurity, and in the natural environment in which the "isolated" nucleic acid molecule is produced, it is usually associated with the impurity nucleic acid molecule. Preferably, the isolated nucleic acid is not associated with all components in its production environment. The form in which the isolated nucleic acid molecule encoding a polypeptide as described herein exists is different from the form in which it naturally exists in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptide described herein that naturally exists in the cell. The isolated nucleic acid includes a nucleic acid molecule that is normally contained in the cell containing the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome, or the position in the chromosome is different from the natural situation.

[0067] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0068] A nucleic acid is "operably linked" when it establishes a functional relationship with another nucleic acid sequence. For example, if the DNA of a polypeptide is expressed in the form of a precursor protein (which participates in the secretion process of the polypeptide), the DNA of a presequence or secretory leader sequence is operably linked to the DNA of a polypeptide; if a promoter or enhancer affects the transcription of the sequence, the promoter or enhancer is operably linked to a coding sequence; or if a ribosome binding site is in a position that facilitates translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the DNA sequences being linked are continuous, and, for a secretory leader sequence, they are not only continuous but also in the reading frame (reading phase). However, enhancers do not have to be continuous. Linking is accomplished by ligating at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used by conventional means.

[0069] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of amplifying another nucleic acid molecule to which it is attached. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that can be integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are attached. Such vectors are referred to herein as "expression vectors."

[0070] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny cells.

[0071] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny produced therefrom, without regard to the number of passages. Progeny cells may not be completely identical to the parental cells in nucleic acid and may contain mutations. Screening or selecting mutant progeny having the same function or biological activity as the original transformed cells is included herein.

[0072] The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation that is in a form effective for the biological activity of the active ingredient and does not contain additional ingredients that are unacceptably toxic to a subject to which the preparation is administered. Such preparations are sterile. A "sterile" preparation is sterile or does not contain any living microorganisms or their spores.

[0073]

[0066] Embodiments of the present invention described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0074] Reference herein to "about" for a value or parameter includes (and describes) variations for that value or parameter itself. For example, descriptions involving "about X" include descriptions for "X" itself.

[0075] As used herein, reference to "not" a value or parameter generally indicates and describes "other than" a value or parameter. For example, the method cannot be used to treat type X disease, which means that the method is generally used to treat other types of diseases except type X.

[0076] As used herein, the term "about XY" is synonymous with "about X to about Y."

[0077] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0078] II. Pharmaceutical Compositions of Factor X Activators

[0079] The inventors of the present application have found that the use of disaccharides (e.g., sucrose and / or trehalose) as stabilizers and amino acid buffers (e.g., arginine and / or histidine) to prepare a composition with an FX activator (e.g., RVV-X) as a single active ingredient not only greatly improves the stability of the product, reduces protein aggregation and the content of insoluble particles, but also avoids the allergic reactions and potential viral contamination risks caused by the use of human serum albumin as a commonly used stabilizer. During the freeze-drying of the FX activator (e.g., RVV-X), the use of sucrose and / or trehalose as stabilizers can make the higher-order structure of the FX activator more stable, prevent it from being deformed due to freeze-drying, maintain the complete structure and function of the FX activator under low-temperature freeze-dehydration conditions, and significantly improve the stability of the FX activator.

[0080] During the freeze-drying of the FX activator (e.g., RVV-X) solution, the concentration of the FX activator gradually increases. When the protein concentration is high, the pH value of the solution may change, which may cause the FX activator to denature and inactivate the FX activator in severe cases. In order to ensure that the active ingredient FX activator has maximum biological activity, the appropriate pH range must be controlled. The inventors of the present application have found that using histidine and / or arginine as a buffer not only has good buffering capacity, but also can maintain the relative stability of the product pH within a certain range. In addition, compared with the commonly used stabilizer HSA, when an amino acid buffer (e.g., arginine and / or histidine) is used in combination with a disaccharide (e.g., sucrose and / or trehalose) as a stabilizer, the formulation scheme of the present application can not only ensure the biological activity of the FX activator, but also further improve the stability of the composition.

[0081] The FX activator pharmaceutical composition (e.g., powder for injection) of the present invention has at least the following beneficial effects: 1) The FX activator pharmaceutical composition or formulation of the present invention uses the FX activator as the single active ingredient, uses sucrose and / or trehalose instead of albumin (e.g., HSA) as a stabilizer, and is combined with a buffer and a surfactant of arginine and / or histidine, which not only significantly improves the stability of the FX activator composition product, but also avoids potential risks caused by other unknown components carried by viruses or albumin; 2) The FX activator pharmaceutical composition or formulation of the present invention uses the FX activator as the single active ingredient, uses sucrose and / or trehalose instead of albumin (e.g., HSA) as a stabilizer, and is combined with a buffer and a surfactant of arginine and / or histidine, which not only significantly improves the stability of the FX activator composition product, but also avoids potential risks caused by other unknown components carried by viruses or albumin; 3) an active ingredient, so the active ingredient and its content are clear, in addition, sucrose and / or trehalose are of high purity and wide source, which is convenient for long-term batch production, and is also convenient for controlling costs and improving product quality; 3) the FX activator pharmaceutical composition or formulation scheme of the present invention can ensure that the FX activator maintains good stability during preparation, transportation and storage, has a low content of insoluble particles, and has better clinical drug safety and quality controllability; and 4) the FX activator pharmaceutical composition or formulation scheme of the present invention does not contain albumin and has higher stability. After being reconstituted with 0.9% sodium chloride injection, it can remain stable for at least about 8 hours at room temperature.

[0082] On the one hand, the present application provides a coagulation factor X activator (FX activator) pharmaceutical composition, hereinafter also referred to as a "FX activator pharmaceutical composition" or "FX activator preparation". In some embodiments, the FX activator pharmaceutical composition (e.g., lyophilized) comprises (or is essentially composed of, or is composed of): i) FX activator (e.g., RVV-X) at a content of 0.1 U / mL to 200 U / mL (e.g., 10 U / mL); ii) stabilizer (e.g., sucrose) at a content of 2 mg / ml to 100 mg / ml (e.g., 30 mg / ml); iii) buffer (e.g., histidine) at a content of 100 mg / ml to 200 mg / ml (e.g., 100 mg / ml); iv) thiazolinone (e.g., thiazolinone ... In some embodiments, the FX activator is RVV-X. In some embodiments, the FX activator is RVV-X, which is isolated from the venom of Vipera rotundifolia.In some embodiments, the RVV-X comprises: a) i) a heavy chain comprising the sequence of SEQ ID NO: 1, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 1; ii) a light chain 1 comprising the sequence of SEQ ID NO: 2, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence of SEQ ID NO: 3, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 3; b) i) a heavy chain comprising the sequence of SEQ ID NO: 1, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: NO:1 has at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity; ii) light chain 1, which comprises the sequence SEQ ID NO:5, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:5; and iii) light chain 2, which comprises the sequence SEQ ID NO:3, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:3; or c) a mixture of a) and b).

[0083] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) FX activator (e.g., RVV-X) at 0.1 U / mL to 100 U / mL; ii) stabilizer (e.g., sucrose) at 15 mg / ml to 60 mg / ml; iii) buffer (e.g., histidine) at 2 mg / ml to 50 mg / ml; iv) surfactant (e.g., polysorbate 20) at 0.005% (w / v) to 0.05% (w / v); and v) excipient (mannitol) at 10 mg / ml to 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.0 to 8.0. In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0084] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) FX activator (e.g., RVV-X) at 5 U / mL to 100 U / mL; ii) stabilizer (e.g., sucrose) at 30 mg / ml to 50 mg / ml; iii) buffer (e.g., histidine) at 2 mg / ml to 15 mg / ml; iv) surfactant (e.g., polysorbate 20) at 0.01% (w / v) to 0.05% (w / v); and v) excipient (mannitol) at 30 mg / ml to 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.3 to 7.3. In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0085] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) FX activator (e.g., RVV-X) at 5 U / mL to 50 U / mL; ii) stabilizer (e.g., sucrose) at 30 mg / ml to 50 mg / ml; iii) buffer (e.g., histidine) at 3 mg / ml to 5 mg / ml; iv) surfactant (e.g., polysorbate 20) at 0.01% (w / v) to 0.03% (w / v); and v) excipient (mannitol) at 30 mg / ml to 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.8 to 7.0. In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0086] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) a FX activator (e.g., RVV-X) at 5 U / mL, 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, or 50 U / mL; ii) a stabilizer (e.g., sucrose) at 30 mg / ml to 50 mg / ml; iii) a buffer (e.g., histidine) at 3 mg / ml to 5 mg / ml; iv) a surfactant (e.g., polysorbate 20) at 0.01% (w / v) to 0.03% (w / v); and v) an excipient (mannitol) at 30 mg / ml to 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.8 to 7.0 (e.g., 6.85). In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0087] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) FX activator (e.g., RVV-X) at 5 U / mL to 50 U / mL; ii) stabilizer (e.g., sucrose) at 30 mg / ml, 40 mg / ml, or 50 mg / ml; iii) buffer (e.g., histidine) at 3 mg / ml to 5 mg / ml; iv) surfactant (e.g., polysorbate 20) at 0.01% (w / v) to 0.03% (w / v); and v) excipient (mannitol) at 30 mg / ml to 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.8 to 7.0 (e.g., 6.85). In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0088] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) a FX activator (e.g., RVV-X) at 5 U / mL to 50 U / mL; ii) a stabilizer (e.g., sucrose) at 30 mg / ml to 50 mg / ml; iii) a buffer (e.g., histidine) at 3 mg / ml, 4 mg / ml, or 5 mg / ml; iv) a surfactant (e.g., polysorbate 20) at 0.01% (w / v) to 0.03% (w / v); and v) an excipient (mannitol) at 30 mg / ml to 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.8 to 7.0 (e.g., 6.85). In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0089] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) FX activator (e.g., RVV-X) at 5 U / mL to 50 U / mL; ii) stabilizer (e.g., sucrose) at 30 mg / ml to 50 mg / ml; iii) buffer (e.g., histidine) at 3 mg / ml to 5 mg / ml; iv) surfactant (e.g., polysorbate 20) at 0.01% (w / v), 0.02% (w / v), or 0.03% (w / v); and v) excipient (mannitol) at 30 mg / ml to 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.8 to 7.0 (e.g., 6.85). In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0090] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, comprising (or consisting essentially of, or consisting of): i) FX activator (e.g., RVV-X) at 5 U / mL to 50 U / mL; ii) stabilizer (e.g., sucrose) at 30 mg / ml to 50 mg / ml; iii) buffer (e.g., histidine) at 3 mg / ml to 5 mg / ml; iv) surfactant (e.g., polysorbate 20) at 0.01% (w / v) to 0.03% (w / v); and v) excipient (mannitol) at 30 mg / ml, 40 mg / ml, 50 mg / ml or 60 mg / ml; wherein the pH of the pharmaceutical composition is 6.8 to 7.0 (e.g., 6.85). In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia.

[0091] In some embodiments, a FX activator pharmaceutical composition (e.g., lyophilized) is provided, which comprises (or consists essentially of, or consists of): i) RVV-X at 10 U / mL; ii) sucrose at 30 mg / ml; iii) histidine at 3 mg / ml; iv) polysorbate 20 at 0.02% (w / v); and v) mannitol at 40 mg / ml; wherein the pH value of the pharmaceutical composition is 6.85 (hereinafter also referred to as "lead formulation" or "RVV-X lead formulation"). In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the RVV-X comprises: a) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; or c) a mixture of a) and b).

[0092] In some embodiments, the pharmaceutical composition is a lyophilized preparation (eg, powder for injection). In some embodiments, the pharmaceutical composition is an aqueous solution.

[0093] The reconstituted formulation can be prepared by dissolving the lyophilized FX activator pharmaceutical composition in a diluent so that the protein is dispersed throughout the solution. Exemplary pharmaceutically acceptable (safe and non-toxic to human administration) diluents suitable for use in the present application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solutions (e.g., 0.9% NaCl injection), dextrose injection (e.g., 5% dextrose injection), Ringer's solution or dextrose solution, or aqueous solutions of salts and / or buffers.

[0094] In some embodiments, the pharmaceutical compositions are sterile. In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical compositions can be sterilized by filtration through a sterile filter membrane. The pharmaceutical compositions described herein are typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

[0095] The pharmaceutical compositions described herein may also contain more than one active compound required for treating a specific indication, preferably compounds with complementary activities that do not adversely affect each other. The content of such molecules in the combination is an amount effective for the intended purpose.

[0096] In some embodiments, the pharmaceutical composition is contained in a disposable vial, such as a disposable sealed vial. In some embodiments, the pharmaceutical composition is contained in a vial that can be used multiple times. In some embodiments, the pharmaceutical composition is bulked in a container. In some embodiments, the pharmaceutical composition is stored frozen.

[0097] In some embodiments, the pharmaceutical composition is contained in a disposable vial. In some embodiments, the vial contains a FX activator (e.g., RVV-X) at a content of 1U to 30U, such as any of 1U to 5U, 1U to 10U, 2U to 18U, 3U to 15U or 5U to 10U. In some embodiments, the vial contains a FX activator (e.g., RVV-X) at a content of 5U. In some embodiments, the pharmaceutical composition (e.g., lyophilized) is reconstituted with 2mL of 0.9% sodium chloride injection for administration.

[0098] In some embodiments, the pharmaceutical composition comprises a homogenous population of FX activators described herein (e.g., RVV-X). A homogenous population means that the FX activators are identical to each other. In some embodiments, at least about 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the FX activators in the pharmaceutical composition are homogenous.

[0099] In some embodiments, the pharmaceutical composition mainly comprises (or consists of) a FX activator as described herein (e.g., RVV-X), and an optional pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition does not contain any host cell (e.g., CHO) or non-FX activator animal (e.g., snake) protein.

[0100] Factor X activator (FX activator)

[0101] Any reagent that can remove the activation peptide from the N-terminus of the FX heavy chain to obtain an activated FXa form can be used as a FX activator. In some embodiments, the FX activator described herein is a protein activator, such as an enzyme with proteolytic properties that acts on FX. In some embodiments, the FX activator can be a non-protein activator.

[0102] In some embodiments, the FX activator is isolated from snake venom, such as species belonging to the families Viperidae and Crotalidae, and some species of cobra. Meanwhile, any venom-derived FX activator useful in the present invention is described in G. Tans and J. Rosing, "Snake venom activators of factor X: an overview" Haemostasis. 2001; 31 (3-6): 225-33 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the FX activator is a metalloproteinase. In some embodiments, the FX activator is a serine protease. In some embodiments, the FX activator is RVV-X, such as RVV-X isolated from the venom of Daboia russellii siamensis. In some embodiments, the FX activator can be prepared by recombination.

[0103] In some embodiments, the FX activator is a wild snake venom derived FX activator. In some embodiments, the FX activator is a natural variant of a snake venom derived FX activator. In some embodiments, the FX activator is a derivative of a snake venom derived FX activator. In some embodiments, the FX activator is a functional fragment of a snake venom derived FX activator. In some embodiments, the FX activator is a mutant of a snake venom derived FX activator. In some embodiments, a functional fragment or mutant form of a snake venom derived FX activator retains at least about 30% (e.g., at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of the activity of a wild snake venom derived FX activator.

[0104] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL, such as 0.1 U / mL to 1 U / mL, 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 80 U / mL, 10 U / mL to 50 U / mL, 1 U / mL to 50 U / mL, 5 U / mL to 50 U / mL, 5 U / mL to 20 U / mL, 5 U / mL to 10 U / mL, 1 U / mL to 30 U / mL, or 5 U / mL to 15 U / mL. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a FX activator (e.g., RVV-X) at 0.1 U / mL, 0.5 U / mL, 1 U / mL, 5 U / mL, 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL or 100 U / mL, such as 10 U / mL. At the same time, the determination of the activity unit (U) is shown in the "Biological Activity" summary below.

[0105] Coagulation factor X (FX) and activated FX (FXa)

[0106] FX or FX polypeptide is a serine protease polypeptide that exhibits catalytic activity (i.e., procoagulant activity) on prothrombin in an activated state. FX is a serine protease that is part of the coagulation pathway, particularly the first serine protease in the common coagulation pathway. In cells, FX is processed from a precursor polypeptide to produce a polypeptide containing a propeptide, which is ultimately cleaved to produce a mature FX polypeptide lacking a signal sequence and a propeptide. The secreted FX polypeptide is a two-chain polypeptide. Activated FXa lacks an activation peptide. FXa is a form of FX that exhibits catalytic activity, and the catalytic activity of activated FX (FXa) is greatly enhanced when it binds to its cofactor Va. The activity of FXa can also be enhanced by adding Ca2+ and phospholipids. Mutations can be introduced to cause the FXa form to undergo a zymogen-like conformational change. When the FVa cofactor is present, FXa is in a fully activated state and exhibits catalytic activity on prothrombin. The FX or FX polypeptide mentioned herein includes all forms thereof, including precursor, single-chain and double-chain forms, including mature forms and zymogen forms.

[0107] FX can be from any organism, such as mammals, including but not limited to livestock (e.g., cattle, sheep, goats, cats, dogs, donkeys and horses), primates (e.g., humans and non-human primates, such as monkeys or chimpanzees), rabbits and rodents (e.g., mice, rats, gerbils and hamsters). FX can also be prepared by recombinant or synthetic methods. In some embodiments, FX is recombinant FX (rFX), such as recombinant mouse FX (rmFX) or recombinant human FX (rhFX).

[0108] In some embodiments, FX is a human FX polypeptide (such as wild-type human FX), including a precursor polypeptide (488aa) and single-chain and double-chain forms thereof, including a mature form (448aa) and a zymogen form. The human zymogen form is a double-chain form comprising a light chain of 139 amino acids and a heavy chain of 306 amino acids. The human FXa form is a double-chain form comprising a light chain of 139 amino acids and a heavy chain of 254 amino acids.

[0109] The FX also includes variants thereof, such as allelic variants and species variants, variants encoded by splice variants and other variants, comprising a polypeptide having at least about 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a precursor polypeptide (e.g., human precursor FX) or a mature form or a zymogen form. Such variants exhibit one or more FX activities, including but not limited to FVa binding, catalytic activity, prothrombin binding, thrombin activity and / or coagulation activity. Compared with the activity of natural or wild-type FX, its activity may be reduced or increased. For example, a FX polypeptide variant exhibits at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of a native or wild-type FX polypeptide.

[0110] The precursor FX polypeptide refers to a non-secretory FX polypeptide, which comprises an N-terminal signal peptide and a propeptide for secreting the protein. The signal peptide is cleaved off in the endoplasmic reticulum.

[0111] "Proregion", "propeptide" or "pro-sequence" refers to a region or fragment that can be cleaved to produce a mature protein. The proregion is an amino acid sequence located at the amino acid terminus of a mature biologically active polypeptide and can be only a few amino acids or a multi-domain structure. For FX polypeptides, the propeptide region is usually about 9 amino acids, but may vary depending on the species (for example, longer or shorter). For FX, the propeptide sequence plays a role in the post-translational modification of the protein and is cleaved before the protein is secreted by the cell. For example, the propeptide is a γ-carboxylation recognition element for vitamin K-dependent carboxylase on the endoplasmic reticulum. This reaction occurs by converting the glutamic acid residue in the Gla domain of FX to γ-carboxyglutamate (Gla). This modification is the most ideal Ca in the blood. 2+ For example, Gla residues allow factor X / Xa to bind to phospholipids (i.e., cell surfaces) in a calcium-dependent manner, which is a requirement for the assembly of the prothrombin complex.

[0112] The propeptide form of FX is a protein that lacks the signal peptide but retains the propeptide.

[0113] "Mature FX polypeptide" refers to a FX polypeptide lacking a signal peptide sequence and a propeptide sequence. The propeptide is removed by proteolytic cleavage in the trans-Golgi apparatus before the polypeptide is secreted. A mature FX polypeptide generally refers to a single-chain form of FX before intrachain proteolysis produces a double-chain polypeptide.

[0114] The activation peptide is a fragment present at the N-terminus of the FX heavy chain, which functions to inhibit proteolytic activity by masking the catalytic machinery, thereby preventing the formation of catalytic intermediates (i.e., by conformationally blocking the substrate binding site). An example of a FX activation peptide is the 52 amino acid residue activation peptide at the N-terminus of the heavy chain of the mature human FX polypeptide.

[0115] The term "wild-type" or "native" FX refers to a FX polypeptide encoded by a natural or naturally occurring FX gene, including allelic variants present in organisms in nature, including humans and other animals. When referring to a wild-type or native FXa polypeptide, it is understood to be an activated or catalytically active portion of the FXa polypeptide. Reference to wild-type factor X without reference to species is intended to cover wild-type factor X of any species. Wild-type FX polypeptides include encoded precursor polypeptides, fragments thereof, and processed forms thereof, such as mature forms lacking signal peptides and propeptides, and any pre-translationally or post-translationally processed or modified forms thereof. Natural FX polypeptides also include post-translationally modified polypeptides, including but not limited to glycosylation, carboxylation, and hydroxylation modifications. Natural FX polypeptides also include double-chain secreted forms, including proenzyme and activated forms, as well as any processed forms or isomers thereof. For example, humans express natural FX.

[0116] Species variants are polypeptide variants that exist between different species, including different mammalian species such as mice and humans. Allelic variants are protein variants that exist between members of the same species. Splice variants are variants that are produced by differential processing of the primary transcript of genomic DNA, which can produce more than one mRNA.

[0117] Factor X circulates in the blood as an inactive precursor, called a zymogen, which requires proteolysis to activate. The zymogen has approximately 10,000-fold or less proteolytic activity than the serine protease produced after activation.

[0118] Activated FX (FXa) or activated FX refers to a two-chain form of the FX polypeptide, whose heavy chain does not contain an N-terminal activation peptide. FXa is activated by cleaving the heavy chain to remove the activation peptide. Therefore, FXa is a heterodimer consisting of two chains connected by a disulfide bond. For human FX, the molecular weight of its light chain is 16,000 Daltons (139aa) and the molecular weight of the heavy chain is 38,000 Daltons (254aa), constituting the serine protease domain. Activation of human FX occurs by cleavage of the Arg194-Ile195 bond, releasing the activation peptide. Activation is achieved by an exogenous factor Xase complex (factor VIIa / TF complex) or an endogenous factor Xase complex (FIXa / FVIIIa complex). Activation generally requires the presence of phospholipids and calcium ions. It can also be activated by Viper venom (RVV-X). FXa has catalytic activity, FVa binding, heparin binding, prothrombin binding, prothrombin activity and / or coagulation activity. For the purposes of this article, the FXa mentioned refers to any FX two-chain form lacking an activation peptide and capable of exhibiting FXa activity (e.g., catalytic activity, FVa binding, heparin binding, prothrombin binding, coagulase activity and / or coagulation activity). The FXa mentioned includes zymogen-like FXa polypeptides that exhibit FXa activity under conditions of saturating FVa concentrations.

[0119] A "catalytically active portion" of a FXa polypeptide refers to a FXa polypeptide comprising a contiguous portion of the heavy chain amino acids, including the catalytic triad residues, but excluding the entire sequence of the heavy chain. The catalytically active portion may also comprise all or part of the FXa light chain. The catalytically active portion of a FXa polypeptide exhibits at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more activity compared to full-length FXa, such as at least about 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500% or more activity. The modified FXa or catalytically active portion thereof referred to herein means that the catalytically active portion comprises a modification (e.g., an amino acid substitution).

[0120] FXa can bypass other defective coagulation factors in the cascade and has the potential to be a therapeutic procoagulant, but direct use of fully functional FX as a therapeutic has proven to be impractical due to overactivation of systemic coagulation. Another limitation of direct use of FXa as a therapeutic is the short half-life of circulating FXa, which is due to its rapid inactivation by plasma protease inhibitors such as antithrombin (AT) III and protein C.

[0121] Round Viper Venom (RVV-X)

[0122] RVV-X is a metalloproteinase from the venom of Russell's viper or Daboiarusselli or Vipera russelli (e.g., Daboia russellii siamensis, Daboiarussellii russellii) that specifically activates factor X (Takeya et al. (1992) J. Biol. Chem., 267: 14109-14117). RVV-X has a molecular weight of 79,000 Daltons and contains a heavy chain and a light chain linked by a disulfide bond. RVV-X does not require phospholipids to activate factor X, but requires exogenous Ca 2+ The presence of the amino-terminal Gla domain enhances activation. In the presence of EDTA, the activity of RVV-X is inhibited. Purified RVV-X preparations are known and available (see Catalog No. RVVX-2010, Haematologic Technologies, Inc.; Catalog No. ab62233, Abcam, Cambridge, Mass.), and their sequences are known (see Takeya et al. (1992) J. Biol. Chem., 267: 14109-14117 and Uniprot No. Q7LZ61, Q4PRD1 and Q4PRD2). RVV-X binds to the Gla domain of FX and removes the heavy chain of FX. Metalloproteinase FX activators from other snake venoms may have similar catalytic mechanisms because their structures are similar to RVV-X.

[0123] RVV-X consists of three polypeptide chains: a heavy chain containing the catalytic domain and two light chains homologous to C-type lectins, which are believed to be involved in the Ca 2+In some embodiments, RVV-X comprises: a) i) a heavy chain (RVV-XH or α chain) comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 1; ii) a light chain 1 (RVV-XL1 or γ chain) comprising the sequence SEQ ID NO: 2, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2; and iii) a light chain 2 (RVV-XL2 or β chain) comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 3. NO:3 having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity; b) i) a heavy chain comprising the sequence of SEQ ID NO:1, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:1; ii) a light chain 1 comprising the sequence of SEQ ID NO:5, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:5; and iii) a light chain 2 comprising the sequence of SEQ ID NO:3, or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: NO:3 has at least 80% (e.g., at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity; or c) a mixture of a) and b). In some embodiments, RVV-X comprises: a) i) a heavy chain comprising the sequence SEQ ID NO:1; ii) a light chain 1 comprising the sequence SEQ ID NO:2; and iii) a light chain 2 comprising the sequence SEQ ID NO:3; b) i) a heavy chain comprising the sequence SEQ ID NO:1; ii) a light chain 1 comprising the sequence SEQ ID NO:5; and iii) a light chain 2 comprising the sequence SEQ ID NO:3; or c) a mixture of a) and b).When RVV-X comprises a mixture of SEQ ID NO: 2 or 5 (or a sequence having at least 80% sequence identity with SEQ ID NO: 2 or 5) as a γ chain, a γ chain of SEQ ID NO: 2 (or a sequence having at least 80% sequence identity with SEQ ID NO: 2) and a γ chain of SEQ ID NO: 5 (or a sequence having at least 80% sequence identity with SEQ ID NO: 2) are present. NO:5) can be in any ratio, such as 99.9:0.1, 99:1, 98:2, 97:3, 96:4, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 25:75, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 1:99, 0.1:99.9, or any ratio in between. In some embodiments, RVV-X comprises (consists essentially of, or consists of): i) a heavy chain comprising the sequence SEQ ID NO:1; ii) a light chain 1 comprising the sequence SEQ ID NO:2; and iii) a light chain 2 comprising the sequence SEQ ID NO:3.

