WO2025119368A1 - Pharmaceutical composition for treating hemophilia and use thereof - Google Patents

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

This application claims the priority of the 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 and invention name “Pharmaceutical composition and use for treating hemophilia”. The full text of the above-mentioned prior application is incorporated into this application by reference.

Technical Field

The present invention 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

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 (activity level of coagulation factor VIII or IX <1%) is characterized by repetitive joint bleeding and life-threatening bleeding, which imposes a great economic burden and life pressure on patients, their families and society.

Although hemophilia replacement therapy has greatly improved the quality of life of hemophilia patients, there are still many shortcomings: 1. Large-scale plasma transfusion not only does not play an effective hemostatic role, but will increase the patient's blood volume; 2. It is easy to induce antigen-antibody reaction; 3. Large-scale infusion is prone to induce venous, 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 infusion, and patient compliance is poor.

When hemophilia patients use replacement therapy, the occurrence of inhibitors is the main 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.

Therefore, there is an urgent need to develop new drugs or treatments to improve the treatment of hemophilia.

Summary of the invention

To improve the existing 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.

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.

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

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

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

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.

According to the embodiments of the present disclosure, the histone deacetylase inhibitors include but are not limited to one or more of the following, or pharmaceutically acceptable salts thereof: valproic acid, vorinostat, romidepsin, belinostat, panobinostat, such as valproate, for example, sodium valproate.

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.

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

According to an embodiment of the present disclosure, in the pharmaceutical composition, the content of the histone deacetylase inhibitor is 0.1g-1.0g, such as 0.3g-0.8g.

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.

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

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

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:

Sirolimus (also called Rapamycin), Everolimus, Temsirolimus, Ibrutinib.

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.

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.

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

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

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

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).

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

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.

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.

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

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

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

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

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.

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

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).

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.

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.

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

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

The mTOR inhibitor includes one or more selected from the following or pharmaceutically acceptable salts thereof: sirolimus, everolimus, temsirolimus and ibrutinib.

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

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 as described above, or the kit as described above to a patient in need thereof.

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.

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

The histone deacetylase inhibitor in the pharmaceutical composition of the present invention 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.

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

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.

FIG. 1 is a schematic diagram of the therapeutic effect of sodium valproate in treating hemophilia, shown in Example 1.

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

FIG3 is a volcano plot of differentially expressed genes in hemophilia patients compared with the normal control group.

FIG4 is a volcano plot of differentially expressed genes in patients after taking sodium valproate compared with before taking the drug.

FIG5 is a volcano plot of differentially expressed genes in patients after taking sirolimus compared with before taking the medicine.

FIG6 is a Veen diagram of differentially expressed genes between hemophilia patients and sodium valproate and sirolimus based on the principle of negative drug regulation.

FIG. 7 is a diagram showing that the sodium valproate-sirolimus combination is involved in regulating the Toll-like receptor signaling pathway to achieve immunosuppressive function.

FIG8 is a diagram showing activation reactions such as the involvement of the sodium valproate-sirolimus combination in regulating the platelet activation signaling pathway and affecting platelet shape changes and degranulation.

FIG. 9 shows that the sodium valproate-sirolimus combination participates in the vascular smooth muscle contraction signaling pathway to alleviate abnormal bleeding in hemophilia patients.

FIG. 10 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.

FIG. 11 is a graph showing the clearance effect of coagulation factor inhibitors in the body of severe hemophilia B patient 1 during the use of the sodium valproate-sirolimus combination for 12 months.

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.

FIG. 13 is a graph showing the clearance effect of coagulation factor inhibitors in the body of severe hemophilia A patient 2 during the use of the sodium valproate-sirolimus combination for 5 months.

DETAILED DESCRIPTION

Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are merely examples of compositions and methods consistent with some aspects of the present disclosure as detailed in the appended claims.

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 (coagulation factor VIII or IX activity level <1%) is characterized by repetitive joint bleeding and life-threatening bleeding, which imposes a great economic burden and life pressure on patients, their families and society.

Although hemophilia replacement therapy has greatly improved the quality of life of hemophilia patients, there are still many shortcomings, including: 1. Large-scale plasma transfusion not only fails to effectively stop bleeding, but increases the patient's blood volume; 2. It is easy to induce antigen-antibody reaction; 3. Large-scale infusion is prone to induce venous, 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 daily or every other day infusion, and patient compliance is poor.

