Preparation for treating cardiovascular diseases and its application and preparation method

Abstract

The present invention discloses a preparation for treating cardiovascular disease and its application and preparation method, wherein the preparation for treating cardiovascular disease comprises the following active ingredients: trichostatin A; and N-suberoylanilide hydroxamic acid. The preparation for treating cardiovascular disease of the present invention can have a more significant therapeutic effect on heart failure through its unique dual active ingredients, trichostatin A and N-suberoylanilide hydroxamic acid, and the preparation can not only effectively alleviate cardiac diastolic dysfunction in heart failure, but also treat and optimize insulin resistance in HFpEF; in addition, it can also significantly reduce the level of ceramide in cardiac tissue, thereby improving mitochondrial functional damage; in addition, the preparation can also treat and improve mitochondrial edema and structural disorder in heart failure, increase the activity of enzyme complexes, and improve cardiac fibrosis associated with heart failure.

Description

Technical Field

[0001] The present invention belongs to the technical field of cardiovascular disease treatment, and specifically relates to a preparation for treating cardiovascular disease and an application and a preparation method thereof. Background Art

[0002] Cardiovascular disease, especially heart failure, is a serious symptom of cardiovascular disease. The pathological mechanism of heart failure is complex, including cardiac diastolic dysfunction, insulin resistance, mitochondrial damage and cardiac fibrosis. Existing treatments have certain limitations in improving the symptoms of heart failure, especially in the treatment of heart failure with preserved ejection fraction (HFpEF). Summary of the invention

[0003] The purpose of the present invention is to provide a new technical solution for a preparation for treating cardiovascular diseases, which can at least solve the technical problem that existing drugs are not effective in treating heart failure.

[0004] The present invention provides a preparation for treating cardiovascular diseases, the preparation comprising the following active ingredients:

[0005] Trichostatin A;

[0006] and N-suberoylanilide hydroxamic acid.

[0007] Optionally, the mass content of the trichostatin A is less than or equal to the mass content of the N-suberylanilide hydroxamic acid.

[0008] Optionally, the preparation further comprises a solvent and an adjuvant, wherein the adjuvant comprises one or more of a co-solvent, an emulsifier, a stabilizer and a solubilizer.

[0009] Optionally, the solvent is water, the auxiliary solvent is polyethylene glycol 300, the emulsifier is Tween 80, the stabilizer is β-cyclodextrin, and the solubilizer is glycerol.

[0010] Optionally, the preparation comprises, by weight:

[0011] 10 to 30 parts of the trichostatin A, 10 to 30 parts of the N-suberylanilide hydroxamic acid, 275 to 550 parts of the auxiliary solvent, 250 to 500 parts of the emulsifier, 5 to 30 parts of the stabilizer, 5 to 30 parts of the solubilizer, and the solvent.

[0012] Optionally, the preparation comprises, by weight:

[0013] 10 to 20 parts of the trichostatin A, 20 to 30 parts of the N-suberylanilide hydroxamic acid, 275 to 550 parts of the auxiliary solvent, 250 to 500 parts of the emulsifier, 5 to 15 parts of the stabilizer, 5 to 15 parts of the solubilizer, and the solvent.

[0014] Optionally, the preparation comprises, by weight:

[0015] 10 to 20 parts of the trichostatin A, 20 to 30 parts of the N-suberylanilide hydroxamic acid, 275 to 550 parts of the auxiliary solvent, 250 to 500 parts of the emulsifier, 15 parts of the stabilizer, 15 parts of the solubilizer, and the solvent.

[0016] Optionally, the content of trichostatin A is 10 mg~30 mg, the content of N-suberylanilide hydroxamic acid is 10 mg~30 mg, the content of the auxiliary solvent is 275 mg~550 mg, the content of the emulsifier is 250 mg~500 mg, the content of the stabilizer is 15 mg~30 mg, the content of the solubilizer is 15 mg~30 mg, and the balance is the solvent.

[0017] Optionally, in every 5 ml of the preparation, the content of trichostatin A is 10 mg~30 mg, the content of N-suberylanilide hydroxamic acid is 10 mg~30 mg, the content of the auxiliary solvent is 275 mg~550 mg, the content of the emulsifier is 250 mg~500 mg, the content of the stabilizer is 5 mg~15 mg, the content of the solubilizer is 5 mg~15 mg, and the balance is the solvent.

[0018] Optionally, the sum of the mass parts of the N-suberylanilide hydroxamic acid and the trichostatin A is 20 to 40 parts.

[0019] Optionally, in every 5 ml of the preparation, the content of trichostatin A is 20 mg, the content of N-suberylanilide hydroxamic acid is 20 mg, the content of the auxiliary solvent is 550 mg, the content of the emulsifier is 500 mg, the content of the stabilizer is 15 mg, the content of the solubilizer is 15 mg, and the balance is the solvent.

[0020] Optionally, the preparation is configured as an injection.

