Pharmaceutical composition for treating hemophilia and use thereof

Abstract

Provided in the present disclosure are a pharmaceutical composition for treating hemophilia and the use thereof, the use of a histone deacetylase inhibitor in the preparation of a drug for treating hemophilia, and the use of a histone deacetylase inhibitor and an mTOR inhibitor in the preparation of a kit. The pharmaceutical composition for treating hemophilia of the present disclosure comprises a histone deacetylase inhibitor and a pharmaceutical carrier. The histone deacetylase inhibitor in the pharmaceutical composition of the present disclosure can activate platelets and promote blood coagulation, thereby alleviating the repeated bleeding in patients with hemophilia.

Description

Pharmaceutical composition for treating hemophilia and its use

[0001] This application claims priority to a prior patent application filed by the applicant with the State Intellectual Property Office of China on December 8, 2023, with patent application number 202311687224.X, entitled “Pharmaceutical Compositions and Uses for Treating Hemophilia.” The entire text of the prior application is incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of medical technology, and specifically relates to a pharmaceutical composition for treating hemophilia and uses thereof, in particular to uses of a histone deacetylase inhibitor in preparing a drug for treating hemophilia and uses of the histone deacetylase inhibitor and an mTOR inhibitor in preparing a kit. Background Art

[0003] Congenital hemophilia (including hemophilia A (A) and hemophilia B (B)) is a rare bleeding disorder caused by mutations in the coagulation factor VIII or factor IX genes. Severe hemophilia (factor VIII or IX activity levels <1%) is characterized by recurrent joint bleeding and life-threatening bleeding, placing a significant financial burden and stress on patients, their families, and society.

[0004] Although hemophilia replacement therapy has greatly improved the quality of life of hemophilia patients, it still has many shortcomings: 1. Large-scale plasma transfusion not only fails to effectively stop bleeding, but will increase the patient's blood volume; 2. It is easy to induce antigen-antibody reactions; 3. Large-scale transfusions can easily induce venous and arterial thrombosis and DIC (disseminated intravascular coagulation); 4. Ordinary coagulation factors have a short half-life. In order to maintain the effective activity of coagulation factors, severe hemophilia patients need daily or every other day transfusions, and patient compliance is poor.

[0005] When hemophilia patients use replacement therapy, the development of inhibitors is the most important complication, and may even cause the clinical manifestations of hemophilia patients to change from mild to severe, endangering the patient's life. For hemophilia A, about 30% of patients develop inhibitors after replacement therapy, and the incidence of inhibitors in hemophilia B is about 1.5-5%. Although some patients can use immune tolerance induction therapy (ITI) to eradicate inhibitors after the development of inhibitors, about 40% of hemophilia patients are still unable to restore normal coagulation factor response levels. Gene therapy is a new type of hemophilia treatment, but it is essentially a replacement therapy; and the long-term efficacy and adverse reactions of this therapy are still unclear, and its high price makes it unaffordable for ordinary family patients.

[0006] Therefore, there is an urgent need to develop new drugs or treatments to improve the treatment of hemophilia. Summary of the Invention

[0007] To improve the current situation, the present disclosure provides a pharmaceutical composition for treating hemophilia, use of a histone deacetylase inhibitor in a drug for treating hemophilia, and use of a histone deacetylase inhibitor and an mTOR inhibitor in the preparation of a kit.

[0008] According to an embodiment of the present disclosure, the present disclosure provides a pharmaceutical composition for treating hemophilia, wherein the pharmaceutical composition comprises: a histone deacetylase inhibitor and a pharmaceutically acceptable carrier.

[0009] According to an embodiment of the present disclosure, the present disclosure further provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor, an mTOR inhibitor and a pharmaceutically acceptable carrier.

[0010] According to an embodiment of the present disclosure, the present disclosure further provides a pharmaceutical composition for treating hemophilia, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor, an mTOR inhibitor and a pharmaceutically acceptable carrier.

[0011] The present disclosure also provides a kit, wherein the kit comprises the above-mentioned pharmaceutical composition.

[0012] The present disclosure also provides a kit, wherein the kit comprises a histone deacetylase inhibitor and an mTOR inhibitor, and optionally a pharmaceutically acceptable carrier.

[0013] According to an embodiment of the present disclosure, the histone deacetylase inhibitor includes but is not limited to one or more of the following, or a pharmaceutically acceptable salt thereof: valproic acid, vorinostat, romidepsin, belinostat, panobinostat, such as valproate, for example, sodium valproate.

[0014] According to an embodiment of the present disclosure, the valproate is present in the form of a salt selected from the group consisting of a sodium salt, a potassium salt, a magnesium salt, a calcium salt or an ammonium salt.

[0015] According to an embodiment of the present disclosure, the sodium salt form of valproic acid includes hemi-sodium valproate or sodium valproate.

[0016] According to an embodiment of the present disclosure, in the pharmaceutical composition, the content of the histone deacetylase inhibitor is 0.1g-1.0g, for example, 0.3g-0.8g.

[0017] According to an embodiment of the present disclosure, in the pharmaceutical composition, the content of sodium valproate is 0.1g-1.0g, for example, 0.15g-0.45g.

[0018] According to an embodiment of the present disclosure, the pharmaceutical composition further comprises an mTOR inhibitor.

[0019] According to an embodiment of the present disclosure, the pharmaceutical composition further comprises an mTOR inhibitor and a pharmaceutically acceptable carrier.

[0020] According to an embodiment of the present disclosure, the mTOR inhibitor includes but is not limited to one or more of the following, or a pharmaceutically acceptable salt thereof:

[0021] Sirolimus (also called rapamycin), everolimus, temsirolimus, and ibrutinib.

[0022] According to an embodiment of the present disclosure, in the pharmaceutical composition, the content of the mTOR inhibitor is 0.1 mg-3 mg, for example, 0.2 mg-3 mg.

[0023] According to an embodiment of the present disclosure, the weight ratio of the histone deacetylase inhibitor to the mTOR inhibitor is 250:1 to 1200:1.

[0024] According to an embodiment of the present disclosure, the pharmaceutical composition is in the form of powder, tablet, lozenge, granule, capsule, suspension or emulsion.

[0025] The present disclosure also provides use of a histone deacetylase inhibitor or the pharmaceutical composition as described above in a drug for treating hemophilia.

[0026] The present disclosure also provides a use of a histone deacetylase inhibitor or the pharmaceutical composition as described above in preparing a medicament, wherein the medicament comprises a histone deacetylase inhibitor and a pharmaceutical carrier for treating hemophilia.

[0027] According to an embodiment of the present disclosure, the medicament is used to treat hemophilia or alleviate symptoms associated with hemophilia (such as bleeding or swelling caused by bleeding).

[0028] According to an embodiment of the present disclosure, the drug includes a histone deacetylase inhibitor and a pharmaceutically acceptable carrier to treat hemophilia.

[0029] According to an embodiment of the present disclosure, the drug includes a histone deacetylase inhibitor to maintain the integrity of blood vessels and activate platelets to promote blood coagulation.

[0030] According to an embodiment of the present disclosure, the histone deacetylase inhibitor includes one or more of the following, or a pharmaceutically acceptable salt thereof: valproic acid, vorinostat, belinostat, romidepsin, and panobinostat.

[0031] According to an embodiment of the present disclosure, the content of the histone deacetylase inhibitor in the drug is 0.3g-0.8g.

[0032] According to an embodiment of the present disclosure, the content of sodium valproate in the histone deacetylase inhibitor is 0.15g-0.45g.