When hemophilia patients use replacement therapy, the occurrence of inhibitors is the main 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 immune tolerance induction therapy (ITI) can be used to eradicate inhibitors after they are produced, 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.

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.

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

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

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).

Histone deacetylase inhibitors have anti-inflammatory, neuroprotective and anticonvulsant functions. It has been reported that histone deacetylase inhibitors can regulate stem cell differentiation and self-renewal. In addition to anti-epileptic, the drug can also be used to treat febrile convulsions, movement disorders, chorea, porphyria, schizophrenia, pain caused by herpes zoster, adrenal dysfunction, and prevent alcohol withdrawal syndrome. Oral absorption is fast and complete, mainly distributed in extracellular fluid, and most of it is bound to plasma proteins in the blood.

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.

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.

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

According to embodiments of the present disclosure, the content of histone deacetylase inhibitor in the pharmaceutical composition is 0.1g-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.

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, such as 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.

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, and its components are: each tablet contains about 0.333g of sodium valproate and about 0.145g of valproic acid (equivalent to 0.5g of sodium valproate).

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

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.

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

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.

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

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. The 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. The protein serves as a target for cell cycle arrest and immunosuppression of the FKBP12-mTOR inhibitor complex.

In this context, mTOR inhibitors are a class of compounds used to inhibit the activity of the mTOR signaling pathway and are often 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 as it has been shown to regulate lifespan and maintain normal glucose homeostasis. It is often used to prevent rejection in organ transplants.

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 replacement therapy for a long time are prone to coagulation factor inhibitors, mTOR inhibitors are included in the regimen.

According to the embodiments of the present disclosure, histone deacetylase inhibitors can maintain the integrity of the blood vessel wall, activate platelets, and promote platelet aggregation. mTOR inhibitors can effectively reduce the concentration of inhibitors in hemophilia patients. The combined use of the two drugs can benefit patients from maintaining the integrity of the blood vessel wall, promoting coagulation and eliminating inhibitors, thereby effectively preventing and treating bleeding in hemophilia patients and achieving the purpose of treatment. Even some existing embodiments of patients have been separated from the transfusion of coagulation factors and gradually recovered.

According to an embodiment of the present disclosure, the pharmaceutical composition may include about 100 mg/d to about 2000 mg/d (such as 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 (such as 0.5 mg/d to about 2 mg/d) of an mTOR inhibitor. The administration of the histone deacetylase inhibitor-mTOR inhibitor composition significantly reduces the concentration of coagulation factor inhibitors in the subject compared to the concentration before administration, and abnormal bleeding is effectively alleviated.

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

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.

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.

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

According to the embodiments of the present disclosure, the pharmaceutical composition can be in the form of powder, tablet, lozenge, granule, capsule, suspension or emulsion. The pharmaceutical carrier of the pharmaceutical composition can be obtained according to the known compounds and known methods used in the relevant art to realize the drug. For example, it can be the solid carrier mentioned above, which will not be repeated here.

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 a histone deacetylase inhibitor and an mTOR inhibitor to treat hemophilia.

The present invention includes a kit suitable for implementing the above-mentioned treatment methods. In one embodiment, the kit includes a first dosage form and a second dosage form for simultaneous administration, and their amounts are sufficient to implement the method of the present disclosure, i.e., for the treatment of hemophilia. The first dosage form contains one or more of the above-mentioned determined forms of histone deacetylase inhibitors, and the second dosage form contains one or more of the above-mentioned determined mTOR inhibitors.

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 the histone deacetylase inhibitor is 0.4g-0.6g. For example, according to embodiments of the present disclosure, the content of the histone deacetylase inhibitor is 0.45g-0.55g. For example, according to embodiments of the present disclosure, the content of the histone deacetylase inhibitor is about 0.25g. For example, according to embodiments of the present disclosure, the content of the histone deacetylase inhibitor is about 0.5g. For example, according to embodiments of the present disclosure, the content of the histone deacetylase inhibitor is about 0.6g.

According to an embodiment of the present disclosure, the content of the mTOR inhibitor in the kit is 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. The content of the mTOR inhibitor can be 0.2 mg-3 mg. For example, according to an embodiment of the present disclosure, the content of the mTOR inhibitor can be 0.5 mg-2 mg. For example, according to an embodiment of the present disclosure, the content of the mTOR inhibitor can be 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.

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.

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

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, panobinostat; and/or the mTOR inhibitor includes one or more of the following: sirolimus, rapamycin, everolimus, temsirolimus.