[0124] In some embodiments, the RVV-X is a wild-type RVV-X. In some embodiments, the RVV-X is a natural variant of RVV-X. In some embodiments, the RVV-X is an analog of RVV-X, such as an RVV-X comprising no more than about 5 amino acid (such as 4, 3, 2 or 1aa) mutation sites. In some embodiments, the RVV-X is a RVV-X derivative. In some embodiments, the RVV-X is glycosylated. In some embodiments, the RVV-X is not glycosylated.

[0125] As used herein, the term "derivative of RVV-X" refers to a molecule having the amino acid sequence of RVV-X or an analog of RVV-X, but having additional chemical modifications on one or more amino acid groups, alpha carbon atoms, amino termini or carboxyl termini. Chemical modifications include, but are not limited to, adding chemical groups, generating new chemical bonds and removing chemical groups. Modifications of amino acid side groups include, but are not limited to, acylation of the epsilon amino group of lysine, N-alkylation of arginine, histidine or lysine, carboxyl alkylation of glutamic acid or aspartic acid, and deamination of glutamine or asparagine. Modifications of the amino terminus include, but are not limited to, deamination, N-low alkyl, N-di-low alkyl and N-acyl modifications. Modifications of the carboxyl terminus include, but are not limited to, amide, low alkyl acyl, dialkyl amide and low alkyl ester modifications. In some embodiments, the low alkyl group is a C1-C4 alkyl group. In addition, one or more side groups or terminal groups can be protected by a person skilled in the art of chemistry using known protecting groups. The alpha carbon of the amino acid can be monomethylated or dimethylated.

[0126] In some embodiments, the RVV-X is a modified RVV-X, such as a pegylated RVV-X, or a covalently modified RVV-X.

[0127] The RVV-X mentioned also includes functional fragments of wild-type RVV-X. In some embodiments, the RVV-X is a mutant RVV-X, such as comprising one or more mutations (e.g., amino acid insertions, deletions, substitutions, and truncations) on one or more polypeptide chains of wild-type RVV-X. When used as FX activators herein, functional fragments of wild-type RVV-X or mutant forms thereof retain at least 30% (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of the activity of wild-type RVV-X activator FX.

[0128] The amino acid sequence variants of the FX activators (e.g., RVV-X) described herein can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the protein or by polypeptide synthesis. The modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the FX activator (e.g., RVV-X). The final construct can be obtained by any combination of deletion, insertion and substitution, as long as the final construct has the desired properties, such as maintaining / improving (e.g., increasing by at least 1.2 times) FX activation, maintaining / extending half-life, reducing (e.g., reducing by at least 1.2 times) FX activation (such as reducing systemic over-activation of coagulation), etc.

[0129] In some embodiments, the FX activator (e.g., RVV-X) has one or more conservative amino acid substitutions. "Conservative substitution" refers to substitution by another amino acid that has the same net charge and approximately the same size and shape as the substituted amino acid. When the total number of carbon atoms and heteroatoms on the side chain differs by no more than 4, the amino acids with aliphatic or substituted aliphatic amino acid side chains are approximately the same size. When the number of branches on their side chains differs by no more than 1, the shape of the amino acids is approximately the same. Amino acids with phenyl or substituted phenyl on the side chain can be considered to have approximately the same size and shape. Unless otherwise indicated, conservative substitutions preferably use natural amino acids.

[0130] Table A shows conservative substitutions. More substantial substitutions are provided in Table A under the heading "Examples of Substitutions," as further described in the section below on amino acid side chain classes. Amino acids can be classified according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain direction: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions require replacing a member of one of these classes with a member of another class. Amino acid substitutions can be introduced into protein constructs and the products screened for the desired activity described above.

[0131] Table A Amino Acid Substitutions

[0132]

[0133] As used herein, the broad term "amino acid" includes both naturally occurring amino acids and non-naturally occurring amino acids, including amino acid analogs and derivatives. The latter include molecules containing amino acid moieties. Based on this broad definition, those skilled in the art will find that the amino acids described herein include, for example, natural L-amino acids that form proteins; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; natural amino acids that do not form proteins, such as norleucine, β-alanine, ornithine, GABA, etc.; and chemically synthesized compounds with amino acid characteristics known in the art. As used herein, the term "protein formation" refers to amino acids that can synthesize peptides, polypeptides, or proteins of cells through metabolic pathways.

[0134] Inserting non-natural amino acids, including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids, into the FX activators of the present invention (e.g., RVV-X) can have a variety of benefits. Compared with polypeptides containing L-amino acids, polypeptides containing D-amino acids and the like exhibit higher stability in vitro and in vivo. Therefore, when better intracellular stability is required, it is particularly useful to construct polypeptides such as by adding D-amino acids. In particular, D-peptides and their analogs are resistant to endogenous peptidase and protease activity, thereby increasing the bioavailability of the molecule and extending its life in vivo when needed. In addition, D-peptides and their analogs cannot be effectively processed because the process of presentation to helper T cells by the type II major histocompatibility complex (MHC) is limited, and therefore it is not easy to induce a humoral immune response in a subject.

[0135] In some embodiments, the FX activator (eg, RVV-X) is recombinantly expressed, such as in prokaryotic cells (eg, E. coli) or eukaryotic cells (eg, CHO cells).

[0136] In some embodiments, the FX activator (e.g., RVV-X) is isolated and / or purified from the venom of Vipera rotundifolia. In some embodiments, the RVV-X is isolated and / or purified as described in CN109943554B (the contents of which are incorporated herein by reference as a whole). In some embodiments, the RVV-X is sterile and / or inactivated by virus. In some embodiments, the purification of the RVV-X includes one or more steps of virus inactivation, anion exchange chromatography, ultrafiltration, hydroxyapatite (HA) chromatography (HAC) and size exclusion chromatography (SEC). In some embodiments, the purity of the RVV-X after separation and / or purification is at least 95% (e.g., at least 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100%). For example, in some embodiments, the purity of the RVV-X after separation and / or purification measured using high performance liquid chromatography (HPLC) is at least about 98%. In some embodiments, the purity of the isolated and / or purified RVV-X is at least 98% (e.g., 100%) as measured by SDS-PAGE. In some embodiments, the biological activity of the isolated and / or purified RVV-X is at least 2×10 4 U / mg (such as at least 2.5×10 4 U / mg, 2.7×10 4 U / mg, 2.9×10 4 U / mg, 3×10 4 U / mg, 4×10 4 U / mg, 5×10 4 U / mg, 1×10 5 U / mg, 1×106 U / mg or more).

[0137] Stabilizer

[0138] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a stabilizer (e.g., sucrose) in an amount of 0.1 mg / ml to 100 mg / ml, such as 0.1 mg / ml to 1 mg / ml, 1 mg / ml to 100 mg / ml, 2 mg / ml to 100 mg / ml, 2 mg / ml to 60 mg / ml, 10 mg / ml to 100 mg / ml, 1 mg / ml to 80 mg / ml, 10 mg / ml to 80 mg / ml, 10 mg / ml to 70 mg / ml, 10 From 1 mg / ml to 60 mg / ml, 1 mg / ml to 60 mg / ml, 1 mg / ml to 50 mg / ml, 10 mg / ml to 50 mg / ml, 10 mg / ml to 40 mg / ml, 10 mg / ml to 30 mg / ml, 15 mg / ml to 50 mg / ml, 20 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, 25 mg / ml to 50 mg / ml or 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a stabilizer (e.g., sucrose) in an amount of 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a stabilizer (e.g., sucrose) in an amount of 25 mg / ml, 30 mg / ml, 40 mg / ml, or 50 mg / ml, such as 30 mg / ml.

[0139] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a stabilizer (e.g., sucrose) in an amount of 0.1 mM to 300 mM, such as 0.1 mM to 1 mM, 1 mM to 300 mM, 1 mM to 200 mM, 1 mM to 100 mM, 10 mM to 300 mM, 10 mM to 200 mM, 10 mM to 100 mM, 50 mM to 300 mM, 50 mM to 200 mM, 50 mM to 100 mM, 50 mM to 90 mM or 70 mM to 90 mM. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a stabilizer (e.g., sucrose) in an amount of 87.64 mM.

[0140] In some embodiments, the stabilizer comprises or is a disaccharide. In some embodiments, the stabilizer is a mixture of sucrose and trehalose. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is an anhydrous form. In some embodiments, the stabilizer is a hydrated form, such as a dihydrated form.

[0141] In some embodiments, stabilizers can protect proteins from denaturation due to lyophilization. In some embodiments, stabilizers are used during lyophilization, and the resulting lyophilized agent and composition include stabilizers. In some embodiments, stabilizers enable pharmaceutical compositions to be free from degradation when exposed to stress (such as warming and / or radiation). In some embodiments, when the drug combination is applied to an injury or bleeding site, stabilizers can enhance the solubility of the pharmaceutical composition. In some embodiments, stabilizers increase the available shelf life of the pharmaceutical composition. In some embodiments, stabilizers provide a shelf life of at least 3 months (e.g., at least 6 months, 1 year, 2 years, 3 years or longer) to the pharmaceutical composition. As used herein, the term available shelf life refers to that the pharmaceutical composition does not show observable degradation when not magnified or magnified (such as a magnifying glass or microscope) is observed.

[0142] As described herein, any agent that can improve the stability and / or extend the shelf life of the pharmaceutical composition can be used as a stabilizer. Examples of stabilizers include, but are not limited to, glycine, alanine, glutamic acid, methionine, arginine, benzoic acid, citric acid, glycolic acid, lactic acid, malic acid, maleic acid, polyols (such as sorbitol, mannitol and trehalose), human serum albumin (HSA), sugars (such as glucose, fructose, galactose, maltose, lactose and sucrose), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethyldiaminetriacetic acid (HEDTA), ethylene glycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), metal ion stabilizers and citrates.

[0143] Buffer

[0144] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml, such as 0.1 mg / ml to 1 mg / ml, 0.1 mg / ml to 5 mg / ml, 1 mg / ml to 50 mg / ml, 1 mg / ml to 40 mg / ml, 1 mg / ml to 30 mg / ml, 2 mg / ml to 20 mg / ml, 1 mg / ml to 10 mg / ml, 2 mg / ml to 15 mg / ml, 1 mg / ml to 8 mg / ml, 3 mg / ml to 5 mg / ml, 2.5 mg / ml to 5 mg / ml, or 1 mg / ml to 5 mg / ml. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a buffer (e.g., histidine) at 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, or 50 mg / ml. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a buffer (e.g., histidine) at 2.5 mg / ml, 3 mg / ml, 4 mg / ml, or 5 mg / ml, such as 3 mg / ml.

[0145] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a buffer (e.g., histidine) in an amount of 0.5 mM to 350 mM, such as 0.5 mM to 1 mM, 1 mM to 300 mM, 1 mM to 200 mM, 1 mM to 100 mM, 1 mM to 50 mM, 10 mM to 50 mM, 50 mM to 300 mM, 100 mM to 300 mM, 5 mM to 40 mM, 5 mM to 30 mM, 10 mM to 30 mM, or 10 mM to 20 mM. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a buffer (e.g., histidine) in an amount of 19.33 mM.

[0146] The buffer or buffers associated with the present invention are compatible with the protein (e.g., RVV-X) in the desired end-use application, provide sufficient buffering capacity at a concentration consistent with an acceptable osmotic pressure, and are inert, stable, and have a maximum buffering capacity at or near the desired pH.

[0147] According to various aspects and embodiments of the present invention, a variety of buffers can be used in the lyophilized solution. The same buffer can be used to redissolve the lyophilized powder. Buffers include, but are not limited to, citrate, citrate-phosphate, phosphate, acetate, succinate, tartrate, maleate, HEPES, Tris, Bicine, glycine, N-glycine, carbonate, bisglycine, lysine, arginine, histidine and / or mixtures thereof. In certain embodiments, the buffer is sodium acetate. In certain embodiments, the buffer includes one or both of histidine and arginine. In certain embodiments, the buffer is histidine.

[0148] Buffers according to the present invention are effective in maintaining a suitable pH. The exact optimal pH will vary from protein to protein. Therefore, different buffer systems may be more or less effective than other buffer systems for different proteins. However, in general, preferred buffers are effective in the pH range of 5 to 8 or 6 to 8, particularly in the range of 5.5 to 7.5.

[0149] Surfactants

[0150] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v), such as 0.001% (w / v) to 0.01% (w / v), 0.005% (w / v) to 0.05% (w / v), 0.005% (w / v) to 0.01% (w / v), 0.01% (w / v) to 0.1% (w / v), 0.01% (w / v) to 0.05% (w / v), or 0.01% (w / v) to 0.03% (w / v). In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a surfactant (e.g., polysorbate 20) at a content (w / v) of 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises a surfactant (e.g., polysorbate 20) at a content of 0.01% (w / v), 0.02% (w / v), or 0.03% (w / v), such as 0.02% (w / v).

[0151] Surfactants can stabilize the formulation during preparation and storage by reducing interfacial interactions and preventing protein adsorption. Surfactants can help dissolve proteins and protect proteins from oscillation-induced aggregation, which makes the formulation not cause active protein denaturation when exposed to shear surface stress. Any known surfactant can be used herein. In certain embodiments, the surfactant includes one or both of polysorbate and poloxamer. In certain embodiments, the surfactant is selected from one or more of polysorbate 20, polysorbate 80 and poloxamer 188. In certain embodiments, the surfactant is polysorbate 20 (PS20).

[0152] In some embodiments, the surfactant is a nonionic surfactant. Nonionic surfactants include, but are not limited to, polysorbate, polysorbate 20. Polysorbate 80. Polysorbate 40. Polysorbate 60; polyoxopolymers, such as poloxamer 184 and 188; Polyols; Span (e.g., sorbitan); Ethoxylates; Alcohol ethoxylates; Alkylphenol polyoxyethylene ethers (e.g., Triton X-100); Polyols; fatty acid ethoxylates; lauromacrogol 400; polyoxyl 40 stearate; polyoxyethylene hydrogenated castor oil 10, 50 and 60; glyceryl monostearate; sucrose fatty acid esters; methylcellulose; carboxymethylcellulose; and other ethylene / polypropylene block polymers. The nonionic surfactant is present in the range of about 0.05 mg / ml to 1.0 mg / ml, preferably 0.07 mg / ml to 0.2 mg / ml.

[0153] In some embodiments, the surfactant is an ionic surfactant, such as an anionic surfactant (e.g., sodium dodecyl sulfate (SDS), sodium dioctyl dibutyrate sulfonate, sodium dioctyl sulfonate) or a cationic surfactant (e.g., benzalkonium chloride, benzethonium chloride).

[0154] In the freeze-drying process of the FX activator composition, the surfactant can not only reduce the freezing and dehydration deformation caused by the ice-water interfacial tension during the freezing and dehydration process, but also play the role of a wetting agent and a heavy crease agent for the active ingredient during the rehydration process. The inventors of the present invention have found that using at least one of polysorbate 20, polysorbate 80 and poloxamer 188 as a surfactant not only has low toxicity, but also can effectively inhibit the surface adsorption and aggregation of the FX activator under various conditions, thereby improving the stability of the composition.

[0155] excipient

[0156] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises an excipient (e.g., mannitol) in an amount of 1 mg / ml to 100 mg / ml, such as 1 mg / ml to 50 mg / ml, 10 mg / ml to 80 mg / ml, 10 mg / ml to 60 mg / ml, 5 mg / ml to 50 mg / ml, 20 mg / ml to 50 mg / ml, 30 mg / ml to 60 mg / ml or 30 mg / ml to 50 mg / ml. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises an excipient (e.g., mannitol) in an amount of 1 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises an excipient (e.g., mannitol) at 30 mg / ml, 40 mg / ml, 50 mg / ml, or 60 mg / ml, such as 40 mg / ml. In some embodiments, the excipient is mannitol.

[0157] In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises an excipient (e.g., mannitol) in an amount of 5mM to 550mM, such as 5mM to 50mM, 10mM to 500mM, 50mM to 400mM, 50mM to 300mM, 100mM to 500mM, 100mM to 400mM, 100mM to 300mM, 150mM to 400mM, 150mM to 300mM, 180mM to 250mM or 200mM to 220mM. In some embodiments, the pharmaceutical composition (e.g., lyophilized) comprises an excipient (e.g., mannitol) in an amount of 219.58mM.

[0158] Pharmaceutical compositions as described herein include one or more excipients. Excipients reduce local irritation caused by pharmaceutical preparations / compositions by preventing osmotic shock at the site of application. Excipients may be present to regulate or maintain the tension of liquids in the composition. When used with charged biomacromolecules (such as proteins), excipients may interact with the charged groups of amino acid side chains to reduce the possibility of intermolecular and intramolecular interactions. Considering the relative content of other ingredients, excipients may be present in a weight ratio of 0.1%-25%, preferably 1%-5%. Examples of excipients include, but are not limited to, sugar alcohols or polyols (such as mannitol or sorbitol), nonionic surfactants (such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80), and sugars (such as sucrose). Preferred excipients include polyhydroxy sugar alcohols, preferably trihydroxy or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0159] pH

[0160] In some embodiments, the pH value of the pharmaceutical composition is 6.0 to 8.0, such as 6.3 to 7.3, 6.8 to 7.0, 6.0 to 7.0, 7.0 to 8.0, 6.5 to 7.5 or 6.5 to 7.0. In some embodiments, the pH value of the pharmaceutical composition is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.85, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. In some embodiments, the pH value of the pharmaceutical composition is 6.8 ± 0.5. In some embodiments, the pH value of the pharmaceutical composition is 6.8 or 6.85.

[0161] Other components

[0162] Acceptable carriers or excipients that are nontoxic to recipients at the dosages and concentrations employed may be used in the pharmaceutical compositions, including antioxidants, including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); isotonic agents (e.g., sodium chloride); metal complexes (e.g., zinc protein complexes); low molecular weight (less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, inositol, inositol, galactose, galactitol, glycerol, cyclic alcohols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; salt-forming counterions such as sodium; and / or chelating agents such as EDTA.

[0163] The addition of preservatives can retard the growth of microorganisms and is generally present in the range of 0.2%-1.0% (w / v). The addition of preservatives can facilitate the production of multiple-use (multiple-dose) formulations. Preservatives suitable for use in the present application include octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol.

[0164] Antioxidants

[0165] In some embodiments, the pharmaceutical composition further comprises an antioxidant, such as methionine, sodium thiosulfate, catalase, platinum, vitamin E, sodium pyrosulfite, etc. In some embodiments, the antioxidant is methionine. In some embodiments, the content of the antioxidant is 0.01mg / ml to 1mg / ml, such as 0.01mg / ml to 0.05mg / ml, 0.01mg / ml to 0.1mg / ml, 0.1mg / ml to 1mg / ml or 0.05mg / ml to 1mg / ml. In some embodiments, the content of the antioxidant is 0.01mg / ml, 0.05mg / ml, 0.1mg / ml, 0.2mg / ml, 0.3mg / ml, 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.7mg / ml, 0.8mg / ml, 0.9mg / ml or 1mg / ml.

[0166] Antioxidants can impart stability to protein formulations and / or resist free radical-induced oxidation or degradation. Free radical oxidation or degradation may occur due to one or more adverse conditions, such as exposure to light, including ultraviolet light, visible light and / or fluorescent light, exposure to free radicals generated from other sources, and / or exposure to metal ions. Any known antioxidant can be used in the pharmaceutical compositions described herein.

[0167] The antioxidant can also be selected from the group consisting of vanillic acid, gallic acid, syringic acid, protocatechuic acid, cinnamic acid, coumaric acid, caffeic acid, ferulic acid, sinapic acid, chlorogenic acid, benzoic acid, methylparaben, ethylparaben, propylparaben, butylparaben, m-cresol, phenoxyethanol, phenol, cytosine, thiamine, pyridinol, fumaric acid, shikimic acid, glycolic acid, p-hydroxybenzoic acid, p-aminobenzoic acid, catechin, epicatechin, furanol, sulfaphenone, pharmaceutically acceptable salts or esters, and mixtures thereof.

[0168] Calcium salt

[0169] In some embodiments, the pharmaceutical composition further comprises a calcium salt. In some embodiments, the calcium salt is calcium chloride. In some embodiments, the content of the calcium salt is 0.1mg / ml to 10mg / ml, such as 0.1mg / ml to 1mg / ml, 0.5mg / ml to 3mg / ml, 1mg / ml to 10mg / ml, 1mg / ml to 5mg / ml, 5mg / ml to 10mg / ml. In some embodiments, the content of the calcium salt is 0.1mg / ml, 0.5mg / ml, 1mg / ml, 2mg / ml, 3mg / ml, 4mg / ml, 5mg / ml, 6mg / ml, 7mg / ml, 8mg / ml, 9mg / ml or 10mg / ml. In some embodiments, the content of the calcium salt is 1mg / ml, 3mg / ml or 10mg / ml.

[0170] Any suitable calcium salt may be used herein, including but not limited to calcium chloride, calcium phosphate, calcium acetate, calcium citrate, calcium carbonate, and the like.

[0171] Bioactivity

[0172] As used herein, "biological activity" refers to the in vivo activity of a compound, or the physiological response resulting from the administration of a compound, composition, or other mixture in vivo. Therefore, biological activity includes the therapeutic effects and pharmaceutical activities of these compounds, compositions, and mixtures. The biological activity can be observed in the in vitro system designed to test or apply these activities. Therefore, for the purposes herein, the biological activity of FX polypeptides includes coagulation activity.

[0173] Bioassays focus on the biological activity of the test protein (e.g., FX activator) and use it as a readout. In a bioassay, the activity of the sample is tested on a sensitive sample (e.g., human plasma) or an animal model / human of a disease associated with the test protein (e.g., a bleeding disorder), and the results of the activity (e.g., hemostasis) are compared with a control (e.g., other hemostatic agents). Other aspects of the biological activity of FX activators include: (i) activating FX and / or promoting FXa production; (ii) increasing thrombin generation (TG) and / or endogenous thrombin generation potential (ETP); (iii) shortening the activated partial thromboplastin time (APTT), prothrombin time (PT) and / or thrombin time (TT); (iv) promoting hemostasis, such as reducing bleeding time and / or bleeding volume; (v) reducing mortality; (vi) promoting wound healing, etc.

[0174] Various in vitro or in vivo methods for determining the biological activity of FX activators as described herein (e.g., FX activators derived from snake venom (e.g., RVV-X)) are known in the art. There are two main techniques for measuring the activity of FX activators. One is the plasma clotting time method, which determines the activity of the FX activator by measuring the plasma clotting time and comparing it with the clotting time of an active control. The other is the chromogenic substrate continuous rate method, which determines the activity of the FX activator by comparing the reaction rate of the test sample with that of the active control. See J. Li et al. ("The activity determination of factor X activator from Vipera russelli by an enzyme rate method using chromogenic substrates," Chinese Journal of New Drugs. 2007; 16 (18); 1491-1495) and CN108089006B, the contents of which are incorporated herein by reference in their entirety. The general mechanism is: the FX activator converts inactive FX into FXa with hydrolysis activity, and the generated FXa in turn hydrolyzes the synthesized chromogenic substrate Suc-Ile-Gly (γPip) Gly-Arg-pNA (SEQ ID NO: 4), and the hydrolysis substrate can release p-nitroaniline with a characteristic absorption peak at 405nm. The reaction rate can be calculated by the amount of product (p-nitroaniline) generated per unit time, and the enzyme activity is proportional to the reaction rate. The activity unit (U) of the test sample (FX activator) can be calculated with reference to the reaction rate of the active reference substance. Unless otherwise specified, the activity unit (U) of the FX activator described herein (e.g., RVV-X) is measured using the chromogenic substrate continuous rate method as described in CN108089006B.

[0175] The products and / or intermediates obtained after administration of FX activators can be subjected to analytical tests such as FX ELISA, TGA (thrombin generation assay), and FXa-specific activity assays. Although the chromogenic substrate for measuring FXa is very specific for the protease, it is not 100%, so a small amount of cleavage of other potential targets will also occur, especially when the protease concentration is high. In the presence of cross-reacting proteins, the FXa-specific activity assay can still be used to measure FXa. To determine FXa-specific activity, a highly specific inhibitor of FXa (i.e., rivaroxaban) can be used to measure the amount of inhibition, which represents the specific activity of the protease. The activation efficiency can be calculated and expressed as a percentage of FXa produced from the total FX added to the reaction. TGA can be performed according to the manufacturer's operating instructions, such as using TGA kit (Technoclone). The readout parameter is the peak concentration of hemocoagulase (PTC), nM.

[0176] The biological activity of FX activators (e.g., RVV-X) can also be measured using a coagulometer, such as measuring the clotting time. Coagulation tests can also be used to assess the coagulation function of a subject, reflecting the biological activity of the FX activator, such as prothrombin time (PT; measuring the extent and time of blood clotting), activated partial thromboplastin time (aPTT; measuring the time of blood clotting) and thrombin time (TT; measuring the working condition of fibrinogen). See Examples 1-3, 6 and 10.

[0177] stability

[0178] The pharmaceutical composition is preferably stable, wherein the protein (FX activator, e.g., RVV-X) contained therein substantially maintains its physical and chemical stability and integrity during storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected time period under selected temperature, humidity, illumination or other conditions. For rapid screening, the formulation can be placed at 40° C. for 2 weeks to 1 month, at which point stability is measured. If the formulation is to be stored at 2-8° C., in general, the formulation should remain stable at 30° C. or 40° C. for at least 1 month and / or remain stable at 2-8° C. for at least 2 years. If the formulation is to be stored at 30° C., in general, the formulation should remain stable at 30° C. for at least 2 years and / or remain stable at 40° C. for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability.

[0179] In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition described herein (e.g., RVV-X lead formulation) has excellent stability, such as physical stability, chemical stability and / or biological stability. In some embodiments, the FX activator pharmaceutical composition described herein has excellent thermal stability. In some embodiments, the FX activator pharmaceutical composition described herein has excellent stability under accelerated stability conditions (e.g., 25°C, RH 65% ± 5%; or 25°C ± 2°C, RH 60% ± 5%), long-term storage conditions (e.g., 2-8°C for at least 3 months) and / or stress conditions (e.g., high temperature (e.g., 40 ± 2°C), high humidity (e.g., 2-8°C, RH 92.5% ± 5%), light (e.g., 2-8°C, 4500 ± 500LX)), such as less or no fragmentation, aggregate formation, insoluble particles (especially insoluble particles greater than 10 μm) and / or aggregation increment compared to FX activator pharmaceutical compositions using HSA as stabilizers.