[0033] According to an embodiment of the present disclosure, the drug is administered twice a day, once a day, or three times a day.

[0034] According to an embodiment of the present disclosure, the administration route of the drug is enteral administration.

[0035] The present disclosure also provides a histone deacetylase inhibitor and an mTOR inhibitor, or use of the above-mentioned pharmaceutical composition in the preparation of a kit.

[0036] According to an embodiment of the present disclosure, the kit includes a histone deacetylase inhibitor and an mTOR inhibitor; and the kit is used for simultaneously co-administering the histone deacetylase inhibitor and the mTOR inhibitor to treat hemophilia.

[0037] According to an embodiment of the present disclosure, the kit is used to treat hemophilia or alleviate symptoms associated with hemophilia (such as bleeding or swelling caused by bleeding).

[0038] According to an embodiment of the present disclosure, the content of the histone deacetylase inhibitor in the kit is 0.1g-1.0g, such as 0.3g-0.8g; and / or the content of the mTOR inhibitor in the kit is 0.1mg-3mg, such as 0.2mg-3mg.

[0039] According to an embodiment of the present disclosure, the histone deacetylase inhibitor in the kit is administered twice a day, once a day, or three times a day; and / or the mTOR inhibitor in the kit is administered once a day, twice a day, or three times a day. Preferably, the histone deacetylase inhibitor in the kit is administered twice a day; and / or the mTOR inhibitor in the kit is administered once a day.

[0040] According to an embodiment of the present disclosure, the content of sodium valproate as the histone deacetylase inhibitor is 0.1g-1.0g, for example, 0.15g-0.45g.

[0041] According to an embodiment of the present disclosure, the histone deacetylase inhibitor includes one or more selected from the following or pharmaceutically acceptable salts thereof: valproic acid, vorinostat, belinostat, romidepsin, panobinostat; and / or

[0042] The mTOR inhibitor includes one or more selected from the group consisting of sirolimus, everolimus, temsirolimus, and ibrutinib, or pharmaceutically acceptable salts thereof.

[0043] According to an embodiment of the present disclosure, the administration route of the kit is enteral administration.

[0044] The present disclosure also provides a method for treating hemophilia or alleviating hemophilia-related symptoms (such as bleeding or swelling caused by bleeding), comprising administering a histone deacetylase inhibitor, the pharmaceutical composition described above, or the kit described above to a patient in need thereof.

[0045] According to an embodiment of the present disclosure, the histone deacetylase inhibitor is administered twice a day, once a day, or three times a day; and / or the mTOR inhibitor is administered once a day, twice a day, or three times a day. Preferably, the histone deacetylase inhibitor is administered twice a day; and / or the mTOR inhibitor is administered once a day.

[0046] According to an embodiment of the present disclosure, in the use or the method, the administration route of the pharmaceutical composition or the kit is enteral administration. Beneficial effects

[0047] The histone deacetylase inhibitor in the pharmaceutical composition of the present disclosure can maintain the integrity of the blood vessel wall, activate platelets, and promote blood coagulation, thereby improving adverse conditions such as repeated bleeding or joint damage (such as knee swelling) in hemophilia patients.

[0048] In addition, the histone deacetylase inhibitor and the mTOR inhibitor in the pharmaceutical composition disclosed herein have a synergistic effect in treating hemophilia, and can significantly improve the therapeutic effect on hemophilia. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0050] FIG1 is a schematic diagram showing the therapeutic effect of sodium valproate in treating hemophilia, as shown in Example 1.

[0051] FIG2 is a schematic diagram showing the therapeutic effect of using a sodium valproate-sirolimus combination to treat hemophilia, as shown in Example 2.

[0052] Figure 3 is a volcano plot of differentially expressed genes in hemophilia patients compared with normal controls.

[0053] Figure 4 is a volcano plot of differentially expressed genes in patients after taking sodium valproate compared with before taking the drug.

[0054] FIG5 is a volcano plot of differentially expressed genes in patients after sirolimus treatment compared with those before treatment.

[0055] Figure 6 is a Veen diagram of differentially expressed genes in hemophilia patients and sodium valproate and sirolimus based on the principle of drug negative regulation.

[0056] FIG7 is a diagram showing that the sodium valproate-sirolimus combination participates in regulating the Toll-like receptor signaling pathway to achieve immunosuppressive function.

[0057] FIG8 is a diagram showing activation reactions such as platelet shape change and degranulation in which the sodium valproate-sirolimus combination participates in regulating the platelet activation signaling pathway.

[0058] FIG9 shows that the sodium valproate-sirolimus combination participates in the vascular smooth muscle contraction signaling pathway to alleviate abnormal bleeding in hemophilia patients.

[0059] FIG10 is a diagram showing that the sodium valproate-sirolimus combination participates in the complement and coagulation cascade contraction signaling pathway to regulate abnormal bleeding in hemophilia patients.

[0060] FIG11 is a graph showing the clearance effect of coagulation factor inhibitors in a patient 1 with severe hemophilia B during 12 months of using the sodium valproate-sirolimus combination.

[0061] FIG12 is a graph showing improvement in the knee joint of patient 1 with severe hemophilia B after using the sodium valproate-sirolimus combination for 12 months.

[0062] FIG13 is a graph showing the clearance effect of coagulation factor inhibitors in the body of patient 2 with severe hemophilia A during 5 months of using the sodium valproate-sirolimus combination. DETAILED DESCRIPTION

[0063] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. In the following description, when referring to the drawings, like numbers in different figures represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all possible embodiments consistent with the present disclosure. Rather, they are merely examples of compositions and methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.

[0064] Congenital hemophilia (including hemophilia A and hemophilia B) is a rare bleeding disorder caused by mutations in the coagulation factor VIII or factor IX genes. Severe hemophilia (factor VIII or IX activity levels <1%) is characterized by recurrent joint bleeding and life-threatening bleeding, placing a significant financial burden and stress on patients, their families, and society.

[0065] Although hemophilia replacement therapy has greatly improved the quality of life of hemophilia patients, it still has many shortcomings, including: 1. Large-scale plasma transfusion not only fails to effectively stop bleeding, but instead increases the patient's blood volume; 2. It is easy to induce antigen-antibody reactions; 3. Large-scale transfusions can easily induce venous and arterial thrombosis and DIC; 4. Ordinary coagulation factors have a short half-life. In order to maintain the effective activity of coagulation factors, hemophilia patients need to infuse them daily or every other day, and patient compliance is poor.

[0066] When hemophilia patients use replacement therapy, the development of inhibitors is the most common complication, which may even cause the clinical manifestations of hemophilia patients to change from mild to severe, endangering the patient's life. For hemophilia A, about 30% of patients develop inhibitors after replacement therapy, and the incidence of inhibitors in hemophilia B is about 1.5-5%. Although inhibitors can be eradicated using immune tolerance induction therapy (ITI), about 40% of hemophilia patients are still unable to restore normal coagulation factor response levels. Gene therapy is a new type of hemophilia treatment, but it is essentially a replacement therapy; and the long-term efficacy and adverse reactions of this therapy are still unclear, and its high price makes it unaffordable for ordinary family patients.

[0067] According to some embodiments of the present disclosure, the present disclosure provides a pharmaceutical composition for treating hemophilia, wherein the pharmaceutical composition comprises: a histone deacetylase inhibitor and a pharmaceutically acceptable carrier.

[0068] According to some embodiments of the present disclosure, the present disclosure further provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor, an mTOR inhibitor and a pharmaceutically acceptable carrier.