According to the embodiments 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.

At present, the treatment of hemophilia (whether hemophilia A or hemophilia B) is to use exogenous plasma infusion or recombinant coagulation factor therapy based on gene editing technology. Although these methods are effective, they also have great side effects (the production of coagulation factor inhibitors greatly affects the efficacy). Although scientists are currently studying methods to remove inhibitors, only a few are effective. In addition, these treatments are expensive.

The present invention proposes for the first time a scheme for treating this disease with a small molecule compound, and reveals the synergistic effect of the sodium valproate-sirolimus combination in the mechanism of treating the disease, providing innovative ideas and strong support for future disease treatment. This research result is not only of guiding significance for the optimization of current disease treatment, but also opens up a new research direction for the development of more effective treatment strategies. In addition, the use of the pharmaceutical composition and kit provided by the present disclosure to treat hemophilia has a low treatment cost.

According to an embodiment of the present disclosure, the mTOR inhibitor refers to a mammalian rapamycin target protein inhibitor, which includes one or more of the following: Sirolimus [also known as Rapamycin], Everolimus, Temsirolimus, and Ibrutinib.

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.

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.

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

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.

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. The composition for oral use can be prepared according to any method for preparing a pharmaceutical composition known in the art.

The solid carrier can be one or more 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.

According to an embodiment of the present disclosure, in powders, the carrier is a finely divided solid, which 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 necessary binding properties in suitable proportions and compressed into the desired shape and size.

According to an embodiment 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.

According to the embodiment of the present disclosure, the present disclosure also includes metformin and statin and the preparation prepared by using capsule forming material as carrier, to provide metformin and statin (with or without other carriers) by the capsule coated by carrier, so that the carrier is combined with metformin and statin. In a similar manner, it also includes bags, tablets, powders, bags and capsules can be used as solid dosage forms suitable for oral administration.

According to the embodiments of the present disclosure, the tablets may not be coated or may be coated by known techniques to delay the disintegration and absorption of the drug in the gastrointestinal tract, and thus provide a sustained effect over a long period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used.

According to an embodiment of the present disclosure, preparations for oral administration may 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).

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.

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

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, flavoring agents, and sweeteners.

According to the embodiments 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 thickener such as beeswax, hard paraffin and cetyl alcohol.

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

According to an embodiment of the present disclosure, a syrup composition containing the novel combination can be prepared using a sweetener. The formulation can also include a demulcent, a preservative, and a flavoring and coloring agent.

According to embodiments of the present disclosure, liquid preparations 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 appropriate flavorings, coloring agents, stabilizers and thickeners as required. 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 ground active compounds and other suspending agents such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other known in the field of pharmaceutical preparations in water.

According to embodiments of the present disclosure, it is preferred that the pharmaceutical formulation is 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, the package containing the divided amount of the formulation is, for example, packaged tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or an appropriate number of any of these packaged forms.

According to the embodiments 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 technology. The suspension can be prepared according to known techniques using the above-mentioned appropriate dispersants, wetting agents and suspending agents or other acceptable substances. 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 non-volatile oils are conventionally used solvents or suspension media. For this purpose, any non-volatile oil with low irritation, including synthetic monoglycerides or diglycerides, can be used. In addition, n-3 polyunsaturated fatty acids are also useful in injectable preparations.

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 an appropriate non-irritating excipient, which is solid at room temperature but liquid at rectal temperature, and thus melts in the rectum to release the drug. The material can be cocoa butter and polyethylene glycol.

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

According to the embodiments of the present disclosure, the daily dose can vary within wide limits and be adjusted according to individual needs in each particular case. Generally, for adult administration, the appropriate daily dose has been described above: but if it is administered as divided doses. For emergency measures, the limits determined as preferred can be exceeded. The daily dose can be administered as a single dose or divided metering.

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:

Sirolimus: 1 mg/day

Sodium valproate: 500 mg/day

Sodium valproate:

The daily dose for mice is 2 mg, and the daily dose for adults is 500 mg.

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

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

Sirolimus:

Mice were given 0.04 mg (concentration: 1 mg/ml) daily.

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

Patient QY: height 190cm, weight 100kg

DuBois formula (body surface area calculation BSA) BSA = 0.007184 × 1900.725 × 1000.425 = 2.329 ^m2

Mouse: body weight 20g

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

BSA ratio: 0.128/2.329=0.055

Sirolimus: 1×0.055=0.055 mg

Sodium valproate: 500×0.055=27.5 mg.