[0021] According to a second aspect of the present invention, there is provided a use of any one of the above-mentioned preparations in the preparation of a drug for treating cardiovascular diseases.

[0022] According to a third aspect of the present invention, there is provided a method for preparing any one of the above-mentioned preparations, the method comprising:

[0023] dissolving the active ingredient in a solvent to obtain a first mixed solution;

[0024] adding a solubilizing agent to the first mixed solution and shear mixing the mixture to obtain a second mixed solution;

[0025] The second mixed solution is homogenized to obtain the preparation.

[0026] Optionally, the step of dissolving the active ingredient in a solvent to obtain a first mixed solution comprises:

[0027] mixing the active ingredient with a solvent, a co-solvent, an emulsifier and a stabilizer to obtain a mixture;

[0028] The mixture is heated and subjected to ultrasonic treatment to obtain a first mixed solution.

[0029] According to the preparation for treating cardiovascular diseases of the present invention, through its unique dual active ingredients, trichostatin A and N-suberylanilide hydroxamic acid, it can have a more significant therapeutic effect on heart failure, especially heart failure with preserved ejection fraction. The preparation can not only effectively alleviate cardiac diastolic dysfunction in heart failure, but also treat and optimize insulin resistance in HFpEF; in addition, it can also significantly reduce the level of ceramide in cardiac tissue, thereby improving mitochondrial functional damage; in addition, the preparation can also treat and improve mitochondrial edema and structural disorder in heart failure, increase the activity of enzyme complexes, and improve cardiac fibrosis associated with heart failure.

[0030] Further features and advantages of the present invention will become apparent from the following detailed description of exemplary embodiments of the present invention with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0032] Figure 1 This is a schematic diagram of verifying the toxicity of different formulations of the preparation for treating cardiovascular diseases (i.e., TS001) according to the embodiment of the present invention in primary neonatal rat cardiomyocytes. CCK-8 kit indicates the cytotoxicity of different formulations by cell activity.

[0033] Figure 2 TS001 of the present invention shows a more significant effect in treating heart failure than TSA (ie, trichostatin A) and SAHA (ie, N-suberylanilide hydroxamic acid). Figure 2 -A is a schematic diagram representing the cardiac contractile function. Figure 2 -B and Figure 2 -C is a marker of cardiac diastolic function ( Figure 2 -C, lung weight / tibia length is an indicator of pulmonary congestion, and a decrease in the ratio indicates improved cardiac diastolic function) Schematic diagram. Statistical data: Mean±SEM. Statistical method: One-way ANOVA. ***p<0.001.

[0034] Figure 3 TS001 of the present invention can improve cardiac diastolic dysfunction caused by HF. Figure 3 -A From top to bottom, the echocardiogram Figure 2 Characteristic images of cusp flow Doppler and mitral valve tissue Doppler. Figure 3 -B is a schematic diagram representing the mitral valve E peak blood flow velocity / A peak blood flow velocity. Figure 3 -C is a schematic diagram representing the mitral valve E peak blood flow velocity / mitral valve e' tissue movement velocity; Figure 3 -D represents the overall strain %, and the increase in absolute value indicates the improvement of cardiac function. Statistical data: Mean±SEM. Statistical method: One-way ANOVA. ***p<0.001, ****p<0.0001.

[0035] Figure 4 TS001 treatment according to an embodiment of the present invention can improve insulin resistance in HF. Figure 4 -A is a schematic diagram showing that TS001 treatment can improve fasting blood glucose in HF mice. Figure 4 -B is a schematic diagram showing that TS001 treatment can improve insulin resistance in HF mice (IPGTT experiment). Statistical data: Mean±SEM. Statistical method: Two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0036] Figure 5 It is a schematic diagram showing that TS001 treatment according to an embodiment of the present invention can inhibit ceramide levels in cardiac tissue and improve mitochondrial function damage. Figure 5 -A is a schematic diagram showing that ELISA detection indicates that TS001 can inhibit the level of ceramide in mouse heart tissue (ELISA method). Figure 5 -B is a schematic diagram representing the results of the Seahorse experiment (suggesting that TS001 significantly improves mitochondrial function). Figure 5 -C is a schematic diagram showing that TS001 does not improve the basal respiratory rate of cardiomyocytes. Figure 5-D is a schematic diagram showing that TS001 can improve the maximum respiratory rate of HF. Statistical data: Mean±SEM. Statistical method: Two-way ANOVA. *p<0.05, ****p<0.0001;

[0037] Figure 6 It is a schematic diagram of TS001 in an embodiment of the present invention for treating and improving mitochondrial edema and structural disorder in HF and improving enzyme complex activity. Figure 6 -A is a schematic diagram characterizing the morphological manifestations of cardiomyocyte mitochondria (electron microscopy). Figure 6 -B is a schematic diagram showing that TS001 can improve the activity of oxidative respiratory chain enzyme complex I when pyruvate is used as a substrate. Figure 6 -C is a schematic diagram showing that TS001 can improve the activity of oxidative respiratory chain enzyme complex I when palmitic acid is used as a substrate. Statistical data: Mean±SEM. Statistical method: Two-way ANOVA. *p<0.05.