[0069] According to some embodiments of the present disclosure, the present disclosure further provides a pharmaceutical composition for treating hemophilia, wherein the pharmaceutical composition comprises a histone deacetylase inhibitor, an mTOR inhibitor, and a pharmaceutically acceptable carrier.

[0070] The hemophilia described in the present invention can be selected from hemophilia A (reduced activity of coagulation factor VIII), hemophilia B (reduced activity of coagulation factor IX), acquired hemophilia A (production of coagulation factor VIII inhibitors in vivo), and acquired hemophilia B (production of coagulation factor IX inhibitors in vivo).

[0071] Histone deacetylase inhibitors (HDIs) have anti-inflammatory, neuroprotective, and anticonvulsant properties. There are reports that HDIs can regulate stem cell differentiation and self-renewal. In addition to anti-epileptic uses, these drugs are also used to treat febrile seizures, movement disorders, chorea, porphyria, schizophrenia, pain associated with herpes zoster, adrenal dysfunction, and to prevent alcohol withdrawal syndrome. Orally, they are rapidly and completely absorbed, primarily distributing in the extracellular fluid and remaining largely bound to plasma proteins in the blood.

[0072] According to an embodiment of the present disclosure, the histone deacetylase inhibitor may include one or more of the following or a pharmaceutically acceptable salt thereof: valproic acid, vorinostat, belinostat, panobinostat. For example, according to an embodiment of the present disclosure, the histone deacetylase inhibitor of the present disclosure may be valproic acid or a pharmaceutically acceptable salt thereof.

[0073] According to an embodiment of the present disclosure, the pharmaceutically acceptable salt of valproic acid is present in the form of a salt selected from the group consisting of a sodium salt, a potassium salt, a magnesium salt, a calcium salt, or an ammonium salt.

[0074] According to an embodiment of the present disclosure, the sodium salt form of valproic acid includes hemi-sodium valproate or sodium valproate.

[0075] According to embodiments of the present disclosure, in the pharmaceutical composition, the content of histone deacetylase inhibitor is 0.1g-1.0g, for example 0.3g-0.8g, for example 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g or 1.0g. For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is 0.4g-0.6g. For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is 0.45g-0.55g. For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is about 0.25g. For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is about 0.5g. For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is about 0.6g.

[0076] According to an embodiment of the present disclosure, the content of sodium valproate in the pharmaceutical composition can be 0.1g-1.0g, such as 0.15g-0.45g, for example, 0.1g, 0.15g, 0.20g, 0.25g, 0.30g, 0.35g, 0.40g, 0.45g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g or 1.0g. For example, according to an embodiment of the present disclosure, the content of sodium valproate can be 0.2g-0.4g. For example, according to an embodiment of the present disclosure, the content of sodium valproate can be 0.3g-0.35g. For example, according to an embodiment of the present disclosure, the content of sodium valproate can be about 0.333g.

[0077] According to an embodiment of the present disclosure, the pharmaceutical composition can be a sustained-release tablet, for example, a sodium valproate sustained-release tablet. According to an embodiment of the present disclosure, the pharmaceutical composition can be a compound preparation, wherein each tablet contains approximately 0.333g of sodium valproate and approximately 0.145g of valproic acid (equivalent to 0.5g of sodium valproate).

[0078] The present disclosure also provides use of a histone deacetylase inhibitor or the pharmaceutical composition as described above in a medicament for treating hemophilia. The medicament comprises a histone deacetylase inhibitor and a pharmaceutically acceptable carrier for treating hemophilia.

[0079] According to the embodiments of the present disclosure, histone deacetylase inhibitors can maintain the integrity of the blood vessel wall and effectively activate platelets, thereby promoting blood coagulation. Therefore, according to the pharmaceutical composition of the present disclosure, a new therapeutic approach for treating hemophilia is provided, which accelerates the coagulation reaction by enhancing the activity of platelets, thereby responding to the development of the disease more quickly. The platelet activation mechanism of histone deacetylase inhibitors provides a biological basis for their unique effects in treatment.

[0080] According to an embodiment of the present disclosure, the drug includes a histone deacetylase inhibitor to activate platelets to promote blood coagulation.

[0081] According to an embodiment of the present disclosure, the histone deacetylase inhibitor includes one or more of the following and pharmaceutically acceptable salts thereof: valproic acid, vorinostat, belinostat, and panobinostat.

[0082] According to an embodiment of the present disclosure, the pharmaceutical composition may further include an mTOR inhibitor and a pharmaceutically acceptable carrier.

[0083] As used herein, the terms "mammalian target of sirolimus (rapamycin)" and "mTOR" refer to protein kinases that are catalytic subunits of mTORC1 and mTORC2, which are involved in various cellular processes. This kinase is a component of two different complexes, mTORC1 controls protein synthesis, cell growth and proliferation, and mTORC2 is a regulator of the actin cytoskeleton, promoting cell survival and cell cycle progression. This protein serves as the target of the cell cycle arrest and immunosuppressive effects of the FKBP12-mTOR inhibitor complex.

[0084] In this context, mTOR inhibitors are a class of compounds used to inhibit the activity of the mTOR signaling pathway and are commonly used in cancer therapy and other disease research. Their primary mechanism of action is inhibition of the mammalian target of sirolimus (rapamycin) (mTOR), a serine / threonine-specific protein kinase that regulates cell growth, proliferation, and survival. mTOR is an important therapeutic target for a variety of diseases because it has been shown to regulate lifespan and maintain normal glucose homeostasis. It is often used to prevent rejection in organ transplants.

[0085] According to the embodiments of the present disclosure, an immunomodulatory strategy using sirolimus (rapamycin) combined with repeated injections of low-dose coagulation factors (FVIII or FIX) prevented the induction of inhibitory antibody responses in hemophiliac mice. Considering that patients who use long-term replacement therapy are prone to coagulation factor inhibitors, mTOR inhibitors were included in the regimen.

[0086] According to the embodiments disclosed herein, histone deacetylase inhibitors can maintain the integrity of blood vessel walls, activate platelets, and promote platelet aggregation. mTOR inhibitors can effectively reduce inhibitor concentrations in hemophilia patients. The combined use of the two drugs can benefit patients by maintaining the integrity of blood vessel walls, promoting coagulation, and eliminating inhibitors, thereby effectively preventing and treating bleeding in hemophilia patients and achieving the purpose of treatment. In some existing examples, patients have even been able to stop transfusing coagulation factors and gradually recover.

[0087] According to an embodiment of the present disclosure, the pharmaceutical composition may comprise about 100 mg / d to about 2000 mg / d (e.g., about 200 mg / d to about 2000 mg / d) of a histone deacetylase inhibitor and about 0.1 mg / d to about 2 mg / d (e.g., 0.5 mg / d to about 2 mg / d) of an mTOR inhibitor. Administration of the histone deacetylase inhibitor-mTOR inhibitor combination significantly reduces the concentration of coagulation factor inhibitors in the subject compared to the concentration before administration, and effectively alleviates abnormal bleeding.

[0088] According to an embodiment of the present disclosure, the mTOR inhibitor includes one or more of the following: sirolimus, rapamycin, everolimus, temsirolimus.

[0089] According to embodiments of the present disclosure, the content of the mTOR inhibitor in the pharmaceutical composition can be 0.1 mg-3 mg, such as 0.2 mg-3 mg, such as 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg or 3 mg. For example, according to embodiments of the present disclosure, the content of the mTOR inhibitor can be 0.5 mg-2 mg, such as 0.5 mg-2 mg. For example, according to embodiments of the present disclosure, the content of the mTOR inhibitor can be 0.8 mg-1.5 mg, such as 0.8 mg-1.5 mg. For example, according to an embodiment of the present disclosure, the content of the mTOR inhibitor may be about 0.5 mg. For example, according to an embodiment of the present disclosure, the content of the mTOR inhibitor may be about 1 mg.