[0180] The stability of proteins, especially the sensitivity to aggregation, depends mainly on the conformation and colloidal stability of protein molecules. It is generally believed that the first step of non-native protein aggregation, i.e., the most common form of aggregation, is a slight disturbance of the molecular structure, for example, partial unfolding of the protein, i.e., conformational change. This is determined by the conformational stability of the protein. In the second step, partially unfolded molecules approach to form aggregates driven by diffusion and random Brownian motion. The second step is mainly determined by the colloidal stability of the molecule (see Chi et al., Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony stimulating factor. Protein Science, 2003 May; 12 (5): 903-913). As used herein, the term "stability" generally refers to maintaining the integrity of a biologically active substance (such as a protein) or minimizing its degradation, denaturation, aggregation or unfolding. As used herein, "improved stability" generally means that a protein of interest (or a protein in a pharmaceutical composition of interest) maintains better stability under conditions known to cause degradation, denaturation, aggregation, or unfolding than a control protein (or the same protein in a control pharmaceutical composition).

[0181] Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) are techniques known in the art for predicting the stability of protein formulations. Specifically, these techniques can be used to determine the unfolding temperature (Tm) of a protein in a given formulation. It is standard practice in the art to associate a high Tm measurement for a protein in a given formulation with a more reliable and stable protein formulation that can be used for long-term, stable storage.

[0182] A "stable" protein (or formulation), e.g., a FX activator (e.g., RVV-X) pharmaceutical composition described herein, substantially maintains its physical stability and / or chemical stability and / or biological activity during manufacturing and / or storage. There are a variety of analytical techniques for measuring protein stability in the art, and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90. For example, in one embodiment, the stability of a protein (or formulation) is determined based on the percentage of monomeric protein in a solution, wherein the percentage of degraded (e.g., fragmented) and / or aggregated protein is low. Preferably, the protein (or formulation) is stable at room temperature or 40°C for at least 1 month and / or at 2-8°C for at least 6 months, or at least 1 year or at least 2 years. Furthermore, the protein (or formulation) is preferably stable after freezing (eg, -70°C to -80°C) and thawing, hereinafter referred to as a "freeze / thaw cycle."

[0183] A protein (or formulation), e.g., a FX activator (e.g., RVV-X) pharmaceutical composition described herein, "retains its physical stability" in a formulation if there is substantially no evidence of instability, such as aggregation, precipitation, and / or denaturation, as measured over time by visual inspection (e.g., color, fullness, smoothness, clarity, adherence) or by ultraviolet light scattering or multi-angle light scattering size exclusion chromatography (SEC-MALS), SEC, SEC-HPLC, reverse phase chromatography (RP-HPLC), or imaged capillary isoelectric focusing (iCIEF). Aggregation is the process by which individual protein molecules or complexes covalently or non-covalently associate to form aggregates. Aggregation can proceed to the extent that a visible precipitate is formed.

[0184] A protein (or formulation), e.g., a FX activator (e.g., RVV-X) pharmaceutical composition described herein, "maintains its chemical stability" in a formulation if the chemical stability over a given period of time is such that the protein still maintains its biological activity (e.g., as described in the "Biological Activity" section above). Chemical stability can be assessed, for example, by detecting and quantifying chemically altered forms of the protein. Chemical changes may involve size changes (e.g., shearing) and can be assessed using size exclusion chromatography (SEC), SDS-PAGE (e.g., reducing or non-reducing), capillary electrophoresis sodium dodecyl sulfate (CE-SDS, reducing or non-reducing), and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes (e.g., changes due to deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.

[0185] A protein (or formulation), e.g., a FX activator (e.g., RVV-X) pharmaceutical composition described herein, "retains its biological activity" in a formulation if the protein in the pharmaceutical formulation is biologically active for its intended purpose. For example, a protein in a formulation retains its biological activity if the biological activity of the protein does not change by more than 30%, 20%, or 10% (within the analytical error range) of the biological activity exhibited when the formulation was prepared.

[0186] Those skilled in the art will appreciate that the stability of proteins (e.g., FX activators, such as RVV-X) depends on other properties in addition to the composition of the formulation. For example, stability may be affected by temperature, stress, humidity, pH, light, and external radiation. The stability of proteins in protein formulations can be determined by a variety of methods. In some embodiments, protein stability is determined by SEC. SEC separates analytes according to the hydrodynamic size, diffusion coefficient, and surface properties of analytes (e.g., macromolecules such as proteins). Thus, for example, SEC can separate the FX activators (e.g., RVV-X) of the native three-dimensional conformation described herein from proteins in various denatured states and / or degraded proteins. In SEC, the stationary phase is typically composed of inert particles filled in a dense three-dimensional matrix in a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, a mixture thereof, or other solvents. The stationary phase particles have pores and / or channels that only allow substances less than a certain size to enter. Therefore, large particles are excluded from these pores and channels, but smaller particles are removed from the mobile phase. The amount of time that particles are held in fixed pores depends in part on how deeply they can penetrate into the pores. Their removal from the mobile phase flow causes them to take longer to elute from the column, thus separating the particles based on their size differences.

[0187] In some embodiments, SEC is combined with an identification technique to identify or characterize a protein (e.g., a FX activator such as RVV-X) or a fragment thereof. Protein identification and characterization can be accomplished by a variety of techniques, including but not limited to chromatographic techniques, such as high performance liquid chromatography (HPLC), capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), immunoassays, electrophoresis, UV / visible / infrared spectroscopy, Raman spectroscopy, surface enhanced Raman spectroscopy, mass spectrometry, gas chromatography, static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS protein binding.

[0188] In some embodiments, the sample preparation (e.g., the FX activator pharmaceutical composition described herein, e.g., sucrose is used as a stabilizer) and the control preparation (e.g., the FX activator pharmaceutical composition or standard containing HSA as a stabilizer) are optionally assayed before the treatment stage to determine the content of monomers, aggregates and / or fragmented proteins (and / or percentage increase in fragments, percentage increase in aggregates, etc.), as described in Example 6 below. Subsequently, each protein preparation undergoes a treatment stage. For example, each protein preparation can be stored at a specific temperature (e.g., 40° C., 25° C., or 5° C.) for a long time (e.g., 3 months, 6 months, 12 months, or longer). In some embodiments, the protein preparation is subjected to a physical stress test, such as a stirring stress test. In some embodiments, the protein preparation is subjected to a reconstitution test or a solvent compatibility test. In some embodiments, the protein preparation is subjected to an accelerated stability test, such as treatment under accelerated stress, including high temperature (e.g., 40° C. or above), high humidity, light, and / or low pH, etc. In some embodiments, the protein preparation is subjected to a freeze and thaw cycle. In some embodiments, samples of the same protein preparation are subjected to different treatments, for example, storage for a period of time at different temperatures / humidities / light exposures. After the treatment period, the protein preparation is assayed to determine the content of protein monomers, aggregates and / or fragments (and / or the percentage increase in fragments, percentage increase in aggregates, etc.). The physical appearance or pH value changes of the protein preparation can also be determined.

[0189] Stability, such as the physical stability of a composition or formulation, can be assessed by methods known in the art, including measuring the apparent optical attenuation (absorbance or optical density) of a sample. Such measurements of optical attenuation are related to the turbidity of the formulation. The turbidity of a formulation is an inherent property of proteins dissolved in solution, typically determined by turbidimetry and measured in nephelometric turbidity units (NTU).

[0190] Turbidity, for example, as a function of the concentration of one or more components in a solution, for example, protein and / or salt concentration, also refers to the "milky white" or "milky appearance" of a formulation. Turbidity can be calculated by using a standard curve generated using a suspension of known turbidity. Reference standards for determining the turbidity of a pharmaceutical composition can be based on European Pharmacopoeia standards (European Pharmacopoeia, 4th edition, European Commission Directorate for the Quality of Medicines (EDQM), Strasbourg, France). According to European Pharmacopoeia standards, a clear solution is defined as a solution having a turbidity less than or equal to that of a control suspension, the control suspension having a turbidity of about 3 according to European Pharmacopoeia standards. In the absence of associated or non-ideal effects, turbidity measurements can detect Rayleigh scattering, which generally varies linearly with concentration. Other methods for evaluating the physical stability of a pharmaceutical protein are well known in the art, for example, size exclusion chromatography or analytical ultracentrifugation.

[0191] In some embodiments, stability refers to formulations comprising a FX activator described herein (eg, RVV-X) having low to undetectable levels of microparticle formation, or low to undetectable levels of formation of microparticles greater than 10 μm in diameter. As used herein, the phrase "low to undetectable level of particle formation" refers to a sample containing less than 2500 particles / ml, 2000 particles / ml, 1800 particles / ml, 1500 particles / ml, 1400 particles / ml, 1200 particles / ml, 1000 particles / ml, 950 particles / ml, 900 particles / ml, 700 particles / ml, 500 particles / ml, 400 particles / ml, 300 particles / ml, 200 particles / ml, 150 particles / ml, 120 particles / ml, 110 particles / ml, 100 particles / ml, 50 particles / ml, 30 particles / ml, 20 particles / ml, 15 particles / ml, 10 particles / ml, 5 particles / ml or less as determined by HIAC analysis, visual analysis or in accordance with the fourth general rule of the Chinese Pharmacopoeia 2015 edition. In some embodiments, the FX activator pharmaceutical compositions described herein (e.g., after reconstitution) have no more than about 350 insoluble particles / mL, such as no more than about 200 or no more than about 150 insoluble particles / mL. In some embodiments, the FX activator pharmaceutical compositions described herein (e.g., after reconstitution) have no more than about 2500 insoluble particles / mL, such as no more than about 1500 or no more than about 1000 insoluble particles / mL after 6 months under accelerated storage conditions.

[0192] "Substantial protein aggregation" refers to a level of protein aggregation in a protein formulation that is significantly higher than the level of protein aggregation in a control protein formulation. The control protein formulation can be the same protein formulation before storage or before treatment (e.g., before being subjected to unstable conditions, such as elevated temperature, humidity, pH, and / or long-term storage). The control protein formulation can be a different protein formulation tested under the same conditions (e.g., other FX activator formulations using HSA as a stabilizer).

[0193] "Substantially free of protein aggregation" refers to a protein (or preparation) of the invention whose level or percentage of protein aggregation is not significantly higher than a control preparation. For example, the term refers to a protein (or preparation) in which the level of protein aggregation is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1%. The level of protein aggregation can be determined using standard techniques known in the art. In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition described herein is substantially free of protein aggregation (e.g., under accelerated stability testing or long-term storage conditions). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) has a maximum of 15% protein aggregation, such as a maximum of 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% protein aggregation (e.g., accelerated stability testing, repeated freeze / thaw (e.g., up to 5 times) or long-term storage conditions). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition described herein (e.g., after reconstitution) has no protein aggregation (e.g., accelerated stability testing, repeated freeze / thaw (e.g., up to 5 times) or long-term storage conditions). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition has no more than 15% increase in aggregation, e.g., no more than 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% increase in aggregation (e.g., accelerated stability testing, repeated freeze / thaw (e.g., up to 5 times) or long-term storage conditions). In some embodiments, stability is measured by SEC or SEC-HPLS. In some embodiments, stability is measured by CE-SDS.

[0194] In some embodiments, stability refers to reduced protein fragmentation. The term "low to undetectable fragment levels" as used herein refers to a sample containing equal to or greater than 80%, 85%, 90%, 95%, 98% or 99% of the total protein, for example, in a single peak determined by HPSEC, or in multiple peaks determined by reduced capillary gel electrophoresis (rCGE) (e.g., peaks with the same number of subunits), representing undegraded protein or its undegraded fragments, and does not contain other single peaks that account for more than 5%, more than 4%, more than 3%, more than 2%, more than 1% or more than 0.5% of the total protein. The term "reduced capillary gel electrophoresis" as used herein refers to capillary gel electrophoresis performed under reducing conditions sufficient to reduce disulfide bonds in the test protein. In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) has 0% to 15% fragments, such as 0% to 12% fragments (e.g., under accelerated stability testing or long-term storage conditions). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) has no more than 30% fragments, such as no more than 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% fragments (e.g., under accelerated stability testing or long-term storage conditions). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) has no fragments (e.g., under accelerated stability testing or long-term storage conditions). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) has a fragment increase of no more than 30%, such as a fragment increase of no more than 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (e.g., under accelerated stability testing or long-term storage conditions). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) has at least 75% of the main peak, such as at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the main peak (e.g., under accelerated stability testing or long-term storage conditions). In some embodiments, stability is measured by SEC. In some embodiments, stability is measured by CE-SDS.

[0195] In some embodiments, the "stability" or "stable" characteristics of a pharmaceutical composition include one or more of appearance, moisture content, reconstitution time, pH, and biological activity.

[0196] In some embodiments, the moisture content of the lyophilized FX activator (e.g., RVV-X) pharmaceutical composition is at most 5%, such as at most 4.5%, 4.0%, 3.5%, 3.0%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.8%, 0.5% or less. In some embodiments, the reconstitution time of the lyophilized FX activator (e.g., RVV-X) pharmaceutical composition is at most 30 seconds, such as at most 28, 26, 24, 22, 20, 18, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 seconds or less. In some embodiments, the shrinkage of the FX activator (e.g., RVV-X) pharmaceutical composition after lyophilization is up to 10% (e.g., compared to a fine / full powder), such as up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. In some embodiments, the lyophilized FX activator (e.g., RVV-X) pharmaceutical composition is a colorless clear liquid after reconstitution. In some embodiments, the pH of the lyophilized FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) changes (e.g., as compared to before lyophilization or before the start of the storage condition with an impact, increases or decreases) by up to 1, such as up to 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or less (e.g., 0). In some embodiments, the biological activity of the lyophilized FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) is altered (e.g., as compared to before lyophilization or before initiation of the influencing storage conditions, is increased or decreased) by up to 30%, such as up to 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0).In some embodiments, the above characteristics are applicable to standard storage conditions (e.g., at least 2 hours at 2-8°C after reconstitution), high temperature conditions (e.g., at least 35°C, 36°C, 38°C, 40°C, 42°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C or higher), high humidity conditions (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 97.5% or higher at 2-8°C), light conditions (e.g., at least 4000lx, 4200lx, 4400lx, 4500lx, 4600lx, 4800lx, 5000lx, 5500lx or higher at 2-8°C), long-term storage conditions (e.g., at least 1 hour at 2-8°C or below -70°C), and high humidity conditions (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 97.5% or higher at 2-8°C). , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36 months or longer), freeze-thaw conditions (e.g., -70°C / 2-8°C freeze-thaw 1, 2, 3, 4, 5 or more times) or accelerated storage conditions (e.g., 25°C ± 2°C, RH at least 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 97.5% or more), such as being placed under the corresponding storage conditions for at least 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 16 days, 20 days, 25 days, 30 days, 1 month, 2 months, 3 months, 6 months, 30 months or longer. In some embodiments, the biological activity of the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) is increased by up to 20% (e.g., up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%)) compared to before the initiation of the influencing storage conditions (e.g., high temperature, high humidity, light, long-term storage or acceleration), such as up to 30 months (e.g., up to 18 months, 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 28 days, 20 days, 14 days, 10 days, 7 days, 5 days, 3 days or 1 day) under the influencing storage conditions.

[0197] In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition described herein may contain less than 10% (preferably less than 5%) of the FX activator in the form of aggregates or insoluble particles in the pharmaceutical composition (e.g., after reconstitution of the lyophilized powder). In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition (e.g., lyophilized) is stable at 25° C. for at least 4 hours (e.g., at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72 hours or longer). In some embodiments, the pharmaceutical composition (e.g., lyophilized) is stable at 2-8° C. (e.g., 2, 3, 4, 5, 6, 7 or 8° C.) for at least 3 months (e.g., at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60 months or longer). In some embodiments, the pharmaceutical composition (e.g., lyophilized) is stable under accelerated stability conditions at 25° C. for at least 3 months (e.g., at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60 months or longer). In some embodiments, the pharmaceutical composition comprises less than 100 (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20 or 10) insoluble particles having a diameter greater than 10 μm (e.g., at least 10, 15, 20, 25, 30 μm or more) after storage under accelerated stability conditions at 25° C. for at least 6 months (e.g., at least 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48 or longer). In some embodiments, the lyophilized pharmaceutical composition is placed at 2-8°C (e.g., 2, 3, 4, 5, 6, 7 or 8°C) for at least 6 months (e.g., at least 9, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60 months or longer), such as at least 30 months, and is stable. In some embodiments, the lyophilized pharmaceutical composition is placed at 25°C ± 2°C (e.g., 23, 24, 25, 26 or 27°C) for at least 1 month (e.g., at least 2, 3, 4, 5, 6, 10, 12 months or longer), such as at least 6 months, and is stable.For example, the biological activity of the FX activator (e.g., RVV-X) is changed (e.g., increased or decreased as compared to before the start of the long-term or accelerated storage conditions) by up to 10% (e.g., up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%)); the pH value is changed (e.g., increased or decreased as compared to before the start of the long-term or accelerated storage conditions) by up to 0.5 ( or a change in moisture content (e.g., an increase or decrease, as compared to before the start of long-term or accelerated storage conditions) of up to 5% (e.g., up to 4%, 3%, 2% 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%)).

[0198] In some embodiments, the FX activator pharmaceutical composition is non-lyophilized. In some embodiments, the pharmaceutical composition (e.g., non-lyophilized) is stable for at least 6 months (e.g., at least 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60 months or longer) at -70°C or below, such as at least 24 months. In some embodiments, the pharmaceutical composition (e.g., non-lyophilized) is stable for at least 1 month (e.g., at least 2, 3, 4, 5, 6, 8, 10, 12 months or longer) at 2-8°C, such as at least 6 months. In some embodiments, the pharmaceutical composition (e.g., non-lyophilized) is stable after at least 1 freeze / thaw (e.g., at least 2, 3, 4, 5, 6, 10 times or more), such as up to 5 times. For example, the protein content thereof is altered (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increased or decreased) by up to 10% (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increased or decreased) by up to 10% (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increased or decreased) by up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%)); the biological activity of the FX activator (e.g., RVV-X) is altered (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increased or decreased) by up to 10% (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increased or decreased) by up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%). the pH value changes (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increases or decreases) by up to 0.5 (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increases or decreases) by up to 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09, 0.08, 0.05, 0.02, 0.01 or less (e.g., 0)); the purity (e.g., as measured by SEC-HPLC) changes (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increases or decreases) by up to 10% (e.g., as compared to before the start of long-term, accelerated or freeze / thaw storage conditions, increases or decreases) by up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%).

[0199] The FX activator (e.g., RVV-X) pharmaceutical compositions described herein can be reconstituted with any suitable solvent, such as a solvent for intravenous administration. In some embodiments, the solvent is sterile water for injection, 0.9% sodium chloride injection, or 5% glucose injection. In some embodiments, the solvent is 0.9% sodium chloride injection. In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition is reconstituted and stored at room temperature for at least 2 hours (e.g., at least 3, 4, 5, 6, 7, 8 or longer), such as after 8 hours, compared to 0 hours after reconstitution, its biological activity changes (e.g., increases or decreases) by up to 30%, such as up to 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%). In some embodiments, the pH of the FX activator (e.g., RVV-X) pharmaceutical composition changes (e.g., increases or decreases) by at most 0.5, such as at most 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.09, 0.08, 0.05, 0.02, 0.01 or less (e.g., 0) after storage at room temperature for at least 2 hours (e.g., at least 3, 4, 5, 6, 7, 8 or longer), such as 8 hours, compared to 0 hours after reconstitution. In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition undergoes a change in osmotic pressure (e.g., an increase or decrease) of up to 5%, such as up to 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (e.g., 0%) after storage at room temperature for at least 2 hours (e.g., at least 3, 4, 5, 6, 7, 8 or longer), such as 8 hours, compared to 0 hours after reconstitution.In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) has an osmotic pressure of 80 mOsm / kg to 450 mOsm / kg, such as 100 mOsm / kg to 400 mOsm / kg, 100 mOsm / kg to 150 mOsm / kg, 150 mOsm / kg to 400 mOsm / kg, 350 mOsm / kg to 400 mOsm / kg, 100 mOsm / kg to 150 mOsm / kg, 150 mOsm / kg to 400 mOsm / kg, 350 mOsm / kg to 400 mOsm / kg, 100 mOsm / kg to 150 mOsm / kg, 200mOsm / kg to 400mOsm / kg, 240mOsm / kg to 400mOsm / kg, 270mOsm / kg to 370mOsm / kg, 300mOsm / kg to 400mOsm / kg, 250mOsm / kg to 350mOsm / kg, 350mOsm / kg to 400mOsm / kg, 300mOsm / kg to 330mOsm / kg or 280mOsm / kg to 320mOsm / kg. The normal osmotic pressure of human plasma is about 280mOsm / kg to 320mOsm / kg. In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., after reconstitution) is isotonic or substantially isotonic, or within a range of 280 mOsm / kg to 320 mOsm / kg with a change of 50% (e.g., 40%, 30%, 20%, 10%, 5% or less). In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition remains a colorless, transparent liquid after storage at room temperature for at least 2 hours (e.g., at least 3, 4, 5, 6, 7, 8 or longer), such as 8 hours after reconstitution.

[0200] In some embodiments, the shelf life of the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., lyophilized) is at least 15 days, such as at least 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or longer, for example, at 2-25° C., such as 2-8° C. As used herein, "shelf life" refers to the storage period during which the active ingredient in the pharmaceutical formulation, such as a therapeutic protein (e.g., an FX activator described herein), undergoes minimal degradation (e.g., no more than 5% degradation, such as no more than 4%, 3%, or 2% degradation) when the pharmaceutical formulation is stored under specific storage conditions, such as 2-8° C. Exemplary techniques for assessing protein or formulation stability include size exclusion chromatography SEC-HPLC to detect, for example, aggregation, reverse phase liquid chromatography (RP)-HPLC to detect, for example, protein fragments, ion exchange HPLC to detect, for example, protein charge changes, mass spectrometry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy to detect protein conformational changes. All of these techniques can be used alone or in combination to assess the degradation of proteins in a pharmaceutical formulation and determine the shelf life of the formulation. In some embodiments, when the pharmaceutical formulation of the invention is stored at 2-8°C, it exhibits no more than 5% (e.g., no more than 4%, 3%, 2% or 1%) degradation (e.g., fragmentation, aggregation or unfolding) for at least 15 days (e.g., at least 20 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or longer).

[0201] III. Methods for preparing a pharmaceutical composition of a factor X activator

[0202] The present invention also provides a method for preparing any FX activator (e.g., RVV-X) pharmaceutical composition described herein (e.g., RVV-X lead formulation). In some embodiments, a method for preparing a FX activator (e.g., RVV-X) pharmaceutical composition is provided, comprising formulating a FX activator (e.g., RVV-X) using any pharmaceutical composition component described herein. In some embodiments, the method further comprises lyophilizing the pharmaceutical composition. In some embodiments, the method further comprises reconstituting the lyophilized pharmaceutical composition. In some embodiments, a stock solution of the pharmaceutical composition is prepared and then diluted as needed. Also, see Examples 5, 7, and 8.

[0203] In some embodiments, a method for preparing a FX activator (e.g., RVV-X) pharmaceutical composition (e.g., RVV-X lead formulation) is provided, comprising formulating a FX activator (e.g., RVV-X) at a concentration of 0.1 U / mL to 200 U / mL (e.g., about 10 U / mL) with i) a stabilizer (e.g., sucrose) at a concentration of 2 mg / ml to 10 mg / ml (e.g., about 30 mg / ml); ii) a surfactant (e.g., histidine) at a concentration of 0.1 U / mL to 200 U / mL (e.g., about 10 U / mL); .1mg / ml to 50mg / ml (e.g., about 3mg / ml); iii) a buffer (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., about 0.02% (w / v)); iv) an excipient (e.g., mannitol) in an amount of 1mg / ml to 100mg / ml (e.g., about 40mg / ml); wherein the pH value of the pharmaceutical composition is 6.0 to 8.0 (e.g., about 6.85). In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the method further comprises lyophilizing the pharmaceutical composition.

[0204] In some embodiments, a method for preparing a FX activator pharmaceutical composition (e.g., a RVV-X lead formulation) is provided, comprising formulating a FX activator RVV-X at 10 U / mL with i) sucrose at 30 mg / ml; ii) histidine at 3 mg / ml; iii) polysorbate 20 at 0.02% (w / v); iv) mannitol at 40 mg / ml; wherein the pH value of the pharmaceutical composition is 6.85. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the method further comprises lyophilizing the pharmaceutical composition.

[0205] The present invention also provides an isolated nucleic acid encoding any FX activator described herein (e.g., RVV-X) and a vector comprising the nucleic acid. At the same time, an isolated host cell (e.g., E. coli, CHO cells, HEK 293 cells, Hela cells or COS cells) comprising a nucleic acid encoding any FX activator described herein (e.g., RVV-X) and a vector is provided. In some embodiments, the isolated nucleic acid further encodes a signal peptide sequence at the N-terminus of the FX activator (e.g., the N-terminus of each RVV-X polypeptide chain).

[0206] In some embodiments, the vector comprising a nucleic acid encoding any FX activator described herein (e.g., RVV-X) is suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK 293 cells, Hela cells, COS cells). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0207] Many virus-based systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors and their derivatives. Viral vector technology is well known in the art, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying construct protein coding sequences can be packaged using experimental methods known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells using methods known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transduction because they allow long-term, stable integration of the transgene and propagation in daughter cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0208] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a transposon, such as the Sleeping Beauty (SB) transposon system or the PiggyBac transposon system. In some embodiments, the vector is a polymer-based non-viral vector, including, for example, poly (lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly (ethyleneimine) (PEI) and dendrimers. In some embodiments, the vector is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions, and solid lipid nanoparticles (SLN). In some embodiments, the vector is a peptide-based non-viral gene vector, such as poly-L-lysine. Any known non-viral vector suitable for genome editing can be used to introduce the encoded nucleic acid into a host cell. See Yin H. et al., Nature Rev. Genetics (2014) 15: 521-555; Aronovich EL et al., "The Sleeping Beauty transposon system: a non-viral vector for gene therapy." Hum. Mol. Genet. (2011) R1: R14-20 and Zhao S. et al., "PiggyBac transposon vectors: the tools of the human gene editing." Transl. Lung Cancer Res. (2016) 5 (1): 120-125, incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding the FX activator described herein (e.g., RVV-X) are introduced into host cells (e.g., CHO, HEK 293, Hela or COS) by physical methods, including but not limited to electroporation, sonoporation, photoporation, magnetofection, and water poration.