Example 1

In the following Example 1, taking sodium valproate as an example of a histone deacetylase inhibitor, the drug 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.

Experimental design: Before the experiment, a vernier caliper was used to measure the diameter of the right knee joint of each mouse (supine position); then a knee joint injury model of hemophilia B mice was constructed, and the experimental group was intervened with sodium valproate 24 hours later, while the model control group was not intervened with drugs, and the degree of knee joint swelling was recorded to evaluate the treatment effect.

Establishment of mouse knee joint injury model: FIX Exon 1-3KO mice were used as hemophilia B mouse models. A 0.8*35mm injection needle was used to perform a brief puncture at the patella of the right knee joint of the mouse. Two hours before the puncture, each mouse was subcutaneously injected with buprenorphine for analgesia, and then buprenorphine was added to the drinking water of the mice within 1 week to prevent the mice from dying due to pain after the injury.

Experimental time point: The right knee joint diameter of the mice was recorded before and 24 hours after the puncture injury, and then the experimental group began drug treatment, once a day. After one week of treatment, the right knee joint diameter of the mice was measured again to evaluate the treatment effect. The dosing regimen for each group is as follows:

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

【Control group】was given an equal volume of normal saline.

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

Experimental results: The diameter of the right knee joint of mice was measured by vernier caliper at different time points after sodium valproate administration. The specific test results are shown in Table 1.

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

According to the data in Table 1 above, 24 hours after the puncture injury, the knee joint diameters of the two groups of mice increased significantly. After one week of treatment, the knee joint diameters of the mice treated with sodium valproate decreased significantly, indicating that sodium valproate has a significant therapeutic effect on joint damage in the hemophilia B mouse model.

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 diameters at different time points. Figure 1 is a schematic diagram of the therapeutic effect of sodium valproate for the treatment of 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).

Example 2A

In the following Example 2A, taking sodium valproate as a histone deacetylase inhibitor and sirolimus as an mTOR inhibitor as examples, 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.

Experimental design: Before the experiment, a vernier caliper was used to measure the diameter of the right knee joint of each mouse (supine position); then a knee joint injury model of hemophilia B mice was constructed, and the drug composition was used for drug intervention 24 hours later, and the degree of knee joint swelling was recorded to evaluate the therapeutic effect.

Establishment of mouse knee joint injury model: FIX Exon 1-3KO mice were used as hemophilia B mouse models. A 0.8*35mm injection needle was used to perform a brief puncture at the patella of the right knee joint of the mouse. Two hours before the puncture, each mouse was subcutaneously injected with buprenorphine for analgesia, and then buprenorphine was added to the drinking water of the mice within 1 week to prevent the mice from dying due to pain after the injury.

Experimental time point: The right knee joint diameter of the mice was recorded before and 24 hours after the puncture injury, and then the drug treatment was started, with medication administered once a day. After one week of treatment, the right knee joint diameter of the mice was measured again to evaluate the therapeutic effect. The medication regimen for each group is as follows:

[Drug combination group] Doses: sirolimus (40 μL, concentration 1 mg/ml) and sodium valproate (50 μL, concentration 40 mg/ml);

【Control group】was given an equal volume of normal saline.

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

Experimental results: The diameter of the right knee joint of the mice at different time points was measured with a vernier caliper and recorded. The results are shown in Table 2A:

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

Example 2B

In the following Example 2B, taking sodium valproate as a histone deacetylase inhibitor and sirolimus as an mTOR inhibitor as examples, 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.

Experimental design: Before the experiment, a vernier caliper was used to measure the diameter of the right knee joint of each mouse (supine position); then a knee joint injury model of hemophilia B mice was constructed, and the drug composition was used for drug intervention 24 hours later, and the degree of knee joint swelling was recorded to evaluate the therapeutic effect.

Establishment of mouse knee joint injury model: FIX Exon 1-3KO mice were used as hemophilia B mouse models. A 0.8*35mm injection needle was used to perform a brief puncture at the patella of the right knee joint of the mouse. Two hours before the puncture, each mouse was subcutaneously injected with buprenorphine for analgesia, and then buprenorphine was added to the drinking water of the mice within 1 week to prevent the mice from dying due to pain after the injury.