[0090] According to an embodiment of the present disclosure, the weight ratio of the histone deacetylase inhibitor to the mTOR inhibitor can be 250: 1 to 1200: 1, such as 300: 1 to 1100: 1, 400: 1 to 1000: 1, 500: 1 to 900: 1 or 600: 1 to 800: 1. For example, according to an embodiment of the present disclosure, the pharmaceutical composition can include about 250 mg of sodium valproate and about 0.5 mg of sirolimus. For example, according to an embodiment of the present disclosure, the pharmaceutical composition can include about 600 mg of sodium valproate and about 1 mg of sirolimus. According to an embodiment of the present disclosure, the pharmaceutical composition can include 500 mg of sodium valproate and 0.5 mg of sirolimus.

[0091] It should be noted that the content in the pharmaceutical composition described herein is a daily dose for one patient / subject during treatment.

[0092] According to the embodiments of the present disclosure, the pharmaceutical composition may be in the form of a powder, tablet, lozenge, granule, capsule, suspension, or emulsion. The pharmaceutical carrier for the pharmaceutical composition may be obtained by using known compounds and methods used in the relevant art to produce pharmaceuticals. For example, it may be the solid carrier described above, which will not be described in detail here.

[0093] The present disclosure also provides a use of a histone deacetylase inhibitor and an mTOR inhibitor or the pharmaceutical composition as described above in the preparation of a kit, wherein the kit comprises a histone deacetylase inhibitor and an mTOR inhibitor; and the kit is used for simultaneously co-administering the histone deacetylase inhibitor and the mTOR inhibitor to treat hemophilia.

[0094] The present invention includes a kit suitable for practicing the above-described treatment methods. In one embodiment, the kit includes a first dosage form and a second dosage form for simultaneous administration, in amounts sufficient to practice the disclosed method, i.e., for treating hemophilia. The first dosage form comprises one or more of the above-identified histone deacetylase inhibitors, and the second dosage form comprises one or more of the above-identified mTOR inhibitors.

[0095] According to embodiments of the present disclosure, the content of the histone deacetylase inhibitor in the test kit is 0.3g-1.0g, such as 0.3g-0.8g, such as 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g or 1.0g.For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is 0.4g-0.6g.For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is 0.45g-0.55g.For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is about 0.25g.For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is about 0.5g.For example, according to embodiments of the present disclosure, the content of histone deacetylase inhibitor is about 0.6g.

[0096] According to embodiments of the present disclosure, the amount of the mTOR inhibitor in the kit is 0.1 mg to 3 mg, such as 0.2 mg to 3 mg, such as 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg or 3 mg. The amount of the mTOR inhibitor may be 0.2 mg to 3 mg. For example, according to embodiments of the present disclosure, the amount of the mTOR inhibitor may be 0.5 mg to 2 mg. For example, according to embodiments of the present disclosure, the amount of the mTOR inhibitor may be 0.8 mg to 1.5 mg. For example, according to an embodiment of the present disclosure, the content of the mTOR inhibitor may be about 0.5 mg. For example, according to an embodiment of the present disclosure, the content of the mTOR inhibitor may be about 1 mg.

[0097] According to an embodiment of the present disclosure, the histone deacetylase inhibitor in the kit is administered twice a day, once a day, or three times a day. According to an embodiment of the present disclosure, the mTOR inhibitor in the kit is administered once a day, twice a day, or three times a day.

[0098] According to an embodiment of the present disclosure, the content of sodium valproate as a histone deacetylase inhibitor is 0.15g-1.0g, such as 0.15g-0.45g, for example, 0.1g, 0.15g, 0.20g, 0.25g, 0.30g, 0.35g, 0.40g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g or 1.0g.

[0099] According to an embodiment of the present disclosure, the histone deacetylase inhibitors include one or more of the following and pharmaceutically acceptable salts thereof: valproic acid, vorinostat, belinostat, panobinostat; and / or the mTOR inhibitors include one or more of the following: sirolimus, rapamycin, everolimus, temsirolimus.

[0100] According to the embodiment of the present disclosure, the administration route of the kit is enteral administration. The specific method of enteral administration is as described above and will not be repeated here.

[0101] Current treatments for hemophilia (whether hemophilia A or hemophilia B) rely on exogenous plasma transfusions or recombinant coagulation factor therapy based on gene editing technology. While these methods are effective, they also have significant side effects (the production of coagulation factor inhibitors, which significantly impacts efficacy). While scientists are currently researching methods to remove inhibitors, only a few have been effective. Furthermore, these treatments are expensive.

[0102] This invention proposes for the first time a small molecule compound treatment for this disease and reveals the synergistic mechanism of the sodium valproate-sirolimus combination in treating the disease, providing innovative insights and strong support for future disease treatments. This research not only provides guidance for optimizing current disease treatments but also opens up new research directions for developing more effective treatment strategies. Furthermore, the pharmaceutical composition and kit provided herein can be used to treat hemophilia at a relatively low cost.

[0103] According to an embodiment of the present disclosure, the mTOR inhibitor refers to a mammalian target of rapamycin inhibitor, which includes one or more of the following: sirolimus (also known as rapamycin), everolimus, temsirolimus, and ibrutinib.

[0104] According to an embodiment of the present disclosure, in the pharmaceutical composition, the content of the mTOR inhibitor is 0.1 mg-3 mg, for example, 0.2 mg-3 mg, for example, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg or 3 mg.

[0105] According to an embodiment of the present disclosure, the weight ratio of the histone deacetylase inhibitor to the mTOR inhibitor is 250:1 to 1200:1, such as 300:1 to 1100:1, 400:1 to 1000:1, 500:1 to 900:1 or 600:1 to 800:1.

[0106] According to an embodiment of the present disclosure, the drug is administered twice a day, once a day, or three times a day.

[0107] According to an embodiment of the present disclosure, the administration route of the drug is enteral administration. For example, it may include oral administration, sublingual administration or rectal administration. According to an embodiment of the present disclosure, oral administration is preferred, but other routes of administration are not excluded.

[0108] According to an embodiment of the present disclosure, the drug is an oral pharmaceutical composition, and the pharmaceutical carrier includes an inert pharmaceutically acceptable carrier, such as a solid carrier or a liquid carrier. For example, solid form preparations include but are not limited to powders, tablets, coated tablets, lozenges, lozenges, dispersible granules, capsules, and bags. Compositions for oral use can be prepared according to any method known in the art for preparing pharmaceutical compositions.

[0109] The solid carrier can be one or more substances selected from the group consisting of a diluent, a flavoring agent, a solubilizer, a lubricant, a suspending agent, a binder, or a tablet disintegrating agent; the solid carrier can also be a capsule-forming material.

[0110] According to embodiments of the present disclosure, in powders, the carrier is a finely divided solid that is present in a mixture with one or more finely divided active ingredients. In tablets, one or more active ingredients are mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size.

[0111] According to embodiments of the present disclosure, suitable carriers may be inert diluents such as magnesium carbonate, calcium stearate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, and the like.