[0209] In some embodiments, the vector comprises a selectable marker gene or reporter gene for selecting cells expressing the FX activator described herein (e.g., RVV-X) from a host cell population transfected with a vector (e.g., a lentiviral vector, a pTT5 vector). The selectable marker and reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. For example, the vector may comprise a transcription and translation terminator, a start sequence, and a promoter for regulating expression of a nucleic acid sequence.

[0210] Can use any molecular cloning method known in the art, comprise, for example, use restriction endonuclease site and one or more selectable markers nucleic acid is cloned in vector.In certain embodiments, nucleic acid is operably connected on the promoter.Developed multiple promoters for carrying out gene expression in prokaryotic cell or eukaryotic cell (for example, mammalian cell), and any promoter known in the art can be used for the present invention.Promoter can be roughly divided into constitutive promoter or regulatable promoter, such as inducible promoter.

[0211] In some embodiments, the nucleic acid encoding the FX activator described herein (e.g., RVV-X) is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Examples of promoters contemplated herein include, but are not limited to, CMV promoter (CMV), human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter coupled to CMV early enhancer (CAGG), Roche sarcoma virus (RSV) promoter, polyoma virus enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site substitution (MND)" promoter. The efficiency of these constitutive promoters in driving transgenic expression has been extensively compared in a large number of studies.

[0212] In some embodiments, the nucleic acid encoding the FX activator described herein (e.g., RVV-X) is operably linked to an inducible promoter. Inducible promoters belong to the category of regulatable promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, an inducer (i.e., an inducer), or a combination thereof. In some embodiments, the inducing conditions do not induce the expression of endogenous genes in the host cell. In some embodiments, the inducing conditions are selected from: an inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), a redox state, and an activation state of the host cell. In some embodiments, the inducible promoter can be a NFAT promoter, Promoter or NFκB promoter.

[0213] Prokaryotic host cells suitable for expressing the protein of the present application include archaea and true bacteria, such as gram-negative bacteria or gram-positive bacteria. Examples of available bacteria include Escherichia coli (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla or Paracoccus. In certain embodiments, gram-negative cells are used. In certain embodiments, Escherichia coli cells are used as hosts of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strains having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA (nmpc fepE) degP41kan R strain 33D3 (US Pat. No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31446), E. coli B, E. coli 1776 (ATCC 31537) and E. coli RV308 (ATCC 31608) are also applicable. These examples are illustrative and not restrictive. Methods for constructing any of the above-mentioned bacterial derivatives of known genotypes are known in the art and are described in detail in, for example, Bass et al., Proteins, 8: 309-314 (1990). Considering the replicability of the replicon in the bacterial cell, it is generally necessary to select a suitable bacterium. For example, when a known plasmid such as pBR322, pBR325, pACYC177 or pKN410 is used to provide the replicon, Escherichia coli, Serratia or Salmonella are suitable for use as hosts.

[0214] Suitable eukaryotic host cells for cloning or expressing the DNA in the vectors described herein include vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) is routine procedure. Examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); COS fibroblast-like cell line derived from monkey kidney tissue; human embryonic kidney line (293 or 293 cell subclones for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC-ccl2); canine kidney cells (MDCK, ATCC-ccl34); buffalo-rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and human liver cancer cell line (Hep G2).

[0215] Host cells are transformed with the above-described expression or cloning vectors to produce the protein construct and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0216] At the same time, a method for preparing any FX activator described herein (e.g., RVV-X) is provided. Therefore, in some embodiments, a method for preparing a FX activator (e.g., RVV-X) is provided, comprising: (a) culturing a host cell (e.g., CHO cell) containing any nucleic acid or vector encoding a FX activator described herein under conditions that can effectively express the encoded FX activator; and (b) obtaining the expressed FX activator from the host cell. In some embodiments, the method of step (a) further comprises producing a host cell, wherein the host cell contains a nucleic acid or vector encoding a FX activator described herein (e.g., RVV-X). The FX activator described herein (e.g., RVV-X) can be prepared using any recombinant preparation method known in the art, or isolated and / or purified (e.g., from snake venom).

[0217] In some embodiments, the FX activator (e.g., RVV-X) is isolated and / or purified from snake venom (e.g., species belonging to the families Viperidae and Crotalidae, and some species of cobra). In some embodiments, the FX activator RVV-X is isolated and / or purified from the venom of Vipera rotundifolia. In some embodiments, the FX activator (e.g., RVV-X) is isolated and / or purified according to the method described in CN109943554B. Any suitable protein purification method can be used for the purification of FX activators, such as ion exchange column fractionation, ethanol precipitation, reverse phase liquid chromatography HPLC, silica gel chromatography, heparin SEPHAROSE. TM Chromatography, anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation, depending on the protein construct to be recovered. In some embodiments, the FX activator is sterile and / or virus-inactivated. In some embodiments, purification of the FX agonist includes one or more steps of virus inactivation, anion exchange chromatography, ultrafiltration, hydroxyapatite (HA) chromatography (HAC) and SEC.

[0218] IV. Methods of Treating Bleeding Disorders

[0219] The FX activators described herein (e.g., RVV-X) or compositions thereof (e.g., RVV-X lead agents) can be used for a variety of purposes, such as diagnosis, molecular detection, and treatment (e.g., on-demand treatment or prophylactic treatment). In some embodiments, the individual to be treated or diagnosed is a livestock animal (e.g., pigs, sheep, goats, cows, bulls, horses, donkeys, mules, ducks, geese). In some embodiments, the individual to be treated or diagnosed is a companion animal (e.g., a pet) or an auxiliary animal, such as a dog, fur, rabbit, hamster, guinea pig, chinchilla, ferret, bird, etc. In some embodiments, the individual to be treated or diagnosed is a human. In some embodiments, the individual to be treated or diagnosed is a primate (e.g., monkey).

[0220] In some embodiments, a method of treating a bleeding disorder (e.g., hemophilia, such as hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of any FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead formulation). In some embodiments, a method of treating a bleeding disorder (e.g., hemophilia, such as hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of any FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead formulation), wherein the FX activator or its pharmaceutical composition is administered at a dose of 0.01 U / kg to 0.48 U / kg (e.g., about 0.08 U / kg to 0.48 U / kg, such as 0.01 U / kg to 0.16 U / kg, 0.08 U / kg to 0.16 U / kg, or 0.16 U / kg). In some embodiments, the FX activator or its pharmaceutical composition is administered intravenously, such as by intravenous injection. When referring to "FX activator pharmaceutical composition administered at a dose of X", it means that the pharmaceutical composition is administered in an effective amount, wherein the dose of the FX activator contained is X. In some embodiments, the FX activator or its pharmaceutical composition is administered once. In some embodiments, the FX activator or its pharmaceutical composition is administered up to 6 times, such as 6, 5, 4, 3, 2 or 1 times. In some embodiments, the FX activator or its pharmaceutical composition is administered 4 times. In some embodiments, the FX activator or its pharmaceutical composition is administered once every 4 hours (q4h) to every 8 hours (q8h), such as 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 hours. In some embodiments, the FX activator or its pharmaceutical composition is administered q4h. In some embodiments, the FX activator or its pharmaceutical composition is administered once at a dose of 0.01U / kg to 0.48U / kg (e.g., 0.01U / kg, 0.04U / kg, 0.08U / kg, 0.16U / kg, 0.32U / kg, or 0.48U / kg). In some embodiments, the FX activator or its pharmaceutical composition is administered at a dose of 0.16U / kg, q8h, up to 6 times (e.g., 6, 5, 4, 3, 2, or 1 times). In some embodiments, the FX activator or its pharmaceutical composition is administered at a dose of 0.16U / kg, q8h, 4 times. In some embodiments, the FX activator or its pharmaceutical composition is administered at a dose of 0.16U / kg, q4h, up to 6 times (e.g., 6, 5, 4, 3, 2, or 1 times). In some embodiments, the FX activator or its pharmaceutical composition is administered at a dose of 0.16U / kg, q4h, 4 times. In some embodiments, the FX activator is RVV-X.In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the purity of the RVV-X is at least about 95%. In some embodiments, the RVV-X comprises a) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; or c) a mixture of a) and b). In some embodiments, the bleeding disorder is hemophilia A, such as hemophilia A with or without FVIII inhibitors. In some embodiments, the bleeding disorder is hemophilia B, such as hemophilia B with or without IX inhibitors. In some embodiments, the bleeding disorder is surgical wound bleeding. In some embodiments, the individual is further given a consolidation treatment, such as an additional administration of the same dose of the FX activator or a pharmaceutical composition thereof, for example within 24 hours of the last administration of the treatment method. In some embodiments, the FX activator (e.g., RVV-X) or its pharmaceutical composition (e.g., RVV-X lead) is administered to an individual within 24 hours (e.g., 20 hours, 18 hours, 16 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes or less) of the onset of bleeding. In some embodiments, the FX activator (e.g., RVV-X) or its pharmaceutical composition (e.g., RVV-X lead) is administered to an individual at least 5 minutes (e.g., at least 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours or longer) before the onset of bleeding (e.g., before surgery). In some embodiments, the FX activator or its pharmaceutical composition is administered to an individual 10 to 15 minutes before the onset of bleeding. In some embodiments, the individual is a human. In some embodiments, the individual is an animal, such as a mouse, rabbit, cat, dog, guinea pig, hamster, horse, cow, sheep, bird (e.g., chicken, duck), etc.

[0221] The dosage and required drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific use. Determining the appropriate dosage or route of administration is well within the skill of the ordinary technician. Animal experiments provide reliable guidance for determining effective doses for human treatment. Interspecies analogies of effective doses can be made based on the principles of Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobie et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0222] In the scope of the present application, different preparations will be effective for different treatment methods and different diseases, and the mode of administration intended to treat a specific organ or tissue may be different from the mode for another organ or tissue. In addition, the dosage can be administered by one or more separate administrations or continuous infusions. For repeated administrations over a few days or longer, depending on the condition, the treatment continues until the disease symptoms reach the desired degree of inhibition (e.g., hemostasis). However, other dosage regimens may be useful. The progress of this treatment is easily monitored by conventional techniques and analysis.

[0223] In some embodiments, the FX activator (e.g., RVV-X) or its pharmaceutical composition (e.g., RVV-X lead formulation) described herein is administered once (e.g., bolus injection). In some embodiments, the FX activator or its pharmaceutical composition is administered multiple times (e.g., 2, 3, 4, 5, 6 or more times). If multiple administrations are given, they can be administered by the same or different routes and can be administered at the same site or at different sites. The FX activator or its pharmaceutical composition can be administered hourly, daily, monthly or on demand. The interval between two administrations can be any time from 1 hour to 1 day. The interval may also be irregular (e.g., with bleeding events). In some embodiments, there is no interruption in the dosing plan. The optimal dose and treatment regimen for a particular patient can be determined by a technician in the medical field by monitoring the patient's disease signs and adjusting accordingly. In some embodiments, the FX activator or its pharmaceutical composition is administered at a frequency of q1h, q2h, q4h, q6h, q8h, q12h, q16h, q18h, once a day (daily administration), once every 2 days, once every 3 days, once a week, once every 2 weeks, once every 3 weeks, once a month. In some embodiments, the FX activator or its pharmaceutical composition is administered at a frequency of q4h to q8h.

[0224] In some embodiments, the FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead formulation) is administered in divided doses, such as 2, 3, 4, 5, or 6 times. In some embodiments, divided doses are administered over 1 day, 2 days, 3 days, 1 week, 1 month, or longer. In some embodiments, the dose is divided equally. In some embodiments, the divided dose is 17%, 20%, 25%, 30%, or 50% of the total dose. In some embodiments, the interval between consecutive divided doses is 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 1 week, 1 month, or longer. For repeated administration over several hours, several days, or longer, depending on the condition, treatment continues until the expected degree of suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this treatment is easily monitored by conventional techniques and analysis.

[0225] In some embodiments, the FX activator (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead) is administered at a dose of 0.01 U / kg to 0.48 U / kg, such as 0.01 U / kg to 0.1 U / kg, 0.02 U / kg to 0.08 U / kg, 0.01 U / kg to 0.05 U / kg, 0.01 U / kg to 0.08 U / kg, 0.05 U / kg to 0.1 U / kg, 0.1 U / kg to 0.48 U / kg, 0.1 U / kg to 0.4 U / kg, 0.1 U / kg to 0.3 U / kg, 0.1 U / kg to 0.2 U / kg, 0.2 U / kg to 0.48 U / kg, 0.2 U / kg to 0.4 U / kg, 0.2 U / kg to 0.3 U / kg, 0.3 U / kg, kg to 0.48 U / kg, 0.08 U / kg to 0.48 U / kg, 0.16 U / kg to 0.48 U / kg, 0.16 U / kg to 0.2 U / kg, 0.01 U / kg to 0.16 U / kg, 0.02 U / kg to 0.14 U / kg, 0.04 U / kg to 0.12 U / kg, 0.06 U / kg to 0.12 U / kg, 0. 0.08 U / kg to 0.1 U / kg, 0.02 U / kg to 0.16 U / kg, 0.04 U / kg to 0.16 U / kg, 0.06 U / kg to 0.16 U / kg, 0.1 U / kg to 0.16 U / kg, 0.12 U / kg to 0.16 U / kg, 0.14 U / kg to 0.16 U / kg, or 0.08 U / kg to 0.16 U / kg. In some embodiments, the subject being treated is a human. In some embodiments, the FX activator (e.g., RVV-X) or a pharmaceutical composition thereof is administered at a dose of 0.01 U / kg, 0.02 U / kg, 0.03 U / kg, 0.04 U / kg, 0.05 U / kg, 0.06 U / kg, 0.07 U / kg, 0.08 U / kg, 0.09 U / kg, 0.1 U / kg, 0.11 U / kg, 0.12 U / kg, 0.1 3U / kg, 0.14U / kg, 0.15U / kg, 0.16U / kg, 0.18U / kg, 0.2U / kg, 0.22U / kg, 0.24U / kg, 0.26U / kg, 0.28U / kg, 0.3U / kg, 0.32U / kg, 0.36U / kg, 0.4U / kg, 0.44U / kg and 0.48U / kg, such as 0.16U / kg.

[0226] In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X), wherein the RVV-X or a pharmaceutical composition thereof is administered once at a dose of 0.01 U / kg to 0.48 U / kg, 0.16 U / kg to 0.48 U / kg, 0.08 U / kg to 0.48 U / kg, 0.01 U / kg to 0.16 U / kg, 0.08 U / kg to 0.16 U / kg, or 0.16 U / kg. In some embodiments, a method of treating hemophilia (e.g., such as hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X), wherein the RVV-X or a pharmaceutical composition thereof is administered once at a dose of 0.01 U / kg to 0.48 U / kg or 0.16 U / kg to 0.48 U / kg. In some embodiments, a method of treating hemophilia (e.g., such as hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X), wherein the RVV-X or a pharmaceutical composition thereof is administered once at a dose of 0.01 U / kg, 0.04 U / kg, 0.08 U / kg, 0.16 U / kg, 0.32 U / kg, and 0.48 U / kg. In some embodiments, a method for treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X), wherein the RVV-X or a pharmaceutical composition thereof is administered once at a dose of 0.16U / kg, 0.32U / kg, or 0.48U / kg. In some embodiments, the RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X) is administered intravenously, such as by intravenous injection. In some embodiments, the RVV-X is isolated from the venom of a viper. In some embodiments, the purity of the RVV-X is at least about 95%. In some embodiments, the RVV-X comprises a) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; or c) a mixture of a) and b).

[0227] In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stabilizer (e.g., sucrose) in an amount of A content of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 5 mg / ml, or 3 mg / ml); iv) a surfactant (e.g. , polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v), or 0.02% (w / v)); and v) an excipient (e.g., mannitol) in an amount of 1 mg / ml to 100 mg / ml (e.g., 10 mg / ml to 60 mg / ml, 30 mg / ml to 60mg / ml or 40mg / ml); wherein the pH value of the pharmaceutical composition is about 6.0 to 8.0 (e.g., 6.3 to 7.3, 6.8 to 7.0 or 6.85); and wherein the dosage of the pharmaceutical composition administered once is 0.01U / kg to 0.48U / kg, 0.16U / kg to 0.48U / kg, 0.08U / kg to 0.48U / kg, 0.01U / kg to 0.16U / kg, 0.08U / kg to 0.16U / kg or 0.16U / kg.In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stabilizer (e.g., sucrose) in an amount of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 50 mg / ml, or 30 mg / ml); g / ml to 5 mg / ml or 3 mg / ml); iv) a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v) or 0.02% (w / v)); and v) an excipient (e.g., mannitol), Its content is 1 mg / ml to 100 mg / ml (for example, 10 mg / ml to 60 mg / ml, 30 mg / ml to 60 mg / ml or 40 mg / ml); wherein the pH value of the pharmaceutical composition is 6.0 to 8.0 (for example, 6.3 to 7.3, 6.8 to 7.0 or 6.85); and wherein the dosage of the pharmaceutical composition for one administration is 0.01 U / kg to 0.48 U / kg or 0.16 U / kg to 0.48 U / kg.In some embodiments, methods are provided for treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human), comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stabilizer (e.g., sucrose) in an amount of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 5 mg / ml, or 30 mg / ml); ml or 3 mg / ml); iv) a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v), or 0.02% (w / v)); and v) an excipient (e.g., mannitol) in an amount of 1 mg / ml to 100 mg / ml (e.g., 10 mg / ml to 60 mg / ml, 30 mg / ml to 60 mg / ml or 40 mg / ml); wherein the pH value of the pharmaceutical composition is 6.0 to 8.0 (e.g., 6.3 to 7.3, 6.8 to 7.0 or 6.85); and wherein the dosage of the pharmaceutical composition for one administration is 0.01 U / kg, 0.04 U / kg, 0.08 U / kg, 0.16 U / kg, 0.32 U / kg and 0.48 U / kg.In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stabilizer (e.g., sucrose) in an amount of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml l, 3 mg / ml to 5 mg / ml or 3 mg / ml); iv) a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v) or 0.02% (w / v)); and v) an excipient (e.g. , mannitol) in an amount of 1 mg / ml to 100 mg / ml (e.g., 10 mg / ml to 60 mg / ml, 30 mg / ml to 60 mg / ml or 40 mg / ml); wherein the pH value of the pharmaceutical composition is 6.0 to 8.0 (e.g., 6.3 to 7.3, 6.8 to 7.0 or 6.85); and wherein the dosage of the pharmaceutical composition administered once is 0.16 U / kg, 0.32 U / kg and 0.48 U / kg. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the method further comprises reconstituting the pharmaceutical composition prior to administration (e.g., with 0.9% sodium chloride injection). In some embodiments, the pharmaceutical composition is administered intravenously, such as by intravenous injection. In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the purity of the RVV-X is at least about 95%.In some embodiments, the RVV-X comprises a) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2, or a sequence having at least 80% sequence identity with SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5, or a sequence having at least 80% sequence identity with SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; or c) a mixture of a) and b).

[0228] In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at 10 U / mL; ii) sucrose at 30 mg / ml; iii) histidine at 3 mg / ml; iv) polysorbate 20 at 0.02% (w / v); and v) mannitol at 40 mg / ml; wherein the pharmaceutical composition has a pH of 6.85; and wherein the pharmaceutical composition is administered once at a dose of 0.01 U / kg to 0.48 U / kg, 0.16 U / kg to 0.48 U / kg, 0.08 U / kg to 0.48 U / kg, 0.01 U / kg to 0.16 U / kg, 0.08 U / kg to 0.16 U / kg, or 0.16 U / kg. In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at a content of 10 U / mL; ii) sucrose at a content of 30 mg / ml; iii) histidine at a content of 3 mg / ml; iv) polysorbate 20 at a content of 0.02% (w / v); and v) mannitol at a content of 40 mg / ml; wherein the pH of the pharmaceutical composition is 6.85; and wherein the pharmaceutical composition is administered once at a dose of 0.01 U / kg to 0.48 U / kg or 0.16 U / kg to 0.48 U / kg. In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at a content of 10 U / mL; ii) sucrose at a content of 30 mg / ml; iii) histidine at a content of 3 mg / ml; iv) polysorbate 20 at a content of 0.02% (w / v); and v) mannitol at a content of 40 mg / ml; wherein the pH of the pharmaceutical composition is 6.85; and wherein the pharmaceutical composition is administered once at a dose of 0.01 U / kg, 0.04 U / kg, 0.08 U / kg, 0.16 U / kg, 0.32 U / kg, and 0.48 U / kg.In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at 10 U / mL; ii) sucrose at 30 mg / ml; iii) histidine at 3 mg / ml; iv) polysorbate 20 at 0.02% (w / v); and v) mannitol at 40 mg / ml; wherein the pH of the pharmaceutical composition is 6.85; and wherein the dosage of the pharmaceutical composition administered once is 0.16 U / kg, 0.32 U / kg, or 0.48 U / kg. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the method further comprises reconstituting the pharmaceutical composition prior to administration (e.g., with 0.9% sodium chloride injection). In some embodiments, the pharmaceutical composition is administered intravenously, such as by intravenous injection. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the purity of the RVV-X is at least about 95%. In some embodiments, the RVV-X comprises a) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; or c) a mixture of a) and b).

[0229] In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X), wherein the RVV-X or its pharmaceutical composition is administered at a dose of 0.01 U / kg to 0.48 U / kg (e.g., 0.16 U / kg to 0.48 U / kg, 0.08 U / kg to 0.48 U / kg, 0.01 U / kg to 0.16 U / kg, 0.08 U / kg to 0.16 U / kg, or 0.16 U / kg), q4h to q8h (e.g., q4h, q5h, q6h, q7h, or q8h), for a maximum of 6 administrations (e.g., 1, 2, 3, 4, 5, or 6 times). In some embodiments, a method for treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X), wherein the RVV-X or its pharmaceutical composition is administered at a dose of 0.16 U / kg, q4h to q8h (e.g., q4h, q5h, q6h, q7h or q8h), for a maximum of 6 administrations (e.g., 1, 2, 3, 4, 5, or 6 times). In some embodiments, a method for treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or its pharmaceutical composition (e.g., a lead formulation of RVV-X), wherein the RVV-X or its pharmaceutical composition is administered at a dose of 0.16 U / kg, q4h, for a maximum of 6 administrations (e.g., 1, 2, 3, 4, 5, or 6 times). In some embodiments, a method for treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of RVV-X or a pharmaceutical composition thereof (e.g., a lead formulation of RVV-X), wherein the dosage of RVV-X or its pharmaceutical composition is 0.16U / kg q4h, administered 4 times. In some embodiments, the RVV-X or its pharmaceutical composition (e.g., a lead formulation of RVV-X) is administered intravenously, such as by intravenous injection. In some embodiments, the RVV-X is isolated from the venom of a viper. In some embodiments, the purity of the RVV-X is at least about 95%. In some embodiments, the RVV-X comprises a) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; or c) a mixture of a) and b).

[0230] In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stabilizer (e.g., sucrose) in an amount of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 5 mg / ml, or 3 mg / ml); iv) a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v). 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v) or 0.02% (w / v)); and v) an excipient (e.g., mannitol) in an amount of 1 mg / ml to 100 mg / ml (e.g., 10 mg / ml to 60 mg / ml, 30 mg / ml to 60 mg / ml or 40 mg / ml); wherein the pharmaceutical composition has a pH of about 6.0 to 8.0 ( For example, 6.3 to 7.3, 6.8 to 7.0 or 6.85); and wherein the dosage of the pharmaceutical composition is 0.01 U / kg to 0.48 U / kg (such as 0.16 U / kg to 0.48 U / kg, 0.08 U / kg to 0.48 U / kg, 0.01 U / kg to 0.16 U / kg, 0.08 U / kg to 0.16 U / kg or 0.16 U / kg), q4h to q8h (such as q4h, q5h, q6h, q7h or q8h), with a maximum of 6 doses (such as 1, 2, 3, 4, 5 or 6 times).In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stable a) a sucrose in an amount of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 5 mg / ml, or iv) a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v), or 0.02% (w / v)); and v) an excipient (e.g., mannitol) in an amount of 1 mg / ml to 100 mg / ml. l (e.g., 10 mg / ml to 60 mg / ml, 30 mg / ml to 60 mg / ml or 40 mg / ml); wherein the pH value of the pharmaceutical composition is 6.0 to 8.0 (e.g., 6.3 to 7.3, 6.8 to 7.0 or 6.85); and wherein the dosage of the pharmaceutical composition is 0.16 U / kg, q4h to q8h (e.g., q4h, q5h, q6h, q7h or q8h), for a maximum of 6 administrations (e.g., 1, 2, 3, 4, 5 or 6 times).In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stabilizer (e.g., sucrose) in an amount of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 15 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); mg / ml to 5 mg / ml or 3 mg / ml); iv) a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v) or 0.02% (w / v)); and v) an excipient (e.g., mannitol ), whose content is 1 mg / ml to 100 mg / ml (e.g., 10 mg / ml to 60 mg / ml, 30 mg / ml to 60 mg / ml or 40 mg / ml); wherein the pH value of the pharmaceutical composition is 6.0 to 8.0 (e.g., 6.3 to 7.3, 6.8 to 7.0 or 6.85); and wherein the dosage of the pharmaceutical composition is 0.16 U / kg, q4h, for a maximum of 6 administrations (e.g., 1, 2, 3, 4, 5 or 6 times).In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) a FX activator (e.g., RVV-X) in an amount of 0.1 U / mL to 200 U / mL (e.g., 1 U / mL to 100 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 10 U / mL); ii) a stabilizer (e.g., sucrose) in an amount of 2 mg / ml to 100 mg / ml (e.g., 2 mg / ml to 60 mg / ml, 15 mg / ml to 60 mg / ml, 30 mg / ml to 50 mg / ml, or 30 mg / ml); iii) a buffer (e.g., histidine) in an amount of 0.1 mg / ml to 50 mg / ml (e.g., 2 mg / ml to 20 mg / ml, 2 mg / ml to 1 iv) a surfactant (e.g., polysorbate 20) in an amount of 0.001% (w / v) to 0.1% (w / v) (e.g., 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.03% (w / v), or 0.02% (w / v)); and v ) excipient (e.g., mannitol) in an amount of 1 mg / ml to 100 mg / ml (e.g., 10 mg / ml to 60 mg / ml, 30 mg / ml to 60 mg / ml, or 40 mg / ml); wherein the pH value of the pharmaceutical composition is 6.0 to 8.0 (e.g., 6.3 to 7.3, 6.8 to 7.0, or 6.85); and wherein the dosage of the pharmaceutical composition once is 0.16 U / kg, q4h, for 4 administrations. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the method further comprises reconstituting the pharmaceutical composition prior to administration (e.g., with 0.9% sodium chloride injection). In some embodiments, the pharmaceutical composition is administered intravenously, such as by intravenous injection. In some embodiments, the FX activator is RVV-X. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the purity of the RVV-X is at least about 95%.In some embodiments, the RVV-X comprises a) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2, or a sequence having at least 80% sequence identity with SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1, or a sequence having at least 80% sequence identity with SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5, or a sequence having at least 80% sequence identity with SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3, or a sequence having at least 80% sequence identity with SEQ ID NO: 3; or c) a mixture of a) and b).