Experimental time point: The right knee joint diameter of the mice was recorded before and 24 hours after the puncture injury, and then the drug treatment was started, with medication administered once a day. After one week of treatment, the right knee joint diameter of the mice was measured again to evaluate the therapeutic effect. The medication regimen for each group is as follows:

[Drug combination group] Doses: sirolimus (40 μL, concentration 1 mg/ml) and sodium valproate (50 μL, concentration 40 mg/ml);

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

【Control group】was given an equal volume of normal saline.

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

Experimental results: The diameter of the right knee joint of the mice at different time points was measured with a vernier caliper and recorded. The results are shown in Table 2B:

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

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

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 diameters 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 for measuring the diameter of the right leg knee joint of the mouse, and the vertical axis represents the measured value of the diameter of the right leg 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).

According to the pharmaceutical composition disclosed in the present invention, the histone deacetylase inhibitor can activate platelets and promote blood coagulation, thereby improving the recurrent bleeding condition of hemophilia patients.

The present invention utilizes the disease gene expression profile data characteristic of hemophilia, and determines the drug based on the Connectivity Map, L1000 public database and EpiMed platform analysis based on the "logic omics" inventor's disease-gene association principle.

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

Method steps:

The hemophilia disease expression profile data obtained in the early stage of the present invention was used, and the edgeR software package was used to screen the differentially expressed genes in the expression profile. The screening criteria for differentially expressed genes were FDR < 0.05, |Log2FC| > 1, and the differentially expressed genes of hemophilia were obtained. Then, the Connectivity Map, L1000 public database and the theoretical algorithms based on "system biology" and "comparative functional genomics" were used to perform multi-omics association analysis on the characteristic expression genes of hemophilia patients to predict potential regulatory drugs; then, the potential therapeutic drugs were determined by comprehensive consideration of the pharmacodynamics and drug toxicity and side effects.

The transcriptome expression data of histone deacetylase inhibitors and mTOR inhibitors obtained in the early stage of the present invention were used to screen differentially expressed genes using the GEO2R online analysis tool. The screening criteria for differential genes were FDR < 0.05, |Log2FC| > 1, and differentially expressed genes before and after the administration of histone deacetylase inhibitors and mTOR inhibitors were obtained. The above two groups of differential expression analysis results were jointly analyzed to determine the intersection genes and perform KEGG enrichment analysis.

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 histone deacetylase inhibitors have similar chemical structures and functions, and the drugs in mTOR inhibitors have similar chemical structures and functions; therefore, sodium valproate (histone deacetylase inhibitor) and sirolimus (also known as: rapamycin, mTOR inhibitor) are preferred for clinical trials.

Figure 3 is a volcano map of differentially expressed genes in hemophilia patients compared with the normal control group. Figure 4 is a volcano map of differentially expressed genes in patients after taking sodium valproate compared with before taking the drug, and Figure 5 is a volcano map of differentially expressed genes in patients after taking sirolimus compared with before taking the drug.

In Figures 3 to 5, Up means "up-regulated gene", Down means "down-regulated gene", Stable means "stable gene (gene with no difference)", log2(fold change) means "log2(differential expression change)", and -log10(PValue) means "-log10(P value)".

Figure 6 is a Veen diagram of differentially expressed genes between hemophilia patients and sodium valproate and sirolimus based on the principle of drug negative regulation. In Figure 6, Hemophilia means hemophilia, Drug combination means drug combination, up and down respectively mean up-regulated genes and down-regulated genes.

Figure 7 is a diagram showing that the sodium valproate-sirolimus combination participates in regulating the Toll-like receptor signaling pathway to achieve immunosuppressive function. Figure 8 is a diagram showing that the sodium valproate-sirolimus combination participates in regulating the platelet activation signaling pathway to affect platelet shape changes, degranulation and other activation reactions. Figure 9 is a diagram showing that the sodium valproate-sirolimus combination participates in the vascular smooth muscle contraction signaling pathway to alleviate abnormal bleeding in hemophilia patients. Figure 10 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.

[Corrected 06.03.2025 in accordance with Rule 26]
In Figures 7 to 10, red indicates upregulation of a gene, and green indicates downregulation of a gene. Indicates upregulation of genes, Indicates down-regulation of the gene.