[0112] According to embodiments of the present disclosure, the present disclosure also includes metformin and statins and preparations prepared using capsule-forming materials as carriers to provide capsules in which metformin and statins (with or without other carriers) are coated with carriers, thereby combining the carrier with metformin and statins. In a similar manner, sachets, tablets, powders, bags, and capsules can be used as solid dosage forms suitable for oral administration.

[0113] According to embodiments of the present disclosure, tablets may be uncoated or coated by known techniques to delay disintegration and absorption of the drug in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used.

[0114] According to an embodiment of the present disclosure, the preparation for oral administration can be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (such as calcium carbonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is present in its own form or mixed with water or an oil medium (such as peanut oil, liquid paraffin or olive oil).

[0115] According to an embodiment of the present disclosure, the pharmaceutical composition may be in the form of powder, tablet, lozenge, granule, capsule, suspension or emulsion.

[0116] According to embodiments of the present disclosure, the pharmaceutical composition can also be prepared as an aqueous suspension comprising the active compound admixed with excipients suitable for the manufacture of aqueous suspensions. The excipients are suspending agents such as carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; dispersants or wetting agents including naturally occurring phospholipids such as lecithin, or condensates of alkylene oxides with fatty acids (such as polyoxyethylene stearate), or condensates of alkylene oxides with fatty acids (such as polyoxyethylene stearate), or condensates of ethylene oxide with partial esters of fatty acids and hexitol anhydrides (such as polyoxyethylene sorbitan monooleate).

[0117] According to an embodiment of the present disclosure, the aqueous suspension may further include one or more preservatives (such as ethylparaben or n-propylparaben), colorants, flavorings, and sweeteners.

[0118] According to an embodiment of the present disclosure, an oily suspension can also be prepared by suspending the active compound in w-3 fatty acids, vegetable oils (such as peanut oil, olive oil, sesame oil, coconut oil) or mineral oils (such as liquid paraffin). The oily suspension can contain a thickening agent such as beeswax, hard paraffin and cetyl alcohol.

[0119] According to embodiments of the present disclosure, sweeteners and flavoring agents may also be added to the pharmaceutical composition to provide a palatable oral formulation. The formulation may be preserved by adding an antioxidant such as ascorbic acid.

[0120] According to an embodiment of the present disclosure, a syrup composition comprising the novel combination can be prepared using a sweetener. The formulation may also include a demulcent, a preservative, and flavoring and coloring agents.

[0121] According to embodiments of the present disclosure, the preparations in liquid form include solutions, suspensions and emulsions suitable for oral administration. Aqueous solutions for oral administration can be prepared by dissolving the active ingredient in water and adding suitable flavorings, coloring agents, stabilizers and thickeners as needed. In order to improve the solubility of the active ingredient, ethanol, propylene glycol and other pharmaceutically acceptable non-aqueous solvents can be added. Aqueous suspensions for oral administration can be prepared by dispersing finely divided active compounds in water together with other suspending agents such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and known in the field of pharmaceutical preparations.

[0122] According to embodiments of the present disclosure, the pharmaceutical formulation is preferably in unit dosage form. In this dosage form, the formulation is divided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form can be a packaged formulation, such as packaged tablets, capsules, and powders in vials or ampoules containing aliquots of the formulation. The unit dosage form can also be a capsule, cachet, or tablet itself, or an appropriate number of any of these packaged forms.

[0123] According to an embodiment of the present disclosure, the pharmaceutical composition can also be administered parenterally in the form of a sterile injectable aqueous or oily suspension, which is administered subcutaneously, intravenously, intramuscularly, intrasternally, or by infusion technique. The suspension can be formulated using the above-mentioned appropriate dispersants, wetting agents and suspending agents or other acceptable substances according to known techniques. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable carriers and solvents, water, Ringer's solution and isotonic sodium chloride solution can be used. In addition, sterile fixed oils are conventionally used solvents or suspending media. For this purpose, any small irritant fixed oil including synthetic monoglycerides or diglycerides can be used. In addition, n-3 polyunsaturated fatty acids are also useful in injectable preparations.

[0124] According to an embodiment of the present disclosure, the pharmaceutical composition can also be administered by inhalation in the form of an aerosol or a solution for a nebulizer, or in the form of a suppository prepared by mixing the active ingredient with a suitable non-irritating excipient, which is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such materials may include cocoa butter and polyethylene glycol.

[0125] According to an embodiment of the present disclosure, preferably, the pharmaceutical composition may be a controlled-release composition.

[0126] According to the embodiments of the present disclosure, the daily dose can vary within wide limits and be adjusted according to the needs of the individual in each particular case. Generally, for adult administration, the appropriate daily dose has been described above; however, if divided doses are administered, the limits specified above may be exceeded for emergency purposes. The daily dose may be administered as a single dose or in divided doses.

[0127] The dosages used for patients and experimental mice in the following examples were obtained through analysis and deduction of effective dosages in early clinical trials, as follows:

[0128] Sirolimus: 1 mg / day

[0129] Sodium valproate: 500 mg / day

[0130] Sodium valproate:

[0131] The daily dosage for mice is 2 mg, and the daily dosage for adults is 500 mg.

[0132] [A daily dose of 500 mg for adults is equivalent to a daily dose of 2 mg per mouse (concentration: 40 mg / ml)]

[0133] Conversion formula for mice and humans: body surface area conversion method (BSA method), mouse dose = 9.1 × human clinical dose (mg / kg).

[0134] Sirolimus:

[0135] Mice were administered 0.04 mg (concentration: 1 mg / ml) daily.

[0136] Examples of body surface area and drug dosage calculations for humans and mice:

[0137] Patient QY: height 190cm, weight 100kg

[0138] DuBois formula (body surface area calculation BSA) BSA = 0.007184 × 1900.725 × 1000.425 = 2.329 m 2

[0139] Mouse: 20g

[0140] Meeh-Rubner formula: BSA=0.00913×200.67=0.128m 2

[0141] BSA ratio: 0.128 / 2.329=0.055

[0142] Sirolimus: 1 x 0.055 = 0.055 mg

[0143] Sodium valproate: 500×0.055=27.5 mg.

[0144] Example 1

[0145] In the following Example 1, sodium valproate, a histone deacetylase inhibitor, was used as an example to test the therapeutic effect of the drug of the present disclosure on joint damage caused by hemophilia by administering the drug of the present disclosure to a hemophilia B mouse model (FIX Exon 1-3KO).

[0146] Experimental Design: Before the experiment, the diameter of the right knee joint of each mouse was measured with a vernier caliper (supine position). A knee joint injury model of hemophilia B mice was then established. The experimental group received sodium valproate intervention 24 hours later, while the model control group received no drug intervention. The degree of knee joint swelling was recorded to evaluate the therapeutic effect.

[0147] Establishment of a mouse knee injury model: FIX Exon 1-3KO mice were used as a hemophilia B mouse model. A brief puncture was performed at the patella of the right knee joint of the mouse using a 0.8 x 35 mm needle. Buprenorphine was administered subcutaneously to each mouse 2 hours prior to the puncture for analgesia. Buprenorphine was then added to the mouse drinking water for 1 week to prevent death from pain after the injury.

[0148] Experimental Time Points: The right knee joint diameter of the mice was recorded before and 24 hours after the puncture injury. The experimental group then began drug treatment, with medication administered once daily. After one week of treatment, the right knee joint diameter of the mice was measured again to assess the therapeutic effect. The dosing schedule for each group was as follows:

[0149] Sodium valproate group: drug dose (50 μL, concentration 40 mg / ml);

[0150]

Control group

[0151] Data analysis: Statistical methods were used to compare the right leg knee joint diameter data before and after puncture and after drug treatment to evaluate the effect of drug treatment.