[0231] In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at a content of 10 U / mL; ii) sucrose at a content of 30 mg / ml; iii) histidine at a content of 3 mg / ml; iv) polysorbate 20 at a content of 0.02% (w / v); and v) mannitol at a content of 40 mg / ml; wherein the pharmaceutical composition The pH value of the pharmaceutical composition is 6.85; and wherein the dosage of the pharmaceutical composition is 0.01 U / kg to 0.48 U / kg (such as 0.16 U / kg to 0.48 U / kg, 0.08 U / kg to 0.48 U / kg, 0.01 U / kg to 0.16 U / kg, 0.08 U / kg to 0.16 U / kg or 0.16 U / kg), q4h to q8h (such as q4h, q5h, q6h, q7h or q8h), and is administered up to 6 times (such as 1, 2, 3, 4, 5 or 6 times). In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at 10 U / mL; ii) sucrose at 30 mg / ml; iii) histidine at 3 mg / ml; iv) polysorbate 20 at 0.02% (w / v); and v) mannitol at 40 mg / ml; wherein the pH of the pharmaceutical composition is 6.85; and wherein the pharmaceutical composition is administered at a dose of 0.16 U / kg, q4h to q8h (e.g., q4h, q5h, q6h, q7h, or q8h), for a maximum of 6 administrations (e.g., 1, 2, 3, 4, 5, or 6 times). In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at a content of 10 U / mL; ii) sucrose at a content of 30 mg / ml; iii) histidine at a content of 3 mg / ml; iv) polysorbate 20 at a content of 0.02% (w / v); and v) mannitol at a content of 40 mg / ml; wherein the pH of the pharmaceutical composition is 6.85; and wherein the pharmaceutical composition is administered at a dose of 0.16 U / kg, q4h, for a maximum of 6 administrations (e.g., 1, 2, 3, 4, 5, or 6 times).In some embodiments, a method of treating hemophilia (e.g., hemophilia A or B, with or without inhibitors) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises i) RVV-X at 10 U / mL; ii) sucrose at 30 mg / ml; iii) histidine at 3 mg / ml; iv) polysorbate 20 at 0.02% (w / v); and v) mannitol at 40 mg / ml; wherein the pH of the pharmaceutical composition is 6.85; and wherein the dosage of the pharmaceutical composition administered once is 0.16 U / kg, q4h, administered 4 times. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the method further comprises reconstituting the pharmaceutical composition prior to administration (e.g., with 0.9% sodium chloride injection). In some embodiments, the pharmaceutical composition is administered intravenously, such as by intravenous injection. In some embodiments, the RVV-X is isolated from the venom of Vipera rotundifolia. In some embodiments, the purity of the RVV-X is at least about 95%. In some embodiments, the RVV-X comprises a) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; b) i) a heavy chain comprising the sequence SEQ ID NO: 1; ii) a light chain 1 comprising the sequence SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence SEQ ID NO: 3; or c) a mixture of a) and b).

[0232] In some embodiments, the method for treating a bleeding disorder (e.g., hemophilia, such as hemophilia A or B, with or without inhibitors; or surgical wound bleeding) has one or more of the following biological activities compared to an individual with a bleeding disorder who has not received the treatment or has received a control agent (e.g., a placebo or other hemostatic agent): (i) activating FX and / or promoting FXa production; (ii) increasing thrombin generation (TG) and / or endogenous thrombin generation potential (ETP); (iii) shortening the activated partial thromboplastin time (APTT), prothrombin time (PT) and / or thrombin time (TT); (iv) promoting hemostasis, improving bleeding symptoms, such as reducing bleeding time and / or bleeding volume; (v) reducing mortality; (vi) promoting wound healing; (vii) prolonging patient survival; and / or (viii) alleviating or relieving pain, etc. In some embodiments, the method of activating FX and / or promoting FXa production can increase FXa by at least about 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 5 times, 10 times, 30 times or more. In some embodiments, the method of increasing TG and / or ETP can increase TG and / or ETP by at least about 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 5 times, 10 times, 30 times or more. In some embodiments, the method of shortening APTT, PT and / or TT can shorten APTT, PT and / or TT by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the method of promoting hemostasis can reduce bleeding time and / or bleeding volume by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the method of reducing mortality can reduce mortality by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the method of promoting wound healing can promote wound healing by at least about 1.1 times (including, for example, at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 times or more). In some embodiments, the method of extending the survival of an individual (e.g., a human) can extend the survival of the individual by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months or 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. In some embodiments, the method of reducing or alleviating pain reduces pain by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%.

[0233] The administration of the FX activator (e.g., RVV-X) or its pharmaceutical composition (e.g., RVV-X lead formulation) described herein can be carried out in any convenient manner, including by injection or infusion. The route of administration is in accordance with known and recognized methods, such as by single or multiple boluses or long-term infusion in an appropriate manner. The FX activator or its pharmaceutical composition is administered to the patient by injection (e.g., subcutaneous injection, intravenous injection or intraperitoneal injection). In some embodiments, the FX activator or its pharmaceutical composition is administered systemically. In some embodiments, the FX activator or its pharmaceutical composition is administered to the individual by infusion (e.g., intravenous infusion). The infusion techniques used for immunotherapy are known in the art (see Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)). In some embodiments, the FX activator or its pharmaceutical composition is administered to the individual by intradermal or subcutaneous (i.e., under the skin) injection. For subcutaneous injection, a syringe can be used to inject the FX activator or its pharmaceutical composition. However, there are other devices for administering FX activators or pharmaceutical compositions thereof, such as injection devices; injection pens; automatic injector devices, needle-free devices; and subcutaneous patch delivery systems. In some embodiments, FX activators or pharmaceutical compositions thereof are administered by intravenous injection. In some embodiments, FX activators or pharmaceutical compositions thereof are applied topically to an injury or wound, such as directly to wound tissue or a site of bleeding. In some embodiments, FX activators or pharmaceutical compositions thereof are applied topically to an injury or wound, such as directly to wound tissue. In some embodiments, FX activators or pharmaceutical compositions thereof are administered by sustained-release or extended-release technology.

[0234] In any embodiment of the method for treating a bleeding disorder described herein, the method further comprises administering one or more additional coagulation factors. For example, the one or more additional coagulation factors can be plasma purified or recombinant coagulation factors, coagulants such as vitamin K, vitamin K derivatives and protein C inhibitors, plasma, platelets, red blood cells or corticosteroids.

[0235] Bleeding disorders

[0236] As used herein, "disease" or "disorder" refers to a pathological condition in an organism caused by a cause of disease or a condition, including but not limited to infection, acquired conditions, hereditary conditions, and characterized by recognizable symptoms. Diseases and disorders of concern herein relate to coagulation, including diseases mediated by coagulation proteins and diseases and disorders in which coagulation proteins play a role in etiology or pathology. Diseases and disorders also include diseases and disorders caused by a lack of protein, such as hemophilia, and of particular concern herein are those diseases in which coagulation does not occur due to coagulation protein defects. Exemplary diseases and disorders, such as but not limited to blood coagulation diseases, hematological diseases, bleeding disorders, hemophilia, coagulation factor deficiencies, and acquired blood diseases, include bleeding associated with trauma and surgery.

[0237] As used herein, "bleeding disorder" refers to a condition in which a patient's ability to control bleeding is reduced due to insufficient blood clotting. A bleeding disorder can be hereditary or acquired, and can be caused by, for example, a defect or deficiency in the coagulation pathway, a defect or deficiency in platelet activity, or a vascular defect. In some embodiments, the bleeding disorder is a congenital bleeding disorder or an acquired bleeding disorder.

[0238] As used herein, "acquired bleeding disorder" refers to a bleeding disorder caused by a clotting defect resulting from liver disease, vitamin K deficiency, or treatment with coumarin (warfarin) or other anticoagulants.

[0239] Bleeding diseases can be diseases caused by lack of coagulation factors, diseases caused by the presence of acquired coagulation factor inhibitors, blood diseases, bleeding diseases, von Willebrand disease, diseases caused by anticoagulation therapy with vitamin K antagonists, hereditary platelet diseases, vitamin K epoxide reductase C1 deficiency, gamma carboxylase deficiency, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC), Bernard-Soulier syndrome, Glanzman thrombosis or storage pool deficiency. In an embodiment, for bleeding diseases due to lack of coagulation factors or due to the presence of acquired coagulation factor inhibitors, coagulation factors can be factor VII, factor IX, factor X, factor XI, factor V, factor XII, factor II or von Willebrand factor. In an embodiment, for bleeding diseases due to lack of coagulation factors, coagulation factors can be factor VII, factor VIII, factor IX, factor XI. In some embodiments, bleeding diseases are due to lack of coagulation factors. In some embodiments, bleeding diseases are hemophilia. For example, the bleeding disorder can be hemophilia A, hemophilia B, or hemophilia C. In some embodiments, the hemophilia is hemophilia with an inhibitor (e.g., an antibody). In some embodiments, the bleeding disorder is hemophilia A without an inhibitor. In some embodiments, the bleeding disorder is hemophilia A with a FVIII inhibitor (anti-FVIII antibodies, e.g., exogenous FVIII). In some embodiments, the bleeding disorder is hemophilia B without an inhibitor. In some embodiments, the bleeding disorder is hemophilia B with a FIX inhibitor (anti-FIX antibodies, e.g., exogenous FIX).

[0240] In the example of the method described herein, the bleeding disease is due to the lack of coagulation factors, and the disease is familial multiple coagulation factor deficiency (FMFD). In the example described herein, the bleeding disease is due to the presence of acquired inhibitors of coagulation factors, and the coagulation factors are factor VII, factor VIII, factor IX, factor X, factor XI and factor XII. In such examples, the acquired inhibitor is an autoantibody. For example, the bleeding disease is acquired hemophilia. In the example of the method described herein, the bleeding disease is an inherited platelet disease, which can be Chediak-Higashi syndrome, Hermansky-Pudlak syndrome, thromboxane A2 dysfunction, Glanzmann thrombosis and Bernard-Soulier syndrome. In other embodiments described herein, when the disease is caused by the anticoagulant treatment of a vitamin K antagonist, the vitamin K antagonist can be heparin, pentasaccharide, warfarin, small molecule antithrombotic drugs and FXa inhibitors.

[0241] In other examples of the methods described herein, the disease is thrombosis, thrombocytopenia, apoplexy and coagulopathy. In further examples of the methods described herein, hemorrhagic conditions are caused by trauma, surgery or wound. For example, in this class example, bleeding is manifested as acute hematoma, chronic hemophilic arthropathy, hematoma, hematuria, central nervous system bleeding, gastrointestinal bleeding or cerebral hemorrhage. In other examples as described herein, bleeding is due to tooth extraction. In other examples as described herein, the hemorrhagic disease is due to surgery, and surgery is heart surgery, angioplasty, lung surgery, abdominal surgery, spinal surgery, brain surgery, vascular surgery, dental surgery or organ transplant surgery. For example, surgery is transplant surgery, by the transplantation of bone marrow, heart, lung, pancreas or liver.

[0242] Congenital bleeding disorders

[0243] The FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead) can be used to bypass any coagulation factor in the intrinsic or extrinsic coagulation pathway and treat coagulopathy caused by its deficiency. Bleeding disorders caused by congenital coagulation factor deficiency include hemophilia A (factor VIII deficiency); hemophilia B (factor IX deficiency); hemophilia C (factor XI deficiency); factor VII deficiency; factor X deficiency; factor XII deficiency; and familial multiple coagulation factor deficiency (FMFD) type I, II, IV, V, and VI (see Roberts, HR and M D Bingham, "Other Coagulation Factor Deficiencies," Thrombosis and Hemorrhage, 2nded. Baltimore, Md.: Williams & Wilkins, 1998: 773-802). FX activators or pharmaceutical compositions thereof may also be used to treat other congenital bleeding diseases and disorders, such as, but not limited to, von Willebrand disease, inherited platelet disorders (e.g., storage pool diseases such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, Glanzmann's thrombosis, and Bernard-Soulier syndrome), hereditary hemorrhagic telangiectasia, also known as Rendu-Osler-Weber syndrome.

[0244] Other congenital bleeding diseases can be treated with the FX activators (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) described herein to promote coagulation. Spontaneous and surgery-related bleeding events associated with von Willebrand disease (vWD) can be treated with FX activators or pharmaceutical compositions thereof. Von Willebrand disease is a bleeding disorder caused by a defect or deficiency in the coagulation protein von Willebrand factor (vWF), which is estimated to occur in 1% to 2% of the population. vWD patients bruise easily, have recurrent nosebleeds, bleed after tooth extraction, tonsillectomy or other surgeries, and female patients have increased menstrual bleeding. FX activators or pharmaceutical compositions thereof can be used to improve spontaneous bleeding and surgery-related bleeding in vWD patients.

[0245] Other platelet-related bleeding disorders are also associated with reduced intrinsic coagulation activity, such as Glanzmann's thrombosis and Hermansky-Pudlak syndrome. Excessive spontaneous or surgery-related bleeding in patients with platelet-related bleeding disorders can also be controlled by therapeutic doses of FX activators (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) described herein. For example, FX activators or pharmaceutical compositions thereof can be used to treat patients with Glanzmann's thrombosis before, during, and / or after surgery to prevent major bleeding.

[0246] Acquired bleeding disorders

[0247] Bleeding disorders can also be acquired rather than congenital. The FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RV-X lead agents) can also be used to treat acquired bleeding disorders. Acquired bleeding disorders include coagulopathy caused by surgery or drug-induced coagulopathy, such as thrombocytopenia caused by chemotherapy regimens. In some examples, FX activators or pharmaceutical compositions thereof can be used as antidotes for excessive anticoagulant therapeutic agents. FX activators or pharmaceutical compositions thereof can also be used to treat acquired coagulation factor deficiencies, such as acquired factor X deficiency and vitamin K deficiency due to liver disease. Other acquired bleeding diseases that may benefit from treatment with FX activators or pharmaceutical compositions thereof include hemolytic uremic syndrome, Henoch Schonlein purpura, and disseminated intravascular coagulation (DIC).

[0248] In some embodiments, the FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) can be used to treat bleeding episodes in subjects due to trauma or surgery or decreased platelet count or activity. For example, FX activators or pharmaceutical compositions thereof can be used to treat, for example, hemodilutional coagulopathy due to transfusion or acute traumatic coagulopathy following trauma. Exemplary methods for surgical patients include preventing bleeding or treatment before, during, or after treatment and surgery, such as, but not limited to, cardiac surgery, angioplasty, lung surgery, abdominal surgery, spinal surgery, brain surgery, vascular surgery, dental surgery, or organ transplant surgery, including bone marrow, heart, lung, pancreas, or liver transplants. In some embodiments, the acquired bleeding disorder is chemotherapy-acquired thrombocytopenia, other coagulation disorders, transplant-acquired bleeding (e.g., severe bleeding following bone marrow transplant (BMT) or stem cell transplant (SCT)), bleeding due to anticoagulant therapy (patients receiving anticoagulant therapy to treat conditions such as thromboembolism may experience bleeding events after acute administration of anticoagulants such as warfarin, heparin, fondaparinux, and rivaroxaban, or develop a bleeding disorder due to long-term use of such therapy), or acquired hemophilia.

[0249] Trauma and surgical bleeding

[0250] The FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) can be used to treat bleeding associated with perioperative blood loss and traumatic blood loss in subjects with a normal coagulation system. For example, FX activators or pharmaceutical compositions thereof can be administered to patients to promote coagulation and reduce blood loss associated with surgery, further reducing the need for transfusions.

[0251] In some examples, the FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) can be administered to patients with normal coagulation function undergoing various types of surgery to achieve rapid hemostasis and prevent blood loss. Treatment with FX activators or pharmaceutical compositions thereof can promote hemostasis at the surgical site and reduce or prevent blood loss, thereby reducing or eliminating the need for blood transfusions. In some embodiments, FX activators or pharmaceutical compositions thereof may exhibit enhanced properties, such as extended half-life, resistance to circulating protease inhibitors, and / or increased catalytic activity, and therefore, can be administered, for example, at lower doses, less frequently, and with fewer adverse reactions.

[0252] The FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RV-X lead formulations) can also be used to promote coagulation and prevent blood loss in trauma subjects. Trauma is defined as damage to biological tissues by exogenous factors and is the fourth leading cause of death in the United States. Trauma is divided into blunt trauma (causing internal compression, organ damage and internal bleeding) or penetrating trauma (agents penetrate the body and destroy tissues, blood vessels and organs, causing external bleeding). Trauma can be caused by a variety of events, including but not limited to car accidents (causing blunt and / or penetrating trauma), gunshot wounds (causing penetrating trauma), stabbings (causing penetrating trauma), mechanical accidents (causing penetrating and / or blunt trauma), and falling from heights (causing penetrating injuries and / or blunt trauma). Uncontrolled bleeding caused by trauma is the cause of most related deaths.

[0253] Diffuse coagulopathy is a relatively common complication associated with trauma patients, occurring in 25-36% of patients. Coagulopathy can develop early after injury and is caused by a variety of factors, such as dilution and consumption of coagulation factors and platelets, fibrinolysis, acidosis, and hypothermia. Conventional treatment includes replacement therapy with transfusions of fresh frozen plasma (FFP) platelets, red blood cells, and / or cryoprecipitate, correction of acidosis, and treatment of hypothermia. These steps are often insufficient to stop bleeding and prevent death.

[0254] hemophilia

[0255] As used herein, "hemophilia" refers to a bleeding disorder caused by a lack of a clotting factor. Hemophilia can be caused by a lack of a clotting factor, reduced expression, or reduced function. The most common type of hemophilia is hemophilia A, which is caused by a lack of factor VIII. The second most common hemophilia is hemophilia B, which is caused by a lack of factor IX. Hemophilia C, also known as FXI deficiency, is a milder and less common hemophilia.

[0256] As used herein, "congenital hemophilia" refers to an inherited type of hemophilia. Congenital hemophilia is caused by mutation, deletion, insertion or other modification of a coagulation factor gene, in which the production of the coagulation factor is absent, reduced or non-functional. For example, inherited mutations in coagulation factor genes, such as factor VIII and factor IX, lead to congenital hemophilia, hemophilia A and hemophilia B, respectively.

[0257] As used herein, "acquired hemophilia" refers to a type of hemophilia that occurs in adulthood due to the production of autoantibodies that inactivate clotting factors such as FVIII. Factor VIII inhibitors can occur spontaneously in otherwise healthy individuals, resulting in "acquired hemophilia." Acquired hemophilia is a rare disease with an incidence of 0.2-1.0 per million per year. Autoantibodies are primarily IgG4 antibodies, which, when bound to FVIII, inhibit FVIII activity by interfering with thrombin cleavage, von Willebrand factor interaction, and / or phospholipid binding. This results in life-threatening bleeding in approximately 87% of affected patients. Common sites of bleeding are the skin, mucous membranes, muscles, and retroperitoneum, while patients with hereditary hemophilia bleed primarily in the joints and muscles. Acquired hemophilia can be treated with activated prothrombin complex concentrate or recombinant activated factor VII ( Novo Nordisk) treatment to control bleeding episodes. The FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) can exhibit enhanced coagulation activity without the need for FVII replacement therapy.

[0258] Hemophilia is a bleeding disorder caused by a deficiency in one or more clotting factors. It is characterized by a reduced ability to form blood clots at sites of tissue damage. Congenital X-linked hemophilia includes hemophilia A and hemophilia B, which are caused by mutations that result in deficiencies in FVIII and FIX, respectively. Hemophilia A affects 1 in 100,000 males, while hemophilia B affects 1 in 50,000 males. Hemophilia A and B are further classified as mild, moderate, or severe. Levels of functional factor VIII or IX in the plasma are mild when they are 5%-25%, moderate when they are 1%-5%, and severe when they are less than 1%. Hemophilia C, commonly known as FXI deficiency, is a relatively mild and rare autosomal recessive disorder that occurs in approximately 1 in 100,000 people.

[0259] Patients with hemophilia experience recurrent bleeding into the joints and muscles, which may occur spontaneously or in response to trauma. Bleeding can cause severe acute pain, limited mobility, and lead to secondary complications, including synovial hypertrophy. In addition, recurrent bleeding into the joints can lead to chronic synovitis, which can cause joint damage and destruction of the synovium, cartilage, and bone. Bleeding is usually treated with transfusions of fresh frozen plasma (FFP), replacement therapy with FXI, or local treatments, such as fibrin glue for external wounds or tooth extractions. The most common treatment for hemophilia A or B is replacement therapy, where FVIII or FIX is administered to the patient. These agents are commercially available as plasma-derived or recombinant products, and recombinant proteins are now a treatment option for previously untreated patients. While these therapies can be very successful, complications can occur if the patient develops inhibitors to the newly administered factor VIII or factor IX.

[0260] Inhibitors are IgG antibodies, mainly of the IgG4 subclass, that react with FVIII or FIX and interfere with procoagulant function. Inhibitors affect about one in five patients with severe hemophilia A. Although subjects may develop inhibitors later, most subjects develop these inhibitors soon after the first injection of factor VIII (usually in early childhood). Inhibitors also affect about one in 15 patients with mild or moderate hemophilia A. These inhibitors usually develop in adulthood and destroy not only the exogenous FVIII administered, but also endogenous FVIII. As a result, the condition of patients with mild and moderate hemophilia worsens further. Clinically, hemophilia A patients with inhibitors are divided into high responders and low responders according to the intensity of the memory reaction they experience when they are exposed to FVIII again. Inhibitors affect about one percent of hemophilia B patients. In most cases, inhibitors develop after the first injection of therapeutic factor IX and may be accompanied by allergic reactions.

[0261] The FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RV-X lead agents) and nucleic acids encoding FX activators provided herein can be generally used to treat hemophilia patients, including hemophilia patients with inhibitors, and can be used to treat bleeding disorders associated with hemophilia. The FX activators described herein or pharmaceutical compositions thereof can be used, for example, to control or prevent spontaneous bleeding events, or to control or prevent bleeding caused by trauma or surgery by enhancing thrombin generation, while no longer requiring FVIIA and / or FIXa.

[0262] The FX activator (e.g., RVV-X) or its pharmaceutical composition (e.g., RVV-X lead formulation) described herein can be tested for therapeutic effectiveness using, for example, an animal model. For example, FVIII or FIX-deficient mice (e.g., M-KOVIII mice), antibody-induced hemophilia mice, or any other known disease model of hemophilia can be treated with a FX activator or its pharmaceutical composition. The progression of disease symptoms and phenotypes is monitored to assess the effect of a FX activator or its pharmaceutical composition. FX activators or their pharmaceutical compositions can also be administered to animal models and subjects, for example, in clinical trials, compared to placebo controls and / or controls using other hemostatic agents to assess in vivo effectiveness.

[0263] The FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) can also be used to treat patients with coagulation disorders caused by deficiencies of coagulation factors other than FVIII and FIX. For example, FX activators or pharmaceutical compositions thereof can be used to treat patients with factor VII deficiency, factor X deficiency, factor XIII deficiency, and familial multiple coagulation factor deficiency (FMFD) of types II, IV, V, and VI (see Roberts, HR and MD Bingham, "Other Coagulation Factor Deficiencies," Thrombosis and Hemorrhage, 2nd ed. Baltimore, Md.: Williams & Wilkins, 1998: 773-802).

[0264] Pharmacokinetics (PK)

[0265] Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., a FX activator such as RVV-X) after administration to a subject. Pharmacokinetic parameters that can be used to determine clinical efficacy include, but are not limited to, serum / plasma concentration, serum / plasma concentration over time, maximum serum / plasma concentration (C max ), time to reach maximum concentration (T max ), half-life (t 1 / 2 ), the area under the concentration-time curve (AUC τ )wait.

[0266] The techniques for obtaining the PK curve of the drug are known in the art. See Heller et al., Annu Rev Anal Chem, 11, 2018; and Ghandforoush Sattari et al., J Amino Acids, Article ID 346237, Volume 2010. In some embodiments, the PK curve of the FX activator (e.g., RVV-X) is measured in the blood, plasma or serum sample of the individual. In some embodiments, the PK curve of the FX activator (e.g., RVV-X) in the individual is measured using mass spectrometry (e.g., LC-MS / MS or ELISA). PK analysis can be performed on the PK curve by any method known in the art, such as non-compartmental analysis, for example, using PKSolver V2 software (Zhang Y. et al., "PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel," Comput Methods Programs Biomed. 2010; 99 (3): 306-1). See also Example 1.

[0267] "C" represents the concentration of the drug in the subject's plasma, serum, or any suitable body fluid or tissue, usually expressed as mass per unit volume, such as nanograms per milliliter or picograms per milliliter. For convenience, the drug concentration in serum or plasma is referred to herein as "serum concentration" or "plasma concentration". The serum / plasma concentration at any time after administration (e.g., FX activator, such as intravenous, intraperitoneal, or subcutaneous injection) is referred to as C time or C t The maximum serum / plasma drug concentration during dosing is called C max ; C min Refers to the minimum serum / plasma drug concentration at the end of the dosing interval; C ave Refers to the average concentration during the dosing interval.

[0268] The term "bioavailability" refers to the extent or rate at which a drug (eg, a FX activator such as RVV-X) passes through the systemic circulation and thereby reaches the site of action.

[0269] “AUC” is the area under the serum / plasma concentration-time curve and is considered the most reliable measure of bioavailability, such as the area under the concentration-time curve during the dosing interval (AUCτ), “total exposure” or “total drug exposure over time” (AUC 0-last or AUC 0-inf), the area under the concentration-time curve (AUC 0-t )wait.

[0270] Peak serum / plasma concentration time (T max ) is the serum / plasma concentration reached after administration (C max ) peak time.