In Figures 7 to 10, TOLL LIKE RECEPTOR SIGNALING PATHWAY represents Toll-like receptor signaling pathway, Peptidoglycan (G+) represents peptidoglycan (G+), Lipoprotein represents lipoprotein, Lipoarabinomannan represents lipoarabinomannan, Mycobactena represents Mycobacterium, Zymosan (Yeast) represents zymosan (yeast), Lipopolysaccharide represents lipopolysaccharide, biosynthesis represents biosynthesis, Flagellar assembly represents flagellar assembly, Flagellin represents flagellar protein, Imidazoquinolin represents imidazoquinolin, anti-viral compounds represent antiviral compounds, ssRNA represents single-stranded RNA, Unmethylated represents unmethylated, PI3K-Akt signaling pathway represents PI3K-Akt signaling pathway, Ubiquitin mediated Proteolysis represents ubiquitin-mediated proteolysis, Inflammatory cytokines represent inflammatory cytokines, and Proinflammaory Effects indicates proinflammatory effects, Chemotactic effects indicates chemotactic effects, Nutrophil, Immnature DC indicates neutrophil, immature dendritic cell, NK cell indicates NK cell, Complement and coagulation cascade indicates complement and coagulation cascade, Costimulatory molecules indicates costimulatory molecules, T cell stimulation indicates T cell stimulation, Inflammatory cytokines indicates inflammatory cytokines, Antiviral effects indicates antiviral effects, cytokine receptor interaction indicates cytokine receptor interaction, Chemotactic effects (Tcell) indicates T cell chemotactic effects, ECM-receptor interaction indicates ECM-receptor interaction, PI3K-Akt signaling pathway indicates PI3K-Akt signaling pathway, Calcium signaling pathway indicates calcium signaling pathway, Rapl signaling pathway indicates Rapl signaling pathway, Arachidonic acid metabolism indicates arachidonic acid metabolism, Complement and coagulation cascade indicates complement and coagulation cascade, VASCULAR SMOOTH MUSCLE CONTRACTION=vascular smooth muscle contraction, Vascular smooth muscle cell=vascular smooth muscle cell, Arachidonic acid metabolism=arachidonic acid metabolism, Intravascual pressure/Stretch=intravascual pressure/stretch, Calcium signaling pathway=calcium signaling pathway, Sarcoplasmic reticulum(SR)=sarcoplasmic reticulum(SR), Reduction of contractile system Ca ⁺ sensitivity=reduction of contractile system Ca ⁺ sensitivity, Myosin=myosin, Extrinsic pathway=extrinsic pathway, Tissue damage=tissue damage, Intrinsic pathway=intrinsic pathway, Contact with damaged vessel=contact with damaged vessel, Kallikrein-kinin system=kallikrein-kinin system, Fibrin monomer=fibrin monomer, Cross-linked fibrin polymer=cross-linked fibrin polymer, Anti-inflammatory responses, vasodilation increased endothelial permeability release of Weibel-Palade bodies=anti-inflammatory response, vasodilators reduce release of endothelial membrane of Webel-Palade bodies, Fibrin degradation products indicates fibrin degradation products, Cell adhesion, migration, proliferation indicates cell adhesion, migration and proliferation, Inflammatory mediator regulation of TRP channels indicates the regulation of tryptophan channels by inflammatory mediators, Classical pathway indicates classical pathway, Antigen-antibody complex anti-n-antibody complex, Lectin pathway indicates lectin pathway, Carbohyrate indicates carbohydrate Alternative 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.

According to the kit provided by the present disclosure, when the patient is admitted to the hospital for diagnosis, peripheral blood is drawn for transcriptome sequencing, and the differentially expressed genes of the patient compared with normal people are explored through bioinformatics methods. The present invention successfully integrates patient data and drug transcriptome data, revealing important evidence of the synergistic effect of the two drugs in the mechanism of treating specific diseases.

First, after precise and detailed research, the present disclosure found that sirolimus (rapamycin) plays a role in clearing coagulation factor inhibitors mainly through immunomodulatory effects. Clearing coagulation factor inhibitors is essential for maintaining normal coagulation function, and the effect of sirolimus (rapamycin) strongly promotes this process.

At the same time, sodium valproate has a unique contribution in treatment. Sodium valproate has been shown to effectively maintain the integrity of blood vessel walls and activate platelets, thereby promoting blood coagulation. This discovery provides us with a completely new treatment approach that 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 sodium valproate provides a biological basis for its unique effect in treatment.

Through a comprehensive analysis of the combined use of sodium valproate and sirolimus (rapamycin), it was found that there is a significant synergistic effect between the two drugs. Sirolimus (rapamycin) removes coagulation factor inhibitors, providing a more favorable environment for sodium valproate to more fully activate platelets. This synergistic effect not only improves the therapeutic effect, but also reduces possible side effects, laying the foundation for individualized and precise disease treatment.