[0152] Experimental results: The diameters of the right knee joints of mice after sodium valproate administration were measured using a vernier caliper and recorded at different time points. The specific test results are shown in Table 1.

[0153] Table 1: Diameter of right knee joint of mice at different time points (unit: mm)

[0154] As shown in Table 1, 24 hours after the puncture injury, the knee joint diameters of both groups of mice increased significantly. After one week of treatment, the knee joint diameters of mice treated with sodium valproate decreased significantly, demonstrating that sodium valproate has a significant therapeutic effect on joint damage in the hemophilia B mouse model.

[0155] In order to better describe the effect of drug treatment, the above data were visualized using a box plot, and the statistical differences in knee joint diameters at different time points were evaluated using a t-test. Figure 1 is a schematic diagram of the therapeutic effect of sodium valproate for treating hemophilia according to Example 1. As shown in Figure 1, the box plot of Figure 1 shows the therapeutic effect of sodium valproate on knee joint injuries in mice: the horizontal axis represents the time point for measuring the diameter of the right knee joint of the mouse, and the vertical axis represents the measured value of the diameter of the right knee joint of the mouse; t-test results: * indicates P < 0.05; ** indicates P < 0.01; ns indicates P > 0.05 (i.e., there is no statistical difference between the two groups of data).

[0156] Example 2A

[0157] In the following Example 2A, sodium valproate was used as a histone deacetylase inhibitor and sirolimus was used as an mTOR inhibitor. The pharmaceutical composition of the present invention was administered to a hemophilia B mouse model (FIX Exon 1-3KO) to test the therapeutic effect of the drug of the present invention on joint damage caused by hemophilia.

[0158] Experimental design: Before the experiment, the diameter of the right knee joint of each mouse was measured using a vernier caliper (supine position); then, a knee joint injury model of hemophilia B mice was established, and drug intervention was performed using the drug composition 24 hours later. The degree of knee joint swelling was recorded to evaluate the therapeutic effect.

[0159] Establishment of a mouse knee injury model: FIX Exon 1-3KO mice were used as a hemophilia B mouse model. A brief puncture was performed at the patella of the right knee joint of the mouse using a 0.8 x 35 mm needle. Buprenorphine was administered subcutaneously to each mouse 2 hours prior to the puncture for analgesia. Buprenorphine was then added to the mouse drinking water for 1 week to prevent death from pain after the injury.

[0160] Experimental Time Points: The right knee joint diameter of the mice was recorded before and 24 hours after the puncture injury. Drug treatment was then initiated, with dosing administered once daily. After one week of treatment, the right knee joint diameter of the mice was measured again to assess the therapeutic effect. The dosing schedule for each group was as follows:

[0161] Combination group: sirolimus (40 μL, 1 mg / ml) and sodium valproate (50 μL, 40 mg / ml).

[0162]

Control group

[0163] Data analysis: Statistical methods were used to compare the right leg knee joint diameter data before and after puncture and after drug treatment to evaluate the effect of drug treatment.

[0164] Experimental results: The diameters of the right knee joints of mice at different time points were measured using a vernier caliper. The results are shown in Table 2A:

[0165] Table 2A: Diameter of right knee joint of mice at different time points (unit: mm)

[0166] Example 2B

[0167] In the following Example 2B, sodium valproate was used as a histone deacetylase inhibitor and sirolimus was used as an mTOR inhibitor. The pharmaceutical composition of the present invention was administered to a hemophilia B mouse model (FIX Exon 1-3KO) to test the therapeutic effect of the drug of the present invention on joint damage caused by hemophilia.

[0168] Experimental design: Before the experiment, the diameter of the right knee joint of each mouse was measured using a vernier caliper (supine position); then, a knee joint injury model of hemophilia B mice was established, and drug intervention was performed using the drug composition 24 hours later. The degree of knee joint swelling was recorded to evaluate the therapeutic effect.

[0169] Establishment of a mouse knee injury model: FIX Exon 1-3KO mice were used as a hemophilia B mouse model. A brief puncture was performed at the patella of the right knee joint of the mouse using a 0.8 x 35 mm needle. Buprenorphine was administered subcutaneously to each mouse 2 hours prior to the puncture for analgesia. Buprenorphine was then added to the mouse drinking water for 1 week to prevent death from pain after the injury.

[0170] Experimental Time Points: The right knee joint diameter of the mice was recorded before and 24 hours after the puncture injury. Drug treatment was then initiated, with dosing administered once daily. After one week of treatment, the right knee joint diameter of the mice was measured again to assess the therapeutic effect. The dosing schedule for each group was as follows:

[0171] Combination group: sirolimus (40 μL, 1 mg / ml) and sodium valproate (50 μL, 40 mg / ml).

[0172] [Single drug (sodium valproate) group] Dose: 50 μL (concentration 40 mg / ml)

[0173]

Control group

[0174] Data analysis: Statistical methods were used to compare the right leg knee joint diameter data before and after puncture and after drug treatment to evaluate the effect of drug treatment.

[0175] Experimental results: The diameters of the right knee joints of mice at different time points were measured using a vernier caliper. The results are shown in Table 2B:

[0176] Table 2B: Diameter of right knee joint of mice at different time points (unit: mm)

[0177] As can be seen from the data in Tables 2A and 2B above, 24 hours after puncture injury, the knee joint diameters of both groups of mice increased significantly. After one week of treatment, the knee joint diameters of mice treated with sodium valproate or the sodium valproate-sirolimus combination decreased significantly, demonstrating that sodium valproate or the sodium valproate-sirolimus combination has a significant therapeutic effect on joint damage in the hemophilia B mouse model.

[0178] In order to better describe the effect of drug treatment, the above data were visualized using a box plot, and the t-test was used to evaluate the statistical differences in knee joint diameter at different time points. Figure 2 is a schematic diagram of the therapeutic effect of a sodium valproate-sirolimus composition for treating hemophilia shown in Example 2. As shown in Figure 2, the box plot of Figure 2 shows the therapeutic effect of the sodium valproate-sirolimus composition on knee joint injuries in mice: the horizontal axis represents the time point at which the diameter of the right knee joint of the mouse is measured, and the vertical axis represents the measured value of the diameter of the right knee joint of the mouse; t-test results: * indicates P < 0.05; ** indicates P < 0.01; ns indicates P > 0.05 (i.e., there is no statistical difference between the two groups of data).

[0179] According to the pharmaceutical composition disclosed herein, the histone deacetylase inhibitor can activate platelets and promote blood coagulation, thereby improving the recurrent bleeding condition of hemophilia patients.

[0180] The present invention utilizes the disease gene expression profile data characteristic of hemophilia, and determines drugs based on the Connectivity Map, L1000 public database and the EpiMed platform based on the "logic omics" inventors' disease-gene association principle.

[0181] The present invention jointly analyzes hemophilia disease expression profile data with drug (histone deacetylase inhibitor, mTOR inhibitor) expression profile data, and comprehensively considers the pharmacodynamics and drug toxicity and side effects. It proposes that the sodium valproate-sirolimus combination can treat hemophilia, and confirms this through experiments.

[0182] Method steps:

[0183] Using the hemophilia disease expression profile data obtained earlier in this study, the edgeR software package was used to screen for differentially expressed genes in the expression profiles. The screening criteria for differentially expressed genes were FDR < 0.05 and |Log2FC| > 1, identifying differentially expressed genes in hemophilia. Multi-omics association analysis was then performed on the characteristically expressed genes in hemophilia patients using the Connectivity Map and L1000 public databases, along with algorithms based on "systems biology" and "comparative functional genomics" theory, to predict potential regulatory drugs. Potential therapeutic drugs were then identified by comprehensively considering pharmacodynamics and drug toxicity and side effects.