[0271] Half-life (t 1 / 2 ) refers to the time required for the concentration of a drug (e.g., a FX activator (e.g., RVV-X)) measured in plasma or serum (or other biological matrix) to fall to half of its concentration or amount at a specific time point. For example, after intravenous administration, the concentration of the drug in plasma or serum decreases due to the distribution and elimination of the drug. In the curve of plasma or serum drug concentration over time after intravenous administration, the first phase or rapid decline is considered to be mainly due to distribution, while the later decline is usually slower and mainly due to elimination, although both processes occur in both phases. After sufficient time, the distribution of the drug is assumed to be complete. In general, the elimination half-life is determined by the terminal phase or elimination (primary) phase of the plasma / serum concentration-time curve. See Michael Schrag and Kelly Regal, "Chapter 3 - Pharmacokinetics and Toxicokinetics" of "A Comprehensive Guide to Toxicology in Preclinical Drug Development", 2013.

[0272] In some embodiments, the FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) have a half-life of at least 5 hours (e.g., intravenous injection, such as injection into humans), such as at least 5.5, 6, 6.5, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48 hours or longer. In some embodiments, the FX activator or its pharmaceutical composition has a half-life of 5 hours to 12 hours (e.g., intravenous injection, such as injection into humans), such as 5 hours to 10 hours, 5 hours to 8.5 hours, 6.5 hours to 9 hours, 6.7 hours to 8.8 hours or 7 hours to 8.5 hours. In some embodiments, the FX activator or its pharmaceutical composition is administered in a single administration, such as a single intravenous injection, such as a dose of 0.16U / kg to 0.48U / kg. In some embodiments, the FX activator or its pharmaceutical composition is administered in multiple administrations (e.g., 4-6 times, such as 5 times), such as intravenous injection, each dose is 0.16U / kg, q4h.

[0273] In some embodiments, the administration of a FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead formulation) in a single or multiple administrations can increase the AUC 0-∞ About 600 h*pg / mL to 4000 h*pg / mL, such as 650 h*pg / mL to 3500 h*pg / mL, 650 h*pg / mL to 1500 h*pg / mL, 900 h*pg / mL to 2000 h*pg / mL, 1500 h*pg / mL to 3500 h*pg / mL, 3200 h*pg / mL to 3500 h*pg / mL, 1200 h*pg / mL In some embodiments, the FX activator or its pharmaceutical composition is administered at a dosage of 0.16 U / kg to 0.48 U / kg (or a single dose), such as 0.16 U / kg each time.

[0274] In some embodiments, the FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead formulation) is administered in a single administration (e.g., intravenous injection, such as injection into a human), such as a dose of 0.16 U / kg to 0.48 U / kg, providing C max About 60pg / mL to 500pg / mL, such as 70pg / mL to 450pg / mL, 70pg / mL to 150pg / mL, 100pg / mL to 500pg / mL, 200pg / mL to 500pg / mL, 300pg / mL to 500pg / mL, 400pg / mL to 500pg / mL, 100pg / mL to 300pg / mL, 90pg / mL to 120pg / mL, 100pg / mL to 250pg / mL, 140pg / mL to 180pg / mL, 150pg / mL to 450pg / mL, or 270pg / mL to 320pg / mL. In some embodiments, C is reached within up to 10 minutes (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, 1 minute) of administration. max , such as administering the drug for 5 minutes.

[0275] Pharmacodynamics (PD)

[0276] The FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) can be evaluated for their biological activity using assays or in vivo parameters, such as in vitro coagulation assays or pharmacodynamic (PD) effects in hemophilia models or individuals.

[0277] PD parameters or tests for detecting coagulation activity are known in the art, including but not limited to, activated partial thromboplastin time (APTT), factor X activity test (FX:C), thrombin generation (TG) (including peak height), calibrated automatic thrombin generation analysis (CAT), endogenous thrombin generation potential (ETP), activated clotting time (ACT), whole blood clotting time (WBCT), etc. The APTT test is to measure and evaluate the endogenous and common pathway coagulation factors of all coagulation cascade reactions by measuring the time (in seconds) required for thrombus formation after adding drugs to plasma samples. APTT can be evaluated using APTT-ACTIN FSL kit (Siemens, Germany), CS-5100 (Sysmex, Japan) automatic blood coagulation analyzer or STA-CKPrest (Stagio, France) and STA-R Max (Stago) automatic coagulation analyzer. FX:C can be confirmed by using an automatic coagulation analyzer (Sysmex) or FX colorimetric activity assay kit for coagulation test. TG can be measured by automated thrombus profiling using a TG (CAT) analyzer (Stago) calibrated with, for example, tissue factor (TF) and phospholipids (Stago) as triggers. See also Example 1.

[0278] In some embodiments, the FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead formulation) is administered as a single dose (e.g., intravenous injection, such as injection into humans), such as a dose of 0.16 U / kg to 0.48 U / kg, and can i) increase the peak height of TG and ETP in a dose-dependent manner; ii) reach the peak value of TG and / or ETP at 5 minutes; iii) maintain TG and / or ETP at a higher level until 4 hours after administration; and / or iv) after about 24 hours of administration, TG and / or ETP returns to the pre-dose level.

[0279] In some embodiments, the FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead) is administered as a single dose (e.g., intravenous injection, such as injection into humans), such as a dose of 0.16 U / kg to 0.48 U / kg, and can i) shorten APTT in a dose-dependent manner; ii) shorten APTT by at least about 5% (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or more); iii) the shortening of APTT reaches a maximum at about 5 minutes after administration; and / or iv) the APTT returns to the pre-dose level after about 24 hours of administration.

[0280] In some embodiments, the FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead) is administered in multiple doses (e.g., intravenous injection, such as injection to humans), such as a dose of 0.16 U / kg, q4h, 2-6 times (e.g., 2, 3, 4, 5 or 6 times), which can i) shorten the APTT within 10 minutes of the first administration; ii) shorten the APTT by at least about 20% (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 13 0%, 60%, 70% or more); iii) shortening APTT by at least about 20 seconds (e.g., at least about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80 or more); iv) APTT reaches a plateau 4 hours after the third dose; v) shortening APTT to about 8 hours after the last dose; and / or vi) APTT returns to pre-dose levels about 28 to 26 hours after the last dose.

[0281] Security

[0282] The safety results of the FX activator described herein (e.g., RVV-X) or its pharmaceutical composition (e.g., RVV-X lead formulation) can be evaluated by vital signs, physical examination, laboratory test results, electrocardiogram (ECG), ultrasound (double lower extremity abdomen and deep veins) and adverse event overview. The severity of adverse events can be determined with reference to the NCI Common Terminology Criteria for Adverse Events (CTCAE v5.0). See Example 1, Table 4-1, Table 4-2, Table S1, Table S2 for safety measurement methods and parameters. For example, dose-limiting toxicity (DLT) events, serious adverse events (SAE), adverse events (AE) leading to drug discontinuation, dose-dependent AEs, drug-related AEs, drug-related SAEs, or suspected unexpected serious adverse reactions (SUSAR) can be detected. Other adverse parameters include, but are not limited to, clinically relevant coagulation abnormalities, deep vein thrombosis (DVT) of the lower extremities, thrombotic events, D-dimers (protein fragments produced when blood clots dissolve) increase, fibrin degradation products (FDP) increase, etc. Adverse parameters also include FX levels and / or fibrinogen (FIB) levels that fall beyond the normal range and / or fail to return to the normal range after a period of time (eg, 3 hours).

[0283] In some embodiments, the methods for treating bleeding disorders described herein (e.g., single or multiple administrations), and the FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) induce no or low levels of adverse reactions, as determined by one or more parameters and / or tests described herein. No or low levels of adverse reactions include, but are not limited to, AEs within grades 1-2 (e.g., drug-related AEs); Grade 3 AEs less than 5% of total AEs (e.g., less than 5%, 4%, 3%, 2%, 1% or 0%); each dose-dependent AE, DLT event, SAE, AE, AE leading to discontinuation, drug-related SAE, SUSAR, clinically relevant coagulation abnormalities, DVT, DIC, D-dimer increase, FDP increase and / or venous thrombosis events less than 5% of total AEs (e.g., less than 5%, 4%, 3%, 2%, 1% or 0%); FX levels and / or FIB levels decreased within the normal range, etc.

[0284] Immunogenicity

[0285] In the context of administering a protein drug to a patient, the term "immunogenicity" is defined as the propensity of the protein drug to produce an immune response in a patient after administration or after repeated administration.

[0286] Anti-FX activator (e.g., anti-RVV-X) binding antibodies in plasma can be detected by an effective electrochemiluminescent assay (Meso Scale Discovery). Antibody titer and neutralizing antibodies (against FX activators) can be further evaluated.

[0287] In some embodiments, the methods of treating a bleeding disorder described herein (e.g., single or multiple administrations), and the FX activators described herein (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) do not induce neutralizing antibodies or induce low titers (e.g., less than 30, 20, 10, 5, or 2) of neutralizing antibodies.

[0288] In some embodiments, the methods of treating a bleeding disorder described herein (e.g., single or multiple administrations), and the FX activators (e.g., RVV-X) or pharmaceutical compositions thereof (e.g., RVV-X lead formulations) described herein i) do not induce anti-FX activator (e.g., anti-RVV-X) binding antibodies in plasma, or induce low titers (e.g., less than 30) of binding antibodies; and / or ii) do not increase inhibitor titers or increase inhibitor titers by less than 10%.

[0289] V. Products and Kits

[0290] Kits, unit doses, and articles of manufacture comprising any FX activator described herein (e.g., RVV-X) or a pharmaceutical composition thereof (e.g., RVV-X lead formulation) are further provided. In some embodiments, a kit comprising any FX activator pharmaceutical composition described herein is provided, and preferably instructions for use thereof are provided, such as for the treatment of a bleeding disorder described herein (e.g., hemophilia A or B, with or without inhibitors).

[0291] In some embodiments, the FX activator (e.g., RVV-X) pharmaceutical composition (e.g., lyophilized, such as the RVV-X lead formulation) is packaged in a vial (e.g., with a stopper that can be pierced by a hypodermic needle). In some embodiments, the pharmaceutical composition is in powder form. In some embodiments, each vial contains 5U of FX activator (e.g., RVV-X). In some embodiments, the kit further provides instructions for reconstituting the pharmaceutical composition with 2ml of 0.9% sodium chloride injection. In some embodiments, the kit further provides instructions for intravenous injection of the pharmaceutical composition. In some embodiments, the kit further comprises an injection needle. In some embodiments, the kit further comprises 0.9% sodium chloride injection in a separate container.

[0292] The kit of the present invention includes one or more containers containing the FX activator (e.g., RVV-X) or its pharmaceutical composition (e.g., RVV-X lead formulation) described herein, for example, for the treatment of bleeding disorders. For example, instructions describing the administration of the FX activator (e.g., RVV-X) or its pharmaceutical composition to treat bleeding disorders (such as hemophilia) are included. The kit may further include a description of identifying whether an individual has a disease and the stage of the disease to select an individual (e.g., human) suitable for treatment. Instructions related to the use of the FX activator (e.g., RVV-X) or its pharmaceutical composition generally include information about the dose, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package) or subunit dose. The instructions provided in the kit of the present invention are generally written instructions written on a label or drug instructions (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions stored on a disk or CD) are also acceptable. The kit of the present application uses suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Also contemplated are packages for use in conjunction with specific devices, such as infusion devices such as micropumps. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an FX activator as described herein (e.g., RVV-X). The container may further comprise a second pharmaceutically active agent. The kit may optionally provide additional components, such as buffers and interpretive information. Generally, the kit comprises a container and a label or package insert on or associated with the container.

[0293] The present application also provides articles, including vials (such as sealed vials), bottles, cans, flexible packages, etc. The article comprises a container and a label or drug instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. In general, the composition contained in the container can effectively treat the diseases or disorders described herein, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or drug instructions indicate that the composition is used to treat a specific condition of an individual. The label or drug instructions further include instructions for administering the composition to an individual. The label may indicate instructions for reconstitution and / or use. The container for holding the pharmaceutical composition can be a vial for multiple use, allowing repeated administration of the reconstituted preparation (for example, 2-6 administrations). Drug instructions refer to instructions usually contained in the commercial packaging of therapeutic products, which contain indications, usage, dosage, administration, contraindications and / or warning information related to the use of such therapeutic products. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. From a commercial and user perspective, other required materials may be further included, including other buffers, diluents, filters, needles and syringes.

[0294] The kit or article of manufacture includes multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. DETAILED DESCRIPTION

[0295] The following examples are intended to be merely exemplary of the present invention and therefore should not be construed as limiting the present invention in any way.The following examples and detailed description are provided for illustration only and not limitation.

[0296] Example 1: First-in-human Phase I study of the FX activator RVV-X in patients with hemophilia A or B with inhibitors

[0297] Hemophilia A (HA) and hemophilia B (HB) are inherited bleeding disorders caused by deficiencies in factor VIII (FVIII) and factor IX (FIX), respectively. Bleeding episodes (BEs) in these patients are usually treated with factor replacement; however, 20%-30% of patients with hemophilia A and 5% of patients with hemophilia B develop exogenous inhibitors to FVIII or FIX. 1Patients with inhibitors have difficult-to-control bleeding episodes. In these patients, hemostasis may not be achieved by replacing the deficient factor (depending on the inhibitor titer), so bypassing agents may be required. The two currently recommended first-line bypassing agents for patients with inhibitors are activated thrombin complex concentrate (aPCC) and activated recombinant factor VII (rFVIIa). 2-4 However, aPCC is not available in China, and the trace amount of factor VIII in aPCC may induce a recall response to FVIII. Due to the high medical cost, rFVIIa has not been widely used, especially in developing countries.

[0298] RVV-X is a 93 kDa heterotrimer purified from the venom of the round-spotted viper (Daboia russelliisiamensis). It consists of an α chain (heavy chain), a β chain (light chain 1), and a γ chain (light chain 2). 8-10 Composition. RVV-X can specifically activate coagulation factor X (FX). The activation process involves cutting FX at the specific "Arg-Ile" peptide bond at the N-terminus of the FX heavy chain, fully exposing the active site to produce coagulation factor Xa (FXa). FXa then forms a prothrombin complex with platelets, coagulation factor Va (FVa) and calcium ions activated at the site of injury, thereby increasing the generation of thrombin and achieving the purpose of hemostasis. 10 Preclinical studies have confirmed that RVV-X can activate FX to FXa and increase the level of thrombin in human plasma and the peak of thrombin generation in a dose-dependent manner. In addition, RVV-X can shorten the APTT of hemophiliac mice and reduce the 24-hour mortality rate of hemophiliac mice after tail amputation. Preclinical toxicology studies have shown that RVV-X has good tolerability and safety.

[0299] In this study, we conducted a phase I, first-in-human, multicenter, open-label, dose-escalation study of RVV-X to evaluate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of RVV-X in patients with hemophilia A or B with factor VIII / IX inhibitors, respectively.

[0300] Chinese patients with inhibitors HA or HB received a single intravenous injection of RVV-X (0.01U / kg, 0.04U / kg, 0.08U / kg, 0.16U / kg, 0.32U / kg and 0.48U / kg) or multiple intravenous injections of RVV-X (0.16U / kg) every 4 hours (q4h) for a maximum of 6 injections in the absence of bleeding. This study is registered at www.clinicaltrials.gov, trial number NCT-04747964 and NCT-05027230.

[0301] Results of the single-dose group: RVV-X showed a linear PK curve. After RVV-X administration, the changes in the peak height of thrombin generation (TG) and the endogenous thrombin generation potential (ETP) increased in a dose-dependent manner. The activated partial thromboplastin time (APTT) was shortened in a dose-dependent manner.

[0302] Results of the multiple-dose group: After the first three administrations of RVV-X, the increase in TG peak height and the shortening of APTT both reached a plateau.

[0303] In summary, both single and multiple injections of RVV-X are safe and well tolerated. Starting from a single dose of 0.16U / kg, RVV-X significantly improved coagulation-related laboratory indicators. In the multiple-dose group, the improvements in APTT and ETP reached a plateau after 4 doses of RVV-X. No serious adverse events (SAEs) or dose-limiting toxicity (DLT) events were reported. No thromboembolic events and disseminated intravascular coagulation (DIC) were observed. No anti-drug antibodies were detected after RVV-X administration. These results suggest that RVV-X has the potential to be used as an on-demand treatment for bleeding events in patients with hemophilia, whether HA or HB.

[0304] · method

[0305] Study design and participants

[0306] This study was a first-in-human, multicenter, open-label, dose-escalation phase I clinical study. This study was conducted at the Hematology Hospital of the Chinese Academy of Medical Sciences (Tianjin, China) and Henan Cancer Hospital (Zhengzhou, China) in accordance with the Declaration of Helsinki and the International Conference on Good Clinical Practice (ICH-GCP). This study was approved by the ethics committee of each participating center (XY2019039-EC-2 and XY2021034-EC-2) and was approved by the www.clinicaltrials.gov All subjects signed informed consent before registration.

[0307] Chinese male patients (age 18-65 years) with moderate or severe hemophilia A or B (factor activity level <5% IU / dL) with inhibitors (factor VIII or IX inhibitors, respectively) were eligible for inclusion. Subjects with a history of clinically significant allergy to any component of drugs or protein blood products, coagulation disorders other than hemophilia A or B, and thromboembolic diseases were excluded.

[0308] In the single-dose group, eligible patients were divided into group 1 (0.01 U / kg, n=2), group 2 (0.04 U / kg, n=2), group 3 (0.08 U / kg, n=3), group 4 (0.16 U / kg, n=3), group 5 (0.32 U / kg, n=3), and group 6 (0.48 U / kg, n=3), and received a single intravenous (IV) injection of RVV-X.

[0309] In the multiple-dose group, eligible patients (n=6) received intravenous RVV-X at a dose of 0.16U / kg every 4 hours (q4h) for up to 6 injections. According to the results of the Phase A study, 0.16U / kg RVV-X can significantly shorten APTT, so 0.16U / kg was selected as the initial dose for multiple dose escalation.

[0310] Dose escalation starts with the lowest dose of RVV-X and follows a step-by-step dose escalation procedure. In the single-dose group, RVV-X is first administered to only one subject each time it is administered. After safety is confirmed for 48 hours, RVV-X is administered to the remaining subjects. Vital signs, laboratory tests (see the various indicators of "Laboratory Tests" in Example 2), electrocardiogram (ECG), ultrasound (abdomen and deep veins of both lower limbs), and adverse events are monitored within 168 hours after administration. After tolerability and safety are confirmed, the Data and Safety Monitoring Committee decides on the next dose level. Multiple dose groups are administered after confirming the tolerability and safety of the single-dose group.

[0311] The results of the study on the effect of RVV-X on APTT in the plasma of FVIII-deficient people, FIX-deficient people and normal people showed that 0.001U / ml~10U / ml RVV-X can shorten the APTT in the plasma of FVIII-deficient and FIX-deficient people in a dose-dependent manner ( Figure 4 ), based on which the minimum expected biological effect dose (MABEL) is determined to be 0.001U / ml. Based on the average healthy human body weight of 60kg and circulating blood volume of 4000ml, the maximum recommended starting dose (MRSD) is 0.067U / kg.

[0312] Study drugs and administration

[0313] The formulation for administration comprises RVV-X (including SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) isolated and purified from the venom of Daboia russellii siamensis and excipients, including sucrose, histidine, polysorbate 20 and mannitol.

[0314] This product is white or white sparse block or powder, sterile, for injection. Each vial contains 5U of freeze-dried RVV-X. Before injection, each vial is reconstituted with 2ml of 0.9% sodium chloride injection. The reconstituted preparation is slowly injected intravenously to the subject, and the injection amount for each subject is calculated based on the dose of each group.

[0315] Measurement results

[0316] The following indices were observed: vital signs, 12-lead electrocardiogram, chest X-ray, ultrasound (abdomen, deep vein of lower limbs), coagulation factor VIII concentration (FVIII:C), coagulation factor IX concentration (FIX:C), FVIII or FIX inhibitors, blood routine, blood biochemistry, urine routine, prothrombin time (PT), APTT, fibrinogen (FIB), thrombin time (TT), coagulation factor X concentration (FX:C), thrombin generation (TG), D-dimer, fibrin degradation product (FDP), PK test of RVV-X and anti-RVV-X antibody.

[0317] Pharmacokinetics (PK)

[0318] Plasma RVV-X concentration was determined by a validated electrochemiluminescence assay (Meso Scale Discovery, USA), with a limit of quantification of 5 pg / mL.

[0319] Pharmacodynamics (PD)

[0320] APTT, factor X activity assay (FX:C), thrombin generation (TG), including peak height, endogenous thrombin generation potential (ETP) were assessed. APTT was determined using the APTT-ACTIN FSL kit (Siemens, Germany), CS-5100 (Sysmex, Japan) automated blood coagulation analyzer or STA-CK Prest (Stago, France) and STA-R Max (Stago) automated blood coagulation analyzers. FX:C was determined by validated coagulation assay using an automated blood coagulation analyzer (Sysmex). TG was determined by calibrated automated thrombograph assay using a TG (CAT) analyzer (Stago) with 1 μM tissue factor (TF) and 4 μM phospholipids (Stago) as triggers.

[0321] Security

[0322] Safety outcomes were assessed by vital signs, physical examination, laboratory tests, electrocardiogram (ECG), ultrasound (abdomen and deep vein of lower extremities), and adverse event profiles. The severity of adverse events was determined according to the NCI Common Terminology Criteria for Adverse Events (CTCAE v5.0).

[0323] Immunogenicity

[0324] Anti-RVV-X binding antibodies in plasma were screened and confirmed by a validated electrochemiluminescence assay (Meso Scale Discovery). For positive samples, antibody titers and neutralizing antibodies were further determined.

[0325] Statistical analysis

[0326] Summary statistics were provided for demographic characteristics, PK, PD, and safety results. In the single-dose group, it was planned to enroll at least two patients in each group to evaluate safety, PK, and PD. PK parameters included the maximum plasma concentration (C max ), the area under the blood drug concentration-time curve (AUC 0-t ), the area under the curve (AUC) from zero to infinity 0-∞ ), time to reach maximum plasma concentration (T max ), half-life (t 1 / 2 ), apparent volume of distribution (V z ), plasma clearance (CL z ), elimination rate constant (λ z The residual area percentage (AUC) extrapolated from the last quantifiable concentration to infinity was calculated from the RVV-X plasma concentration-time curve for each subject using PhoenixWinNonlin software version 8.1. ％Extrap ). The power function model was used to analyze the relationship between AUC and dose, C max In the multiple dose group, the AUC was calculated. 0-t , AUC 0-∞ ,t 1 / 2 .

[0327] · result

[0328] Research group

[0329] Single-dose group: A total of 16 Chinese hemophilia patients were included in this trial, of which 15 patients were hemophilia A (HA) with inhibitors and 1 patient was hemophilia B (HB) with inhibitors (Table 1).

[0330] Multiple-dose group: A total of 7 Chinese hemophilia patients were included in this trial, of which 5 subjects received 5 times, 1 subject received 6 times, and 1 patient received 2 times. Among them, 5 patients were hemophilia A (HA) with inhibitors, and 2 patients were hemophilia B (HB) with inhibitors (Table 1).

[0331] All patients were of Han nationality and completed RVV-X administration as required by the protocol.

[0332] Table 1 Demographic data and clinical characteristics of the enrolled subjects

[0333]

[0334] ·PK

[0335] Single-dose group: At doses of 0.16U / kg, 0.32U / kg, and 0.48U / kg, the plasma concentration of RVV-X increased in a dose-dependent manner. max Increase with linear PK characteristics, ( Figures 1A-1C ). C max The C of RVV-X at single doses of 0.16 U / kg, 0.32 U / kg and 0.48 U / kg was significantly higher than that of RVV-X at 5 minutes and gradually returned to the pre-dose level 24 hours after administration. max The AUC values ​​were 110.04±32.03pg / mL, 163.51±50.79pg / mL and 296.48±122.65pg / mL, respectively. 0-∞ The AUC values ​​were 988.38±325.97 hours*pg / mL, 1419.10±497.46 hours*pgs / mL, and 2478.88±944.98 hours*pg / mL, respectively. 0-∞ It also increases with increasing dose. 1 / 2 They were 7.33±0.55h, 8.33±1.41h and 7.57±0.68h respectively.

[0336] Table 2 PK parameters after a single injection of RVV-X

[0337]

[0338]

[0339] Multiple-dose group: After 5 consecutive administrations, the plasma concentration of RVV-X gradually increased, but the increasing trend gradually became flat ( Figure 1D ). The PK parameter analysis results of multiple injections of 0.16U / kg showed that AUC 0-∞ 3342.02h*pg / mL, T 1 / 2 It is 8.40 hours.

[0340] The PK data for single-dose and multiple-dose regimens were consistent and comparable.

[0341] ·PD

[0342] Single-dose group: After RVV-X administration, the peak height of TG and the change of ETP increased in a dose-dependent manner, reaching a peak at 5 minutes, remaining at a high level in the first 4 hours, and gradually returning to the pre-dose level after 24 hours ( Figure 2A and 2B ). APTT was shortened in a dose-dependent manner, with the maximum shortening of APTT observed at 5 minutes, which lasted until 12 hours and gradually returned to the pre-dose level after 24 hours (Table 3-1).

[0343] In the single-dose group, as the dose of the study drug increased, the improvements in APTT and ETP became more and more obvious. Starting from a dose of 0.16 U / kg, RVV-X significantly improved coagulation-related experimental indicators (see the indicators in "Laboratory Tests" in Example 2).

[0344] Table 3-1 APTT of single-dose group

[0345]

[0346]

[0347] Multiple-dose group: After the first three doses of RVV-X, the peak height of TG continued to increase, and there was no significant increase after the fourth dose. After RVV-X administration, APTT was rapidly shortened (shortened to within 10 minutes after the first dose, and an average shortening of about 22 seconds compared with the baseline level), and dropped to a plateau 4 hours after the third dose. This indicates that RVV-X has achieved maximum efficacy after 4 doses. The shortening of APTT lasted until 8 hours after the last dose, and gradually returned to the pre-dose level 28-36 hours after the last dose (Table 3-2).

[0348] Multiple doses of 0.16U / kg also significantly improved coagulation-related laboratory indicators.

[0349] Table 3-2 APTT (0.16U / kg) in multiple dose groups

[0350]

[0351] Safety results

[0352] Single-dose group: During the study, RVV-X was well tolerated at doses up to 0.48U / kg. A total of 36 adverse events were reported by 14 of 16 patients (88%) (Table 4-1), of which 32 (89%) events were grade 1 in severity and 4 (11%) events were grade 2. No dose-limiting toxicity (DLT) events, serious adverse events (SAEs), or adverse events (AEs) leading to study discontinuation were reported during the trial. In addition, the incidence of adverse events did not increase with increasing doses. All drug-related adverse events judged by the researchers were grade 1, and no drug-related SAEs or suspected unexpected serious adverse reactions (SUSARs) were reported.