The present invention successfully reveals the synergistic effect of the sodium valproate-sirolimus combination in the treatment mechanism of diseases, providing innovative ideas and strong support for future disease treatment. This research result not only has guiding significance for the optimization of current disease treatment, but also opens up a new research direction for the development of more effective treatment strategies.

Example 3

This study was approved by the Ethics Committee. Patient 1 was diagnosed with hemophilia B upon admission and had a history of exogenous coagulation factor infusion. Coagulation factor inhibitors were produced in the body, which greatly hindered traditional treatment methods.

Medication regimen: sodium valproate (500 mg/d), sirolimus (rapamycin) (1 mg/d)

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

Route of administration: Oral.

During the entire treatment cycle, the route of administration and dosage of the drug remained unchanged.

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

Clinical manifestations: Patient 1, the patient's FIX inhibitor decreased from 4.8BU/ml to 0.6BU/ml, and the HAL scale score increased from 127 points to 187 points. After 12 months of treatment, the patient's knee swelling symptoms were significantly reduced. The patient improved from needing to rely on a wheelchair to being able to stand and walk without a wheelchair. Currently, the patient can use bicycles and other means of transportation to travel.

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

Table 3

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

Figure 11 is a graph showing the effect of blood coagulation factor inhibitor clearance in the body of severe hemophilia B patient 1 within 12 months of using the sodium valproate-sirolimus composition. Figure 12 is a graph showing the improvement of the knee joint of severe hemophilia B patient 1 after using the sodium valproate-sirolimus composition for 12 months.

As shown in Figures 11 and 12, after 12 months of treatment, the knee swelling symptoms of Patient 1 were significantly alleviated, and the number of bleeding injuries was significantly reduced. Patient 1 improved from being dependent on a wheelchair to being able to stand and walk without a wheelchair, and is now able to use bicycles and other means of transportation to travel.

Example 4

This study was approved by the Ethics Committee. Patient 2 was diagnosed with a history of exogenous coagulation factor infusion during admission. Coagulation factor inhibitors were produced in the body, which greatly hindered traditional treatment methods. The admission diagnosis was (hemophilia A).

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.

Medication regimen: sodium valproate (500 mg/d), sirolimus (rapamycin) (1 mg/d)

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

Route of administration: Oral.

During the entire treatment cycle, the route of administration and dosage of the drug remained unchanged.

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

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

Table 4

FIG. 13 is a graph showing the clearance effect of coagulation factor inhibitors in the body of severe hemophilia A patient 2 during the use of the sodium valproate-sirolimus combination for 5 months.

Patient 2 before treatment: All the large joints in the body were prone to bleeding. The slightest fatigue or heavy activity would cause bleeding and swelling. Most activities were not suitable for him. He did not receive systematic treatment and his condition was intermittent. He was infused with FVIII when bleeding occurred.

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 and abdominal cavity.

Example 5

This study was approved by the Ethics Committee. In this example, the pharmaceutical composition was used to conduct relevant clinical experiments on the following patients. The patient's relevant conditions and experimental results are shown in Table 5 below.

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).

In the above table, "combination" means that the administration method is the same as that in Example 3.

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.

The term "treat" as used herein means to reverse, alleviate, inhibit the progression of, or prevent the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

Those skilled in the art will readily appreciate other embodiments of the present disclosure 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 that are not disclosed in the present disclosure. The specification and examples are intended to be exemplary only, and the true scope and spirit of the present disclosure are indicated by the following claims.

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

Claims (11)

A pharmaceutical composition for treating hemophilia, comprising: a histone deacetylase inhibitor and a pharmaceutically acceptable carrier; 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. 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. 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. 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. A kit, wherein the kit comprises the pharmaceutical composition according to any one of claims 1 to 4. Use of a histone deacetylase inhibitor or the pharmaceutical composition according to any one of claims 1 to 4 in the treatment of hemophilia. Use of a histone deacetylase inhibitor or the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for treating hemophilia or in the preparation of a kit. The use according to claim 7, wherein the medicine or kit is used to treat hemophilia or alleviate symptoms associated with hemophilia (such as bleeding or swelling caused by bleeding). 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 according to any one of claims 1 to 4, or the kit according to claim 5 to a patient in need thereof. 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. 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.