[0184] The transcriptome expression data of histone deacetylase inhibitors and mTOR inhibitors obtained earlier in this study were used to screen for differentially expressed genes using the GEO2R online analysis tool. The screening criteria for differentially expressed genes were FDR < 0.05 and |Log2FC| > 1. Differentially expressed genes before and after administration of histone deacetylase inhibitors and mTOR inhibitors were obtained. The results of the two differential expression analyses were combined to identify intersecting genes and perform KEGG enrichment analysis.

[0185] In the present invention, histone deacetylase inhibitors and mTOR inhibitors are both a class of drugs; based on the results of bioinformatics analysis, the drugs in the histone deacetylase inhibitors have similar chemical structures and functions, and the drugs in the mTOR inhibitors have similar chemical structures and functions; therefore, sodium valproate (a histone deacetylase inhibitor) and sirolimus (also known as: rapamycin, an mTOR inhibitor) are preferred for clinical trials.

[0186] Figure 3 is a volcano plot of differentially expressed genes in hemophilia patients compared to normal controls. Figure 4 is a volcano plot of differentially expressed genes in patients after sodium valproate treatment compared to before treatment, and Figure 5 is a volcano plot of differentially expressed genes in patients after sirolimus treatment compared to before treatment.

[0187] In Figures 3 to 5, Up represents "up-regulated genes", Down represents "down-regulated genes", Stable represents "stable genes (no differentially expressed genes)", log2 (fold change) represents "log2 (differential expression change)", and -log10 (PValue) represents "-log10 (P value)".

[0188] Figure 6 is a Veen plot of differentially expressed genes in hemophilia patients, sodium valproate, and sirolimus based on the principle of drug negative regulation. In Figure 6, "Hemophilia" represents hemophilia, "Drug combination" represents drug combination, and "up" and "down" represent upregulated and downregulated genes, respectively.

[0189] Figure 7 shows the sodium valproate-sirolimus combination participating in the regulation of the Toll-like receptor signaling pathway to achieve immunosuppressive function. Figure 8 shows the sodium valproate-sirolimus combination participating in the regulation of the platelet activation signaling pathway to affect platelet shape changes, degranulation and other activation reactions. Figure 9 shows the sodium valproate-sirolimus combination participating in the vascular smooth muscle contraction signaling pathway to alleviate abnormal bleeding in hemophilia patients. Figure 10 shows the sodium valproate-sirolimus combination participating in the complement and coagulation cascade contraction signaling pathway to regulate abnormal bleeding in hemophilia patients.

[0190] [Corrected 06.03.2025 according to Rule 26] In Figures 7 to 10, red indicates up-regulation of genes and green indicates down-regulation of genes. Indicates upregulation of genes, Indicates downregulation of the gene.

[0191] In Figures 7 to 10, TOLL LIKE RECEPTOR SIGNALING PATHWAY indicates Toll-like receptor signaling pathway, Peptidoglycan (G+), Lipoprotein, Lipoarabinomannan, Mycobactena, Zymosan (Yeast), Lipopolysaccharide, biosynthesis, Flagellar assembly, Flagellin, Imidazoquinolin, anti-viral compounds, ssRNA, single-stranded RNA, Unmethylated, PI3K-Akt signaling pathway, Ubiquitin mediated Proteolysis, Inflammatory cytokines, and Proinflammatory cytokines. Effects means pro-inflammatory effects, Chemotactic effects means the effects of chemotaxis, Nutrophil,Immnature DC denotes neutrophil, immature dendritic cell, NK cell denotes NK cell, Complement and coagulation cascade denotes complement and coagulation cascade, Costimulatory molecules denotes costimulatory molecules, T cell stimulation denotes T cell stimulation, Inflammatory cytokines denotes inflammatory cytokines, Antiviral effects denotes antiviral effects, cytokine receptor interaction denotes cytokine receptor interaction, Chemotactic effects (T cell) denotes T cell chemotaxis, ECM-receptor interaction denotes ECM-receptor interaction, PI3K-Akt signaling pathway denotes PI3K-Akt signaling pathway, Calcium signaling pathway denotes calcium signaling pathway, Rapl signaling pathway denotes Rapl signaling pathway, Arachidonic acid metabolism denotes arachidonic acid metabolism, Complement and coagulation cascade denotes complement and coagulation cascade, VASCULAR SMOOTH MUSCLE CONTRACTION denotes vascular smooth muscle contraction, Vascular smooth muscle cell denotes vascular smooth muscle cell, Arachidonic acid metabolism indicates arachidonic acid metabolism, Intravascual pressure / Stretch indicates intravascular pressure / stretch, Calcium signaling pathway indicates calcium signaling pathway, Sarcoplasmic reticulum (SR) indicates sarcoplasmic reticulum (SR), Reduction of contractile system Ca, + sensitivity indicates that the contractile system Ca +Decreased sensitivity, Myosin, Extrinsic pathway, Tissue damage, Intrinsic pathway, Contact with damaged vessel, Kallikrein-kinin system, Fibrin monomer, Cross-linked fibrin polymer, Anti-inflammatory responses, vasodilation increased endothelial permeability, Release of Weibel-Palade bodies, Fibrin degradation products, Cell adhesion, migration, proliferation, Inflammatory mediator regulation of TRP channels, Classical pathway, Antigen-antibody complex, Lectin pathway, Carbohyrate pathway indicates alternative pathway, Tick-over indicates selection, Degranulation,chemotaxis indicates degranulation and chemotaxis, Phagocytosis indicates phagocytosis, and B cell receptor signaling pathway indicates B cell receptor signaling pathway.

[0192] The kit provided herein treats hemophilia by drawing peripheral blood for transcriptome sequencing upon admission. Bioinformatics methods are then used to identify differentially expressed genes in patients compared to healthy controls. This method successfully integrates patient data with drug transcriptome data, revealing significant evidence of synergistic effects between the two drugs in treating specific diseases.

[0193] First, through precise and detailed research, the present disclosure discovered that sirolimus (rapamycin) plays a role in clearing coagulation factor inhibitors primarily through immunomodulatory effects. Clearing coagulation factor inhibitors is crucial for maintaining normal coagulation function, and the action of sirolimus (rapamycin) effectively promotes this process.

[0194] At the same time, sodium valproate offers a unique therapeutic contribution. It has been shown to effectively maintain blood vessel wall integrity and activate platelets, thereby promoting blood coagulation. This discovery provides a novel therapeutic approach that accelerates coagulation by enhancing platelet activity, thereby more rapidly addressing disease progression. Sodium valproate's platelet activation mechanism provides the biological basis for its unique therapeutic effects.

[0195] A comprehensive analysis of the combined use of sodium valproate and sirolimus (rapamycin) revealed significant synergy between the two drugs. Sirolimus (rapamycin) eliminates coagulation factor inhibitors, creating a more favorable environment for sodium valproate to fully exert its platelet-activating effect. This synergistic effect not only enhances therapeutic efficacy but also reduces potential side effects, laying the foundation for personalized and precise disease treatment.

[0196] This study successfully reveals the synergistic effect of the sodium valproate-sirolimus combination in treating diseases, providing innovative insights and strong support for future disease treatments. This research not only provides guidance for optimizing current disease treatments but also opens up new research directions for developing more effective treatment strategies.