[0353] Based on adverse event reports, no evidence of clinically relevant coagulation abnormalities was found, no deep vein thrombosis (DVT) of the lower extremities was found, and no thromboembolic events were reported (Table 4-1). Within 24 hours of RVV-X administration, each group showed a certain degree of reduction in FX, but the change was within 10% compared with the baseline ( Figure 3A ), the actual measured value is above 90% of the normal value ( Figure 3B ). This result indicates that FX activation by RVV-X is very safe and that the consumption of FX is minimal.

[0354] Table 4-1 All AEs in the single-dose group

[0355]

[0356]

[0357] MedDRA V24.0 was used for coding. Note: SOC: System Organ Class, PT: Preferred Term.

[0358] Multiple-dose group: Multiple injections of RVV-X are safe and tolerable. A total of 26 adverse events (AEs) were reported by 7 of 7 patients (100%) (Table 4-2), of which 24 (92%) events were grade 1, 1 (4%) event was grade 2, and 1 (4%) was grade 3. Adverse events led to the discontinuation of continuous dosing in 6 subjects, of which 1 withdrew from the trial. The specific reasons were: an increase in D-dimers (protein fragments produced when blood clots dissolve) and fibrin degradation products (FDP) in 4 cases, a decrease in fibrinogen in 1 case, and coldness in the right dorsal foot in 1 case. After 3-4 doses of RVV-X, D-dimers increased significantly, but the maximum values ​​in most patients did not exceed 5 mg / L, and they decreased automatically after stopping dosing. No thromboembolic events or disseminated intravascular coagulation (DIC) were reported. Within 24 hours of RVV-X administration, FX showed a certain degree of reduction, but the change was within 20% compared with baseline.

[0359] A total of 18 adverse events related to the study drug RVV-X were reported by 6 subjects, of which 17 were grade 1 and 1 was grade 3. Most adverse events recovered without treatment after stopping RVV-X. The only grade 3 AE was a decrease in fibrinogen (FIB) levels. The course of this adverse event: After 5 doses of RVV-X, FIB decreased from 1.91g / L to 0.92g / L; without treatment, the FIB level returned to 1.84g / L after 3 hours. In other subjects, the FIB decreased by no more than 1g / L compared with baseline and was within the normal range. No RVV-X-related SAEs and SUSARs were reported.

[0360] In the multiple-dose group at 0.16 U / kg, up to 6 doses of RVV-X did not cause thrombotic events, indicating that RVV-X has a good safety profile in improving coagulation disorders in hemophilia patients. However, after administration of 3-4 doses of RVV-X, D-dimer increased significantly, so it is safer not to exceed 4 consecutive doses.

[0361] Table 4-2 All AEs in multiple-dose groups

[0362]

[0363] The target of RVV-X is FX, so activation of FX by RVV-X will lead to a certain amount of FX consumption. The above results of the single-dose group and the multiple-dose group indicate that activation of FX by RVV-X will not lead to excessive consumption of FX.

[0364] Immunogenicity

[0365] Binding Antibody

[0366] Single-dose group: 1 of 16 subjects had a lower titer of binding antibodies (<30) before and on day 8 after RVV-X administration. However, no increase in inhibitor titer was observed in this subject. Therefore, the positive result of binding antibodies was considered unrelated to RVV-X, but may be related to the previous use of other procoagulant drugs. Both APTT and TG of this subject improved after administration.

[0367] Multiple-dose group: 1 of 7 subjects had a lower titer of binding antibodies (<30) before and on day 7 after RVV-X administration. However, no significant increase in inhibitor titer was observed in this subject. Therefore, the positive result of binding antibodies was considered unrelated to RVV-X, but may be related to the previous use of other procoagulant drugs. Both APTT and TG of this subject improved after administration.

[0368] Neutralizing antibodies

[0369] In both the single-injection group and the multiple-injection group, all subjects were negative for neutralizing antibodies.

[0370] These findings lead to a preliminary conclusion that RVV-X has low immunogenicity and that hypersensitivity reactions will not be obtained with the administration of RVV-X.

[0371] In summary, this first in-human RVV-X study showed that intravenous RVV-X single doses up to 0.48U / kg and multiple injections of 0.16U / kg were safe and well tolerated. Starting from a single dose of 0.16U / kg, RVV-X significantly improved coagulation-related laboratory indicators. With multiple injections of RVV-X, the improvements in APTT and ETP reached a plateau after four doses. In summary, these results suggest that RVV-X has the potential to be an on-demand treatment for bleeding events in patients with hemophilia, whether HA or HB. The findings of this study will push RVV-X into subsequent clinical trials to explore its effectiveness and safety in treating bleeding events in patients with hemophilia with inhibitors.

[0372] References

[0373] 1.Astermark J.Overview of inhibitors.Semin Hematol.2006;43:S3-S7.

[0374] 2. Chevreux G, Faid V, Scohyers JM, Bihoreau NN- / O-glycosylation analysis of human FVIIa produced in the milk of transgenicrabbits. Glycobiology. 2013; 23:1531-1546.

[0375] 3. Fenaille F, Groseil C, Ramon C, et al. Mass spectrometric characterization of N-and O-glycans of plasma-derived coagulation factor VII. Glycoconj J. 2008; 25:827-842.

[0376] 4.Grandoni J,Perret G,Forier C.Kinetic analysis and binding studiesof a new recombinant human factor VIIa for treatment ofhaemophilia.Haemophilia.2017;23:300-308.

[0377] 5.Baxter HC.Anti-Inhibitor Coagulant Complex For Intravenous Use Label.2013.11.

[0378] 6.Parameswaran R,Shapiro AD,Gill JC,Kessler CM.Dose effect andefficacy of rFVIIa in the treatment of haemophilia patients with inhibitors:analysis from the Hemophilia and Thrombosis Research SocietyRegistry.Haemophilia 2005;11:100–6.

[0379] 7.Santagostino E,Mancuso ME,Rocino A,Mancuso G,Scaraggi F,MannucciPM.A prospective randomized trial of high and standard dosages of recombinantfactor VIIa for treatment of hemarthroses in hemophiliacs with inhibitors.JThromb Haemost 2006;4:367–71.

[0380] 8. Gowda DC, Jackson CM, Hensley P, Davidson EA. Factor X-activatingglycoprotein of Russell's viper venom. Polypeptide composition and characterization of the carbohydrate moieties. J Biol Chem. 1994.269(14):10644-50.

[0381] 9. Takeda S, Igarashi T, Mori H. Crystal structure of RVV-X: an example of evolutionary gain of specificity by ADAM proteinases. FEBS Lett. 2007.581(30):5859-64.

[0382] 10.Tans G,Rosing J.Snake venom activators of factor X:anoverview.Haemostasis.2001.31(3-6):225-33.

[0383] 11.Drug levels,anti-drug antibodies,and clinical efficacy of theanti-TNFαbiologics in rheumatic diseases.[J].Clinical Rheumatology,2013,32(10):1429-1435.

[0384] Example 2: Phase II efficacy study of the FX activator RVV-X in hemophilia A or B patients with inhibitors

[0385] After review by the investigators, the 0.16 U / kg multiple-dose group was determined to be safe in the Phase I study, in which APTT was significantly improved after administration of the study drug RVV-X. Therefore, a Phase II trial (on-demand treatment of bleeding events efficacy observation trial) was conducted using the 0.16 U / kg multiple-dose regimen.

[0386] Research Description

[0387] The subjects enrolled in the Phase II clinical trial are hemophiliacs with inhibitors. The subjects receive the study drug after a bleeding event and the efficacy is evaluated. According to the protocol requirements, no new subjects will be enrolled after 80 bleeding events have been treated with the study drug.

[0388] Subjects who have completed the Phase I trial can continue to participate in the Phase II trial after evaluation by the researcher and obtaining the subject's informed consent.

[0389] If the subject does not have bleeding at enrollment, follow-up is allowed. During the follow-up period, subjects who have bleeding events (non-fatal or disabling bleeding events, excluding central nervous system bleeding and gastrointestinal bleeding) should immediately go to the research center for RVV-X treatment. Subjects who have bleeding events at enrollment should receive RVV-X hemostatic treatment immediately after enrollment.

[0390] Dosage regimen : Continuous administration, with at least 4 hours between two doses, until bleeding stops, with a maximum of 6 doses. Consolidation therapy of the study drug can be performed after clinical judgment of hemostasis (8 hours after the last dose, up to 1 time, and no additional consolidation therapy for patients who have been administered 6 times). If the bleeding event is still not effectively controlled 4 hours after the end of 6 consecutive doses and further treatment is required, the study drug will no longer be continued, and rescue treatment (such as recombinant human coagulation factor VIIa or PCC) recommended by current clinical guidelines should be used and recorded. If RVV-X is administered continuously for more than 2 times but less than 6 times, but the subject's bleeding continues to worsen during the administration period, rescue treatment may also be performed if the researcher determines that continued administration of RVV-X may not stop the bleeding and there may be significant medical risks.

[0391] After the subjects are enrolled, they will be followed up until week 12. After evaluation by the investigator and informed consent of the subjects, they can be re-enrolled. For bleeding events that occur during the follow-up period, all patients will be treated with RVV-X on demand according to the dosing principle for initial hemostasis, and the dose and frequency of administration will be recorded to evaluate the hemostatic effect. If the subjects receive treatment for bleeding events outside the research center, the bleeding events and medical treatment should be recorded in detail in the form of a diary card.

[0392] Participants

[0393] Participants in the Phase II study were patients with hemophilia A or hemophilia B who had inhibitors to factor VIII or IX, respectively.

[0394] Inclusion criteria: 1) Male, aged 18-65 years; 2) Hemophilia A or B patients; 3) Hemophilia patients with high inhibitor titer or high memory response: In at least 1 previous test, the inhibitor titer was >5BU / ml and the inhibitor test was positive at the time of enrollment; 4) Ability to establish appropriate intravenous access; 5) Subjects have no fertility plans during the trial and within 3 months after the end of the follow-up, and agree to take appropriate contraceptive measures to prevent their partners from becoming pregnant; 6) Voluntary participation and signing of the informed consent form.

[0395] Exclusion criteria : 1) Other coagulation disorders other than hemophilia A and B; 2) Hemophilia patients who plan to receive immune tolerance induction therapy or coagulation factor prevention during the trial, or stop immune tolerance induction therapy or coagulation factor treatment for less than 4 weeks; 3) Those who received anticoagulant or antifibrinolytic therapy within 7 days before the study drug administration, or plan to use these drugs during the trial; 4) Those with a history of arteriovenous thrombosis, disseminated intravascular coagulation, myocardial infarction, cerebral infarction, thrombotic microangiopathy, autoimmune diseases, malignant tumors, cirrhosis, etc., who are judged by the researchers to be unsuitable for inclusion; 5) Thrombocytopenia (PLT<100×10 9 / L), moderate to severe anemia (hemoglobin <90g / L); 6) patients with liver disease who meet one of the following indicators: a) total bilirubin ≥ 2 times the upper limit of normal, and b) alanine aminotransferase (ALT) and aspartate aminotransferase ≥ 2 times the upper limit of normal; patients with kidney disease (serum creatinine ≥ 1.5 times the upper limit of normal); 7) severe bleeding before screening (fatal or disabling bleeding, central nervous system bleeding or gastrointestinal bleeding); 8) major surgery and blood or blood component transfusion within 4 weeks before screening; 9) positive anti-human immunodeficiency virus (HIV) antibody test, or known HIV infection, with CD4 count <200 cells / μL; 10) severe allergy to any component of the composition or other protein blood products; 11) participated in other new drug clinical trials within one month; 12) used procoagulant drugs (such as prothrombin complex, recombinant human coagulation factor VIIa) within 7 days before administration of study drugs; or 13) the investigator believes that it is not suitable to participate in this trial.

[0396] Stop drug administration reference standard

[0397] During multiple dosing, if the test results of the subject meet any of the following criteria, the dosing can be stopped at the discretion of the investigator: i) fibrinogen (FIB) decreases by more than 50% of the lower limit of normal, or decreases by more than 1 g / L compared with baseline; ii) APTT returns to below the upper limit of normal; iii) D-dimer is significantly higher than the baseline level and greater than 5 mg / L; and iv) thromboembolic events occur.

[0398] During the medication process, attention should be paid to the reporting of laboratory test results, and safety judgments should be made promptly after obtaining the test results.

[0399] Study drugs and administration

[0400] The formulation for administration includes RVV-X (including SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) isolated and purified from the venom of Daboia russellii siamensis and excipients. Excipients include sucrose, histidine, polysorbate 20 and mannitol. Each vial contains 5U of lyophilized RVV-X. Before injection, each vial is reconstituted with 2ml of 0.9% sodium chloride injection. The reconstituted formulation is slowly intravenously injected into the subject, and the injection amount for each subject is calculated according to the dosage regimen.

[0401] Study design

[0402] Screening period : The study started after the subjects signed the informed consent and baseline data were collected.

[0403] Follow-up and treatment period : Subjects entered the research center immediately to receive RVV-X treatment after a bleeding event (non-fatal or disabling bleeding event).

[0404] The follow-up period is 12 weeks. Once the subject completes the follow-up content in the 12th week after enrollment, the subject is defined as having completed the trial.

[0405] ·Test completed

[0406] The completion of the trial is determined when the subjects participating in the trial complete the treatment and follow-up of 80 bleeding events and complete the follow-up content at week 12 after enrollment.

[0407] Monitoring indicators

[0408] Daily safety monitoring indicators

[0409] Physical examination: A comprehensive physical examination.

[0410] Vital signs: blood pressure, pulse, respiration, temperature.

[0411] Laboratory testing

[0412] Complete blood test:Measure white blood cell (WBC) count and classification (absolute neutrophil count (NEUT#), absolute lymphocyte count (LYMP#), absolute monocyte count (MONO#), absolute eosinophil count (EOS#), absolute basophil count (BASO#)), hemoglobin (HGB), red blood cell (RBC) count, hematocrit (HCT) and platelet (PLT) count.

[0413] Blood biochemistry : Determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), blood glucose (GLU), triglycerides (TG), low-density lipoprotein (LDL-C), lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CK-MB), creatinine (Cr), urea (Urea), uric acid (UA), potassium (K), sodium (Na), calcium (Ca), phosphorus (P) and chloride (Cl).

[0414] Urinalysis :Urine specific gravity (SG), pH value, glucose (GLU), ketone bodies (KET), urine protein (PRO), white blood cells (LEU), occult blood (BLD), nitrite (NIT), urobilinogen (URO), and urine bilirubin (BIL) were measured in morning urine.

[0415] Routine coagulation function : Detect prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB).

[0416] Coagulation factor X (FX:C) and thrombin generation (TG) assay .

[0417] Fibrinolytic index test : Detection of D-dimer and fibrin degradation products (FDP).

[0418] FVIII and FIX activity and inhibitor assays .

[0419] 12-lead ECG .

[0420] Chest X-ray : Check once during the screening period.

[0421] Infection Screening : Check once during screening.

[0422] B-ultrasound : Includes abdominal B-ultrasound and deep vein color Doppler ultrasound of the lower limbs.

[0423] Immunogenicity screening : Detection of anti-drug antibodies.

[0424] Blood concentration .

[0425] Efficacy endpoints

[0426] Primary Efficacy End Point

[0427] Effective hemostasis rate (all bleeding events): judged according to the efficacy evaluation table (Table 5), and the efficacy level of good or above was rated as effective.

[0428] Secondary Efficacy End Points

[0429] Clinical remission rate at 4h / 8h / 24h after the first dose (all bleeding events): Clinical remission is defined as significant relief of pain and / or improvement of bleeding signs in patients after the first dose in each bleeding event.

[0430] Statistical analysis

[0431] The effective hemostasis rate and its 95% CI were calculated. The clinical remission rate and its 95% CI were calculated 4h, 8h and 24h after the first administration of each bleeding event.

[0432] ·result

[0433] The participants were Chinese male patients (18-65 years old) with moderate or severe hemophilia A or B and inhibitors to factor VIII or IX (factor activity level <5% IU / dL). After a bleeding event, the subjects were treated with the study drug RVV-X, and the treatment effect was then evaluated. RVV-X was administered at 0.16U / kg every 4 hours for 4-6 doses.

[0434] A total of 8 subjects experienced 19 bleeding events, all of which were stopped within 4 doses of RVV-X, and 16 of them met the "effective hemostasis" standard specified in the clinical trial protocol ("effective hemostasis" is achieved when the efficacy level is excellent or good, see Table 5).

[0435] Table 5 Hemophilia efficacy evaluation table

[0436]

[0437]

[0438] The results of the study found that the therapeutic effect of RVV-X was significantly better than that of prothrombin complex concentrate (PCC; effective hemostasis rate was 50%) [see JM Lusher, "Controlled clinical trials with prothrombin complex concentrates," Prog Clin Biol Res. 1984; 150: 277-290]). It was also found that the therapeutic effect of RVV-X was comparable to that of injectable recombinant human coagulation factor VIIa (effective hemostasis rate was about 90% [see C. Knight et al., "Systematic review of efficacy of rFVIIa and aPCC treatment for hemophilia patients with inhibitors," Adv Ther. 2009; 26 (1): 68-88]).

[0439] Example 3: Study of RVV-X on M-KOFVIII hemophiliac mice

[0440] FDA-approved activated recombinant factor VII (rFVIIa, e.g., RT) acts by forming a complex with tissue factor, activating FX to FXa through the extrinsic pathway, and then converting prothrombin to thrombin to achieve hemostasis. RT is commonly used to treat HA and HB patients with inhibitors. However, it is extremely expensive, so its application is limited. In addition, nonclinical studies have shown that RT has a short half-life in vivo: in rats, 1 / 2 0.5 hours in dogs and monkeys 1 / 2 About 3 hours. Mice that were acutely administered RT showed pulmonary vascular coagulation; and repeated injections Pulmonary thrombosis and local irritation at the injection site occurred in rats and cynomolgus monkeys treated with RT. See NovoSeven: EPAR–SCIENTIFIC DISCUSSION (https: / / www.ema.europa.eu / en / medicines / human / EPAR / novoseven) and RT Prescribing Information (revised 7 / 2020; https: / / www.novomedlink.com / rare-bleeding-disorders / products / treatments / novosevenrt / about / why-novosevenrt.html ).

[0441] This example proves that Compared with RT, RVV-X has comparable or even superior therapeutic effects in vivo.

[0442] Preliminary study on the procoagulant efficacy of RVV-X for injection on M-KOFVIII hemophilia mice

[0443] The experiment used M-KOFVIII (FVIII gene knockout) hemophilia mice. RVV-X (containing SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5) was administered by tail vein injection, twice on D1 and D5, with doses of 0.1U / kg, 1U / kg, 10U / kg and 100U / kg. The amount of bleeding and bleeding time were detected 30 minutes after the first administration, and the prothrombin time (PT) and activated partial thromboplastin time (APTT) were detected 30 minutes after the second administration. Served as positive control. RVV-X dilution served as placebo (PBO).

[0444] As shown in Table 6, the APTT of M-KOFVIII mice was significantly higher than that of normal mice. The bleeding time and bleeding volume of M-KOFVIII mice treated with Noci were reduced (data not shown), and the APTT returned to the level of normal animals (Table 6). Compared with untreated animals, RVV-X showed a tendency to shorten the APTT of M-KOFVIII hemophiliac mice at a single intravenous injection of 0.1U / kg (41.7±14.9s vs 55.7±10.8s). A single intravenous injection of RVV-X at a dose of 0.1 to 100U / kg shortened the APTT in a dose-dependent manner (Table 6). RVV-X also reduced the bleeding time and bleeding volume of hemophiliac mice to varying degrees (data not shown). 0.1U / kg is the pharmacologically active dose (PAD) of RVV-X for M-KOFVIII hemophiliac mice.

[0445] Table 6 Effects of RVV-X on APTT and PT in M-KOFVIII hemophiliac mice

[0446]

[0447] △ APTT was measured 30 minutes after a single injection of RVV-X and expressed as mean ± standard deviation. Compared with group 1, * Indicates P ≤ 0.05; compared with the 2 groups, ▲ indicates P ≤ 0.05; APTT, activated partial thromboplastin time; Nociceptin, activated recombinant factor VII (rFVIIa), which has been approved by the FDA for the treatment of hemophilia A or B with inhibitors.

[0448] Effect of RVV-X for injection on APTT in M-KOFVIII hemophilia mice

[0449] C57BL / 6 mice and M-KOFⅧ hemophilia mice were used in the experiment. 30 minutes after a single administration of the test drug or control via the tail vein, blood was collected from the abdominal aorta and the APTT was determined by the coagulation method. Noci was used as a positive control. RVV-X dilution was used as a placebo (PBO).

[0450] As shown in Table 7, the APTT of the M-KOFⅧ hemophiliac mouse control group was significantly higher than that of the normal control group. After treatment with Noci, the APTT of M-KOFⅧ mice was significantly reduced, close to but still higher than the level of normal animals. A single intravenous injection of 0.25U / kg-4U / kg of RVV-X significantly shortened the APTT of M-KOFVIII mice in a dose-dependent manner. In addition, a single intravenous injection of 4U / kg of RVV-X shortened the APTT, which was even better than Noci, and reduced the APTT to normal levels. A single dose of 1U / kg of RVV-X significantly shortened the APTT of M-KOFVIII hemophiliac mice, which was comparable to the effect of 3mg / kg Noci.

[0451] Table 7 Effect of RVV-X for injection on APTT of M-KOFⅧ hemophilia mice

[0452]

[0453] △ APTT was measured 30 minutes after a single injection of RVV-X; NA, not significant; compared with group 1, * Indicates P ≤ 0.05; compared with the 2 groups, ▲ Indicates P≤0.05; compared with the other 3 groups, □ Indicates P ≤ 0.05.

[0454] Effects of RVV-X for injection on bleeding volume, bleeding time and 24h mortality of M-KOFVIII hemophiliac mice

[0455] The amount of bleeding and bleeding events are affected by many factors, such as the number and quality of platelets, the structure and function of capillaries, the interaction between platelets and capillaries, and coagulation factors. The 24-hour mortality rate after tail docking is less affected by these factors and is closer to clinical evaluation indicators, so it is used as the main evaluation indicator of this experiment.

[0456] C57BL / 6 mice and M-KOFⅧ hemophiliac mice were used in the experiment. The test drug or placebo was injected into the tail vein once, and the bleeding volume and bleeding time were measured by tail cutting 30 minutes after administration. The animals were then returned to the original cages for continued feeding, and the mortality of the test animals was recorded the next day. Noci was used as a positive control. RVV-X dilution was used as a placebo (PBO).

[0457] As shown in Table 8, the mortality rate of normal mice after tail docking for 24 hours was 0%, while the mortality rate of M-KOFⅧ hemophilia mice was 66.7%. After administration of Noci, the mortality rate of M-KOFⅧ hemophilia mice after tail docking was significantly reduced to 8.3%. A single intravenous injection of 0.25U / kg of RVV-X has been shown to be able to reduce the mortality rate of M-KOFVIII hemophilia mice after tail docking for 24 hours. A single intravenous injection of 0.25U / kg-4U / kg of RVV-X significantly inhibited the bleeding tendency of M-KOFⅧ hemophilia mice (data not shown), and the reduction in 24h mortality was dose-dependent. In addition, the mortality rate of M-KOFVIII hemophilia mice injected with a single intravenous injection of 4U / kg of RVV-X was the same as that of normal mice, which was 0%, and this effect was significantly better than Noci.

[0458] Table 8 Effect of RVV-X for injection on the mortality rate of M-KOFⅧ hemophiliac mice after tail docking for 24 hours

[0459]

[0460] Effects of RVV-X injection at different time points on APTT in M-KOFⅧ hemophilia mice

[0461] This study investigated the time-effect relationship of RVV-X injection on the coagulation function of M-KOFⅧ hemophilia mice. C57BL / 6 mice (normal control group) and M-KOFⅧ hemophilia mice were selected for the study. Hemophilia mice were injected with 1U / kg RVV-X via tail vein once, and blood was collected at 5min, 10min, 30min, 1h, 2h, 4h, 6h and 8h after administration. Eight mice / time point were used to detect APTT.

[0462] As shown in Table 9, compared with normal mice, the APTT of M-KOFⅧ hemophilia mice increased significantly. A single intravenous injection of 1U / kg RVV-X significantly shortened the APTT of M-KOFVIII hemophilia mice 5 minutes after administration, and the effect lasted until 2 hours after administration, and basically returned to the pre-drug level 4 hours after administration, showing a significant time-effect relationship.

[0463] Table 9 Effects of RVV-X injection at different time points on APTT of M-KOFⅧ hemophilia mice

[0464]

[0465] Note: * Indicates p < 0.05 compared with the C57BL / 6 pre-drug group; # Indicates p<0.05 compared with the pre-drug group of hemophilia mice.

[0466] Based on the above in vivo efficacy test, a single intravenous injection of RVV-X in M-KOFVIII hemophilia mice can significantly shorten APTT, and there is a trend of shortening APTT at a dose of 0.1U / kg. The APTT of hemophilia mice is significantly shortened in a dose-dependent manner within the dose range of 0.25-4U / kg, and can inhibit bleeding tendency and reduce mortality after tail docking. Time-effect studies have shown that intravenous injection of 1U / kg of RVV-X can significantly shorten the APTT of hemophilia mice 5min-2h after administration, and basically return to the level before administration 4h after administration.

[0467] Toxicology studies on M-KOFⅧ hemophiliac mice repeatedly injected intravenously with RVV-X at a dose of 5U / kg per day for 4 consecutive weeks showed that there were no other systemic toxic reactions except for local thrombosis at the injection site. Therefore, the safe dose for mice was determined to be 5U / kg.

[0468] Example 4: Phase I clinical study of multiple doses of FX activator for injection in surgical patients

[0469] The safety and tolerability of multiple doses of the FX activator RVV-X were studied in surgical patients. Patients with category II and III surgery were selected for the study and divided into several dose groups. The drug was administered 2 times: once 10-15 minutes before surgery and once during surgery (1.5 hours after the first dose), by intravenous injection. The patients were observed for 3 days after surgery, and then the study was completed.

[0470] A total of 18 subjects were enrolled in the trial, including 6 men and 12 women, of whom 2 patients underwent general surgery, 5 patients underwent urological surgery, and 11 patients underwent gynecological surgery.