[0197] Example 3

[0198] This study was approved by the Ethics Committee. Patient 1, upon admission, was diagnosed with hemophilia B. He had a history of exogenous coagulation factor transfusions, which resulted in the development of coagulation factor inhibitors, significantly hindering traditional treatment.

[0199] Medication regimen: Sodium valproate (500 mg / day), sirolimus (rapamycin) (1 mg / day)

[0200] Medication schedule: Sodium valproate (sodium valproate sustained-release tablets) twice a day (morning and afternoon), sirolimus (capsules) once a day (morning).

[0201] Route of administration: Oral.

[0202] The route of administration and dosage of the drug remained unchanged throughout the treatment cycle.

[0203] Patient 1 was treated according to the above medication regimen for 12 months.

[0204] Clinical Presentation: Patient 1's FIX inhibitor level decreased from 4.8 BU / ml to 0.6 BU / ml, and his HAL score improved from 127 to 187. After 12 months of treatment, his knee swelling significantly subsided. He improved from wheelchair dependence to being able to stand and walk without a wheelchair, and now uses a bicycle and other means of transportation.

[0205] The results of the coagulation factor IX inhibitor test for patient 1 during the treatment period are recorded in the following Table 3:

[0206] Table 3

[0207] As can be seen from Table 3 , the FIX inhibitor of patient 1 decreased from 4.8 BU / ml to 0.6 BU / ml.

[0208] Figure 11 shows the effect of clearing coagulation factor inhibitors in the body of severe hemophilia B patient 1 after using the sodium valproate-sirolimus combination for 12 months. Figure 12 shows the improvement of the knee joint of severe hemophilia B patient 1 after using the sodium valproate-sirolimus combination for 12 months.

[0209] As shown in Figures 11 and 12, after 12 months of treatment, Patient 1's knee swelling symptoms were significantly reduced, and the number of bleeding injuries decreased significantly. Patient 1 improved from being wheelchair-dependent to being able to stand and walk without a wheelchair, and now uses a bicycle and other means of transportation.

[0210] Example 4

[0211] This study was approved by the Ethics Committee. Patient 2, upon admission, was diagnosed with a history of exogenous coagulation factor transfusions, which resulted in the development of coagulation factor inhibitors, significantly hindering traditional treatment. The admission diagnosis was hemophilia A.

[0212] On April 10, 2023, patient 2 went to the outpatient department for genetic sequencing. After the sequencing results came out on April 30, 2023, the drugs sodium valproate and sirolimus (rapamycin) were used.

[0213] Medication regimen: Sodium valproate (500 mg / day), sirolimus (rapamycin) (1 mg / day)

[0214] Medication schedule: Sodium valproate (sodium valproate sustained-release tablets) twice a day (morning and afternoon), sirolimus (capsules) once a day (morning).

[0215] Route of administration: Oral.

[0216] The route of administration and dosage of the drug remained unchanged throughout the treatment cycle.

[0217] Patient 1 was treated according to the above medication regimen for 12 months.

[0218] The results of the coagulation factor VIII inhibitor test for patient 1 during the treatment period are recorded in the following Table 4:

[0219] Table 4

[0220] FIG13 is a graph showing the clearance effect of coagulation factor inhibitors in the body of patient 2 with severe hemophilia A during 5 months of using the sodium valproate-sirolimus combination.

[0221] Before treatment, Patient 2 experienced frequent bleeding in major joints. Even the slightest exertion or strenuous activity would cause bleeding and swelling. Most activities were unsuitable for him. He received no systematic treatment and his condition was intermittent. He received FVIII infusions whenever bleeding occurred.

[0222] After treatment with the kit disclosed herein: Patient 2 had basically no bleeding after taking the medicine. Sometimes, there was a little bleeding in the mouth when brushing teeth, but there was no bleeding in other joints or abdominal cavity.

[0223] Example 5

[0224] This study was approved by the ethics committee. In this example, the pharmaceutical composition was also used to conduct clinical trials on the following patients. The patient information and experimental results are shown in Table 5.

[0225] Table 5 Note: "Single drug" in the above table refers to the administration of sodium valproate (500 mg / d) twice a day (once in the morning and once in the afternoon).

[0226] In the above table, "combined" means that the administration method is the same as that in Example 3.

[0227] The term "subject" or "patient" refers to an animal, preferably a mammal, most preferably a human, that has been the object of treatment, observation or experiment. In any embodiment described herein, the subject can be a human.

[0228] The terms "treat," ...

[0229] Other embodiments of the present disclosure will readily occur to those skilled in the art after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present disclosure that follow the general principles of the present disclosure and include common knowledge or customary techniques in the art not disclosed herein. The description and examples are to be considered as exemplary only, with the true scope and spirit of the present disclosure being indicated by the following claims.

[0230] It should be understood that the present disclosure is not limited to the exemplary technical solutions described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from the scope thereof. Such modifications and changes should also fall within the scope of protection of the claims attached to the present disclosure.

Claims

1. A pharmaceutical composition for treating hemophilia, wherein: include: Histone deacetylase inhibitors and pharmaceutically acceptable carriers; Preferably, the histone deacetylase inhibitor includes one or more selected from the following, or a pharmaceutically acceptable salt thereof: valproic acid, vorinostat, romidepsin, belinostat, panobinostat; More preferably, the pharmaceutically acceptable salt of valproic acid exists in the form of a salt selected from the group consisting of sodium salt, potassium salt, magnesium salt, calcium salt or ammonium salt; for example, the sodium salt form of valproic acid includes hemi-sodium valproate or sodium valproate.

2. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition further comprises an mTOR inhibitor; The mTOR inhibitors include one or more of the following, or pharmaceutically acceptable salts thereof: sirolimus, everolimus, temsirolimus and ibrutinib.

3. The pharmaceutical composition according to claim 1 or 2, wherein The content of the histone deacetylase inhibitor in the pharmaceutical composition is 0.1g-1.0g, for example 0.3g-0.8g; For example, in the pharmaceutical composition, the content of sodium valproate is 0.1g-1.0g, such as 0.15g-0.45g; and / or The content of the mTOR inhibitor in the pharmaceutical composition is 0.2 mg-3 mg, for example 0.2 mg-3 mg; and / or The weight ratio of the histone deacetylase inhibitor to the mTOR inhibitor is 250:1 to 1200:

1.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The pharmaceutical composition is in the form of powder, tablet, lozenge, granule, capsule, suspension or emulsion.

5. A kit, wherein the kit comprises the pharmaceutical composition according to any one of claims 1 to 4.

6. Use of a histone deacetylase inhibitor or the pharmaceutical composition according to any one of claims 1 to 4 in the treatment of hemophilia.

7. Use of a histone deacetylase inhibitor or the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a medicament for treating hemophilia or in the preparation of a kit.

8. The use according to claim 7, wherein The medicament or kit is used to treat hemophilia or alleviate symptoms associated with hemophilia (such as bleeding or swelling caused by bleeding).

9. A method for treating hemophilia or alleviating symptoms associated with hemophilia (such as bleeding or swelling caused by bleeding), comprising administering a histone deacetylase inhibitor, the pharmaceutical composition according to any one of claims 1 to 4, or the kit according to claim 5 to a patient in need thereof.

10. The use according to claim 7 or 8 or the method according to claim 9, wherein The histone deacetylase inhibitor is administered twice a day; and / or The mTOR inhibitor is administered once a day.

11. The use according to claim 7 or 8 or the method according to claim 9, wherein The administration route of the pharmaceutical composition or kit is enteral administration.

Images