[0471] During the trial, a total of 9 cases (13 times) of adverse events occurred, all of which were mild, including 7 cases of leukocytosis, 1 case of erythrocyte reduction, 1 case of hemoglobin decrease, 1 case of increased international normalized ratio (INR) of coagulation function, 1 case of prolonged prothrombin time (PT) of coagulation function, 1 case of rash, and 1 case of left upper lung nodule shadow. Among them, prolonged PT and rash may be related to the study drug, and the others are not related to the study drug. Except for the shadow of the left upper lung nodule, all other adverse events returned to normal at the end of the trial. No serious adverse events (SAE) occurred during the trial. No adverse events that led to the termination of the trial occurred.

[0472] Thus, all doses of injectable FX activators were well tolerated by surgical patients.

[0473] The efficacy of multiple injections of the FX activator RVV-X has also been studied in surgical patients. Dozens of surgical patients were enrolled in the study and RVV-X was administered intravenously. They were observed after surgery.

[0474] RVV-X showed therapeutic efficacy in surgical patients (data not shown).

[0475] Example 5: Preparation of Factor X Activator Injection Powder

[0476] The coagulation factor X (FX) activator stock solution is taken from the venom of Vipera rotundus and is prepared by the method of Example 6 in CN109943554A (the contents of which are incorporated herein by reference in their entirety). The remaining excipients are of injection grade unless otherwise specified.

[0477] According to the prescription in Table 10, weigh the prescribed amount of stabilizer, buffer, excipient, surfactant, antioxidant, and divalent calcium salt, add an appropriate amount of water for injection and stir until completely dissolved (when the surfactant is a liquid, other components can be dissolved first, and then the liquid surfactant is added and stirred evenly), add the prescribed amount of FX activator stock solution, adjust the pH with HCl or NaOH, and finally add water for injection to the scale. After mixing evenly, filter through a 0.22μM filter membrane into a sterile container to prepare a semi-finished product solution.

[0478] Table 10FX activator preparation prescription 1-10

[0479]

[0480] According to Table 10, FX activator preparations 1-10 (preparation 1 was used as a control group) were prepared, and dispensed into medium borosilicate glass injection bottles at 0.53±0.03 ml / bottle, half-stoppered, and freeze-dried to obtain FX activator injection powder (hereinafter referred to as "FX activator preparations 1-10 injection powder").

[0481] Example 6: Performance test of coagulation factor X activator preparation

[0482] I) Appearance, moisture, reconstitution time, pH, and biological activity testing

[0483] The appearance, moisture content, reconstitution time, pH value and biological activity of FX activator preparation 1-10 injection powder were tested. The biological activity was tested using a coagulometer. The results are shown in Table 11.

[0484] Table 11 Test results of FX activator preparations 1-10

[0485]

[0486] In order to describe the appearance more intuitively, the appearance is divided into the following 4 levels. Level 1: Delicate, plump, white loose body. Level 2: Slightly atrophied, slightly rough surface, with particles, slightly separated from the wall. Level 3: Moderately atrophied, separated from the wall, and the block is slightly reduced. Level 4: Severely atrophied, the block is significantly reduced, and it is seriously separated from the wall.

[0487] As shown in Table 11, the appearance, moisture content, reconstitution time, pH value, and biological activity of the tested preparations all meet the corresponding product standards. Except for preparation 3, the appearance of other injection powder preparations is a delicate, full, white loose body, and they all quickly dissolve into a colorless clear liquid after adding 2 ml of reconstitution solvent. Since the injection powder of preparation 3 uses a higher amount of trehalose and a lower amount of mannitol, its appearance is slightly shrunk, the moisture content is close to 3.0%, and the reconstitution time is slightly longer than other preparations, but all within 30 seconds, and they are all colorless clear liquids after dissolution. There was no significant difference in pH value and biological activity before and after freeze-drying of all preparations.

[0488] II) Stability test under high temperature, high humidity and light conditions

[0489] Since the preparation may deviate from the preset conditions during storage, we used the influencing factor test to examine the impact of the samples on the key indicators when the samples were exposed to high temperature (40±2℃), high humidity (2-8℃, RH 92.5%±5%) and light (2-8℃, 4500±500LX) in a short period of time. The test results are shown in Tables 12 and 13.

[0490] Table 12 Some indicators under influencing factors

[0491]

[0492] As shown in Table 12, under high temperature conditions, as the storage time increased, all formulations met the corresponding product standards. The appearance of formulations 3 and 10 showed a shrinking trend, and the reconstitution time was slightly prolonged. Under other test conditions, the appearance, moisture, and pH of formulations 2, 4-9 did not change significantly compared with 0 days. In particular, formulations 2, 4, 6, and 8 always maintained a level 1 state in appearance.

[0493] Table 13 Biological activity under influencing factors (%; compared with the labeled amount)

[0494]

[0495] As shown in Table 13, the activity of preparations 3, 4, and 10 decreased slightly after 14 days of high temperature or light exposure, with a reduction rate between 3.41% and 9.45%, but was still better than the control group (preparation 1). The activity of preparations 2, 5-9 remained basically stable under the influencing factors, and the activity reduction rate was only less than 1.60% after 14 days of high temperature or light exposure. Compared with the technical solution of the present invention (preparations 2-10), the biological activity of the FX activator in the control group (preparation 1) was significantly reduced. This result shows that under high temperature and light conditions, the stabilization and protection effect of human serum albumin (HSA) on the active ingredient (FX activator) is insufficient, while the technical solution of the present invention can provide more effective stabilization and protection.

[0496] The above results show that the technical solution of the present invention can significantly and effectively stabilize and protect the activity of the coagulation factor X activator under the conditions of high temperature, high humidity and light.

[0497] III) Accelerated and long-term stability tests

[0498] Stability studies were conducted on all preparations to examine the biological activity and changes in insoluble particles.

[0499] 1) Biological activity detection

[0500] Preparations 1-10 were placed under accelerated (25±2°C, RH 65±5%) and long-term storage (2-8°C) conditions for stability testing to detect changes in biological activity. The results are shown in Table 14.

[0501] Table 14 Biological activity under accelerated and long-term storage conditions (%; compared with the labeled amount)

[0502]

[0503] The results of biological activity test showed that after 6 months of accelerated or long-term storage, the stability of the freeze-dried preparations (preparations 2-10) prepared according to the technical scheme of the present invention was significantly better than that of the control group (preparation 1), especially the activity reduction rate of preparations 2, 5-9 after 6 months of accelerated or long-term storage was less than 1.60% or 0.80%. The above results show that the technical scheme of the present invention can make the FX activator more stable under accelerated and long-term storage conditions.

[0504] 2) Insoluble particle detection

[0505] Since the diameter of human capillaries is 7-12μm, insoluble particles may cause vascular thrombosis, induce phlebitis, granuloma, pulmonary hypertension and pyrogen-like reactions during intravenous administration. For drugs administered intravenously, the content of insoluble particles should be avoided and reduced. Therefore, insoluble particles need to be detected during the preparation of related drugs.

[0506] Insoluble particles mainly come from the production process, compatibility process and administration process of drugs. In addition, protein drugs may also produce insoluble particles during mixing, freeze-drying and storage.

[0507] The present invention adopts The 8000 sub-visible particle imaging analysis system uses an electronic imaging system to capture particles dynamically flowing through a flow cell, and then uses software to analyze parameters such as the size, morphology, and refractive index of insoluble particles, and then distinguish all images, such as protein particles, silicone oil, bubbles, fibers, etc. The detectable particle size range is 2-100μm.

[0508] All injectable powder preparations were dissolved in 2 ml of normal saline at 0 month and after 6 months of acceleration, and the insoluble particles were tested according to the insoluble particle inspection method in 0903 of the fourth general chapter of the 2015 edition of the Chinese Pharmacopoeia. The results are shown in Tables 15 and 16 (the data in the tables have been deducted from the background particles).

[0509] Table 15 Results of insoluble particles in 0 month

[0510]

[0511] As shown in Table 15, the number of insoluble particles in each freeze-dried preparation sample meets the requirements of the pharmacopoeia, but the number of insoluble particles of each particle size in the control sample (preparation 1) is significantly higher than that of other freeze-dried preparations. The number of insoluble particles in the freeze-dried preparations of the present invention is at a relatively low level, especially in preparations 2, 5-10, the total number of insoluble particles is even less than 260. These results show that the formulation of the present invention can significantly reduce insoluble particles. See also the attached Figure 5 .

[0512] Table 16 Accelerated 6-month insoluble particle results

[0513]

[0514]

[0515] As shown in Table 16, after 6 months of accelerated storage, the insoluble particles in the control sample (Formulation 1) increased significantly, among which the number of particles with a particle size range of 2-10 μm was the largest. Figure 6 As shown. It may be that the human albumin added to the preparation partially aggregated during its own storage process, resulting in an increase in the content of insoluble particles. Although the number of insoluble particles in the control sample is still within the qualified range, it is much higher than the number in other preparation samples of the present invention. The total amount of insoluble particles in the preparation samples of the present invention is at a relatively low level, especially in preparations 2, 5-10, the total amount of insoluble particles is even less than 2000. The above results once again show that compared with the control preparation, the technical solution of the present invention can effectively improve the content of insoluble particles, thereby further reducing the risks brought by insoluble particles and improving the safety of clinical use.

[0516] In summary, the present invention uses disaccharides (i.e., sucrose or / and trehalose) as stabilizers and uses them in combination with amino acid buffers (i.e., arginine or / and histidine). The resulting preparation not only significantly improves the stability of the FX activator composition product, but also can avoid the potential risks of viruses or other unknown components caused by using albumin as a stabilizer. In particular, when the active concentration of the FX activator is 5U / ml-50U / ml, the mass volume concentration of the stabilizer is 30mg / ml-50mg / ml, the mass volume concentration of the buffer is 3mg / ml-5mg / ml, the mass volume concentration of the surfactant is 0.1mg / ml-0.3mg / ml, the mass volume concentration of the excipient is 30mg / ml-60mg / ml, and the pH of the preparation is 6.8-7.0, the appearance of the composition is delicate and full, the water content, reconstitution time, pH value, and biological activity all meet the product standards, and the stability of the composition is maintained well under high temperature, high humidity, light, accelerated and long-term conditions.

[0517] Example 7: Formulation Optimization Test

[0518] To further optimize the FX activator preparation, the preparation was prepared according to Table 17 and the method described in Example 5 to prepare a factor X activator semi-finished solution with a concentration of 10 U / ml and 0.5 ml / bottle (i.e., 5 U / bottle). Subsequently, the prepared preparation was prepared into a sterile powder for injection by a lyophilization process.

[0519] Table 17 Optimization of the formulation formula of FX activator

[0520]

[0521]

[0522] The samples at 0 month and after 6 months of acceleration were reconstituted with 2 ml of normal saline and tested for insoluble particles according to the insoluble particles inspection method in General Chapter 0903 of Part IV of the 2015 edition of the Chinese Pharmacopoeia. The re...

Claims

1. A pharmaceutical composition comprising: i) Factor X activator (FX activator), having a content of 0.1 U / mL to 200 U / mL; ii) a stabilizer, the content of which is 2 mg / ml to 100 mg / ml; iii) a buffer in an amount of 0.1 mg / ml to 50 mg / ml; iv) a surfactant in an amount of 0.001% (w / v) to 0.1% (w / v); and v) excipients, the content of which is 1 mg / ml to 100 mg / ml; in, The pH value of the pharmaceutical composition is 6.0 to 8.

0.

2. The pharmaceutical composition according to claim 1, wherein the FX activator is RVV-X.

3. The pharmaceutical composition according to claim 2, wherein the RVV-X is isolated from the venom of Daboiarussellii siamensis.

4. The pharmaceutical composition according to any one of claims 2-3, wherein the purity of the RVV-X in the pharmaceutical composition is at least 95%. 5 . The pharmaceutical composition according to claim 1 , wherein the content of the FX activator is 1 U / mL to 100 U / mL. 6 . The pharmaceutical composition according to any one of claims 1 to 5 , wherein the content of the FX activator is 5 U / mL to 100 U / mL.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the content of the FX activator is 5 U / mL to 50 U / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the content of the FX activator is 10 U / mL.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the stabilizer comprises one or both of sucrose and trehalose.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the stabilizer is sucrose.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the content of the stabilizer is 2 mg / ml to 60 mg / ml.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the content of the stabilizer is 15 mg / ml to 60 mg / ml.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the content of the stabilizer is 30 mg / ml to 50 mg / ml.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the content of the stabilizer is 30 mg / ml.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the buffer comprises one or both of histidine and arginine.

16. The pharmaceutical composition according to any one of claims 1-15, wherein the buffer is histidine.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the content of the buffer is 2 mg / ml to 20 mg / ml.

18. The pharmaceutical composition according to any one of claims 1-17, wherein the content of the buffer is 2 mg / ml to 15 mg / ml.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the content of the buffer is 3 mg / ml to 5 mg / ml.

20. The pharmaceutical composition according to any one of claims 1-19, wherein the content of the buffer is 3 mg / ml.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the surfactant comprises one or both of polysorbate and poloxamer.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the surfactant is selected from one or more of polysorbate 20, polysorbate 80 and poloxamer 188.

23. The pharmaceutical composition according to any one of claims 1-22, wherein the surfactant is polysorbate 20.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the content of the surfactant is 0.005% (w / v) to 0.05% (w / v).

25. The pharmaceutical composition according to any one of claims 1 to 24, wherein the content of the surfactant is 0.01% (w / v) to 0.05% (w / v).

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the content of the surfactant is 0.01% (w / v) to 0.03% (w / v).

27. The pharmaceutical composition according to any one of claims 1 to 26, wherein the content of the surfactant is 0.02% (w / v).

28. The pharmaceutical composition of any one of claims 1-27, wherein the excipient is mannitol.

29. The pharmaceutical composition according to any one of claims 1-28, wherein the content of the excipient is 10 mg / ml to 60 mg / ml.

30. The pharmaceutical composition according to any one of claims 1-29, wherein the content of the excipient is 30 mg / ml to 60 mg / ml.

31. The pharmaceutical composition according to any one of claims 1-30, wherein the content of the excipient is 40 mg / ml.

32. The pharmaceutical composition according to any one of claims 1-31, wherein the pH value of the pharmaceutical composition is 6.3 to 7.

3.

33. The pharmaceutical composition according to any one of claims 1-32, wherein the pH value of the pharmaceutical composition is 6.8 to 7.

0.

34. The pharmaceutical composition according to any one of claims 1-33, wherein the pH value of the pharmaceutical composition is 6.

85.

35. The pharmaceutical composition of any one of claims 1-34, wherein the pharmaceutical composition further comprises an antioxidant.

36. The pharmaceutical composition of claim 35, wherein the antioxidant is methionine.

37. The pharmaceutical composition according to claim 35 or 36, wherein the content of the antioxidant is 0.01 mg / ml to 1 mg / ml.

38. The pharmaceutical composition according to any one of claims 35-37, wherein the content of the antioxidant is 0.05 mg / ml to 1 mg / ml.

39. The pharmaceutical composition of any one of claims 1-38, wherein the pharmaceutical composition further comprises a calcium salt.

40. The pharmaceutical composition of claim 39, wherein the calcium salt is calcium chloride.

41. The pharmaceutical composition according to claim 39 or 40, wherein the content of the calcium salt is 0.01 mg / ml to 10 mg / ml.

42. A pharmaceutical composition according to any one of claims 1-41, wherein the pharmaceutical composition comprises: i) RVV-X, the content of which is 10 U / mL; ii) sucrose, the content of which is 30 mg / ml; iii) histidine, the content of which is 3 mg / ml; iv) polysorbate 20 in an amount of 0.02% (w / v); and v) mannitol, the content of which is 40 mg / ml; in, The pH value of the pharmaceutical composition is 6.

85.

43. The pharmaceutical composition of any one of claims 1-42, which is lyophilized.

44. The pharmaceutical composition of any one of claims 1-43, which is sterile.

45. A pharmaceutical composition according to claim 43 or 44, wherein the pharmaceutical composition is stable under the following conditions: i) After reconstitution, place at 25°C for at least 4 hours; ii) kept at 2-8°C for at least 3 months; and / or iii) placed under accelerated stability conditions at 25°C for at least 3 months.

46. ​​The pharmaceutical composition according to any one of claims 43-45, wherein the pharmaceutical composition contains less than 5% insoluble particles having a diameter greater than 10 μm after being stored at 25°C under accelerated stability conditions for 6 months and then reconstituted.

47. The pharmaceutical composition of any one of claims 2-46, wherein the RVV-X comprises: a) i) a heavy chain comprising the sequence of SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1; ii) a light chain 1 comprising the sequence of SEQ ID NO: 2, or a sequence having at least 80% sequence identity to SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to SEQ ID NO: 3; b) i) a heavy chain comprising the sequence of SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1; ii) a light chain 1 comprising the sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity to SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to SEQ ID NO: 3; or c) A mixture of a) and b).

48. Use of the pharmaceutical composition of any one of claims 1-47 in the preparation of a medicament for treating a bleeding disorder in an individual.

49. Use of FX activation in the preparation of a medicament for treating a bleeding disorder in an individual, wherein the FX activation is administered at a dose of 0.01 U / kg to 0.48 U / kg.

50. The use according to claim 49, wherein the FX activator is administered at a dose of 0.08 U / kg to 0.48 U / kg.

51. The use according to claim 49, wherein the FX activator is administered at a dose of 0.01 U / kg to 0.16 U / kg.

52. The use according to any one of claims 49-51, wherein the FX activator is administered at a dose of 0.08 U / kg to 0.16 U / kg.

53. The use according to any one of claims 49-52, wherein the FX activator is administered at a dose of 0.16 U / kg.

54. The use according to any one of claims 49 to 53, wherein the FX activator is administered once.

55. The use according to any one of claims 49-53, wherein the FX activator is administered up to 6 times.

56. The use according to any one of claims 49-53 and 55, wherein the FX activator is administered four times.

57. The use according to any one of claims 49-53, 55 and 56, wherein the FX activator is administered once every 4 hours (q4h) to once every 8 hours (q8h).

58. The use according to any one of claims 49-53 and 55-57, wherein the FX activator is administered at an interval of q4h.

59. The use according to claim 49, wherein the single administration dose of the FX activator is 0.01 U / kg to 0.48 U / kg.

60. The use according to claim 49, wherein the FX activator is administered at a dosage of 0.16 U / kg q4h, for a maximum of 6 administrations.

61. The use according to claim 49 or 60, wherein the FX activator is administered at a dosage of 0.16 U / kg q4h, for 4 times.

62. The use according to any one of claims 49-61, wherein the FX activator is RVV-X.

63. The use according to claim 61 or 62, wherein the RVV-X is isolated from the venom of Daboiarussellii siamensis.

64. The use according to claim 63, wherein the purity of the RVV-X is at least 95%.

65. The use according to any one of claims 49-64, wherein the FX activator is contained in a pharmaceutical composition.

66. The use according to claim 65, wherein the concentration of the FX activator in the pharmaceutical composition is 0.1 U / mL to 200 U / mL.

67. The use according to claim 65 or 66, wherein the concentration of the FX activator in the pharmaceutical composition is 1 U / mL to 100 U / mL.

68. The use according to any one of claims 65-67, wherein the concentration of the FX activator in the pharmaceutical composition is 5 U / mL to 100 U / mL.

69. The use according to any one of claims 65-68, wherein the concentration of the FX activator in the pharmaceutical composition is 5 U / mL to 50 U / mL.

70. The use according to any one of claims 65-69, wherein the concentration of the FX activator in the pharmaceutical composition is 10 U / mL.

71. The use according to any one of claims 65-70, wherein the pharmaceutical composition further comprises one or more of a stabilizer, a buffer, a surfactant and an excipient.

72. The use according to claim 71, wherein the stabilizer comprises one or both of sucrose and trehalose.

73. Use according to claim 71 or 72, wherein the stabilizer is sucrose.

74. The use according to any one of claims 71-73, wherein the content of the stabilizer is 2 mg / ml to 100 mg / ml.

75. The use according to any one of claims 71-74, wherein the content of the stabilizer is 2 mg / ml to 60 mg / ml.

76. The use according to any one of claims 71-75, wherein the content of the stabilizer is 15 mg / ml to 60 mg / ml.

77. The use according to any one of claims 71 to 76, wherein the content of the stabilizer is 30 mg / ml to 50 mg / ml.

78. The use according to any one of claims 71-77, wherein the content of the stabilizer is 30 mg / ml.

79. The use according to any one of claims 71-78, wherein the buffer comprises one or both of histidine and arginine.

80. The use according to any one of claims 71-79, wherein the buffer is histidine.

81. The use according to any one of claims 71-80, wherein the buffer is present in an amount of 0.1 mg / ml to 50 mg / ml.

82. The use according to any one of claims 71-81, wherein the buffer is present in an amount of 2 mg / ml to 20 mg / ml.

83. The use according to any one of claims 71-82, wherein the buffer is present in an amount of 2 mg / ml to 15 mg / ml.

84. The use according to any one of claims 71-83, wherein the buffer is present in an amount of 3 mg / ml to 5 mg / ml.

85. The use according to any one of claims 71-84, wherein the buffer is present in an amount of 3 mg / ml.

86. The use according to any one of claims 71-85, wherein the surfactant comprises one or both of a polysorbate and a poloxamer.

87. The use according to any one of claims 71-86, wherein the surfactant is selected from one or more of polysorbate 20, polysorbate 80 and poloxamer 188.

88. The use according to any one of claims 71-87, wherein the surfactant is polysorbate 20.

89. The use according to any one of claims 71 to 88, wherein the surfactant is present in an amount of 0.001% (w / v) to 0.1% (w / v).

90. The use according to any one of claims 71-89, wherein the surfactant is present in an amount of 0.005% (w / v) to 0.05% (w / v).

91. The use according to any one of claims 71-90, wherein the surfactant is present in an amount of 0.01% (w / v) to 0.05% (w / v).

92. The use according to any one of claims 71-91, wherein the surfactant is present in an amount of 0.01% (w / v) to 0.03% (w / v).

93. The use according to any one of claims 71-92, wherein the surfactant is present in an amount of 0.02% (w / v).

94. The use according to any one of claims 71-93, wherein the excipient is mannitol.

95. The use according to any one of claims 71-94, wherein the content of the excipient is 1 mg / ml to 100 mg / ml.

96. The use according to any one of claims 71-95, wherein the content of the excipient is 10 mg / ml to 60 mg / ml.

97. The use according to any one of claims 71-96, wherein the content of the excipient is 30 mg / ml to 60 mg / ml.

98. The use according to any one of claims 71-97, wherein the content of the excipient is 40 mg / ml.

99. The use according to any one of claims 65-98, wherein the pH of the pharmaceutical composition is 6.0 to 8.

0.

100. The use according to any one of claims 65-99, wherein the pH of the pharmaceutical composition is 6.3 to 7.

3.

101. The use according to any one of claims 65-100, wherein the pH value of the pharmaceutical composition is 6.8 to 7.

0.

102. The use according to any one of claims 65-101, wherein the pH value of the pharmaceutical composition is 6.

85.

103. The use according to any one of claims 65-102, wherein the pharmaceutical composition further comprises an antioxidant.

104. The use according to claim 103, wherein the antioxidant is methionine.

105. The use according to claim 103 or 104, wherein the content of the antioxidant is 0.01 mg / ml to 1 mg / ml.

106. The use according to any one of claims 103-105, wherein the content of the antioxidant is 0.05 mg / ml to 1 mg / ml.

107. The use according to any one of claims 65-106, wherein the pharmaceutical composition further comprises a calcium salt.

108. The use according to claim 107, wherein the calcium salt is calcium chloride.

109. The use according to claim 107 or 108, wherein the content of the calcium salt is 0.01 mg / ml to 10 mg / ml.

110. The use according to any one of claims 65-109, wherein the pharmaceutical composition comprises: i) RVV-X, the content of which is 10 U / mL; ii) sucrose, the content of which is 30 mg / ml; iii) histidine, the content of which is 3 mg / ml; iv) polysorbate 20 in an amount of 0.02% (w / v); and v) mannitol, the content of which is 40 mg / ml; in, The pH value of the pharmaceutical composition is 6.

85.

111. The use according to any one of claims 65-110, wherein the pharmaceutical composition is lyophilized.

112. The use according to any one of claims 65-111, wherein the pharmaceutical composition is sterile.

113. The use according to claim 111 or 112, wherein the pharmaceutical composition is stable under the following conditions: i) After reconstitution, place at 25°C for at least 4 hours; ii) kept at 2-8°C for at least 3 months; and / or iii) placed under accelerated stability conditions at 25°C for at least 3 months.

114. The use according to any one of claims 111-113, wherein the pharmaceutical composition contains less than 5% insoluble particles with a diameter greater than 10 μm after being reconstituted after storage at 25°C accelerated stability conditions for 6 months.

115. The use according to any one of claims 49-114, wherein the FX activator is administered intravenously.

116. The use according to any one of claims 48-115, wherein the bleeding disorder is a congenital bleeding disorder or an acquired bleeding disorder.

117. The use according to any one of claims 48-116, wherein the bleeding disorder is selected from the group consisting of a disease caused by a lack of a coagulation factor, a disease caused by the presence of an acquired coagulation factor inhibitor, a blood disease, a bleeding disorder, von Willebrand's disease, a disease caused by anticoagulation therapy with a vitamin K antagonist, an inherited platelet disease, vitamin K epoxide reductase C1 deficiency, gamma-carboxylase deficiency, bleeding associated with trauma or injury or surgery, thrombosis, thrombocytopenia, stroke, coagulation disorders, disseminated intravascular coagulation (DIC), Bernard-Soulier syndrome, Glanzmann's thrombosis, and a storage pool deficiency.

118. The use according to any one of claims 48-117, wherein the bleeding disorder is due to a lack of a coagulation factor.

119. The use according to any one of claims 48-118, wherein the bleeding disorder is hemophilia.

120. The use according to any one of claims 48-119, wherein the hemophilia is hemophilia A or hemophilia B.

121. The use according to claim 119 or 120, wherein the hemophilia is hemophilia with inhibitors.

122. The use according to any one of claims 48-121, wherein the subject is a human.

123. The use according to any one of claims 62-122, wherein the RVV-X comprises: a) i) a heavy chain comprising the sequence of SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1; ii) a light chain 1 comprising the sequence of SEQ ID NO: 2, or a sequence having at least 80% sequence identity to SEQ ID NO: 2; and iii) a light chain 2 comprising the sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to SEQ ID NO: 3; b) i) a heavy chain comprising the sequence of SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1; ii) a light chain 1 comprising the sequence of SEQ ID NO: 5, or a sequence having at least 80% sequence identity to SEQ ID NO: 5; and iii) a light chain 2 comprising the sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to SEQ ID NO: 3; or c) A mixture of a) and b).

Images