[0038] Figure 7 This is a schematic diagram showing that TS001 of the present invention can inhibit the transcriptional expression of ceramide synthesis metabolism-related enzymes (RT-qPCR). Statistical data: Mean±SEM. Statistical method: Two-way ANOVA. *p<0.05, ***p<0.001;

[0039] Figure 8 TS001 treatment of an embodiment of the present invention does not improve myocardial hypertrophy in HF. Figure 8 -A is a cardiac tissue pathology staining image, specifically, the upper part is a hematoxylin-eosin (HE) staining image, and the lower part is a wheat germ agglutinin (WGA) immunofluorescence staining image. bar: 50 um. Figure 8 -B is a statistical diagram of the cross-section of cardiomyocytes under WGA staining conditions. Statistical data: Mean±SEM. Statistical method: Two-way ANOVA. ****p<0.0001;

[0040] Fig. 9 TS001 treatment of an embodiment of the present invention does not improve myocardial hypertrophy in HF. Fig. 9 -A is a schematic diagram representing the characteristic exposure bands of Western Blot. Fig. 9 -B is a schematic diagram showing the statistical data of relative expression levels of myocardial hypertrophy-related proteins. Fig. 9 -C is a schematic diagram of the relative expression of mRNA of myocardial hypertrophy-related factors. Statistical data: Mean±SEM. Statistical method: Two-way ANOVA. *p<0.05, ****p<0.0001;

[0041] Fig.10 This is a schematic diagram of TS001 in an embodiment of the present invention for treating and improving myocardial fibrosis in HF. Fig.10 -A is a cardiac tissue pathology staining image, specifically, the upper part is a PSR (Picro Sirius Red Stain) staining image, and the lower part is a Masson (Masson's Trichrome Stain) staining image. bar: 50 um. Fig.10 -B is the statistical data of myocardial tissue fibrosis under PSR and MT staining conditions. Fig.10 -C is the statistical data of relative expression level of mRNA of fibrosis-related factors. Statistical data: Mean±SEM. Statistical method: Two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0043] The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.

[0044] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered as part of the specification.

[0045] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0046] It should be noted that like reference numerals and letters refer to similar items in the following figures, and therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0047] An embodiment of the present invention provides a preparation for treating cardiovascular diseases, the preparation comprising the following active ingredients: trichostatin A; and N-suberanoylanilide hydroxamic acid.

[0048] In this embodiment, the preparation can have a more significant therapeutic effect on heart failure, especially heart failure with preserved ejection fraction, through its unique dual active ingredients, trichostatin A and N-suberylanilide hydroxamic acid. The preparation can not only effectively alleviate cardiac diastolic dysfunction in heart failure, but also treat and optimize insulin resistance in HFpEF; in addition, it can significantly reduce the level of ceramide in cardiac tissue, thereby improving mitochondrial functional damage; in addition, the preparation can also treat and improve mitochondrial edema and structural disorder in heart failure, increase the activity of enzyme complexes, and improve cardiac fibrosis associated with heart failure.

[0049] According to one embodiment of the present invention, the mass content of the trichostatin A is less than or equal to the mass content of the N-suberanoylanilide hydroxamic acid.

[0050] Specifically, the mass content of trichostatin A and the mass content of N-suberylanilide hydroxamic acid have a corresponding relationship with whether the preparation has significant toxicity. In other words, if the mass content of trichostatin A in the active ingredient of the preparation is too large, the preparation will have significant toxicity. Therefore, in this embodiment, the mass content of trichostatin A is less than or equal to the mass content of N-suberylanilide hydroxamic acid, thereby avoiding the presence of significant toxicity in the preparation and ensuring that the preparation still has a significant therapeutic effect on heart failure.

[0051] In some specific embodiments of the present invention, the preparation further comprises a solvent and an adjuvant, wherein the adjuvant comprises one or more of a co-solvent, an emulsifier, a stabilizer and a solubilizer.

[0052] That is to say, the preparation can be configured as a liquid preparation, which mainly includes trichostatin A, N-suberylanilide hydroxamic acid and a solvent. Trichostatin A and N-suberylanilide hydroxamic acid can be dissolved in the solvent to form a liquid preparation, so as to be injected into the patient as an injection.

[0053] Furthermore, the preparation may also include some adjuvants. For example, when the solubility of trichostatin A and N-suberylanilide hydroxamic acid is poor, the adjuvant may include a cosolvent and / or a solubilizer, etc., so as to improve the solubility of trichostatin A and N-suberylanilide hydroxamic acid, which is conducive to the formation of a liquid preparation. In addition, the adjuvant may also include an emulsifier and / or a stabilizer, so as to effectively improve the stability of the liquid preparation.

[0054] It should be noted that the specific components and contents of the adjuvant can be determined according to trichostatin A, N-suberylanilide hydroxamic acid and the solvent used, which will not be elaborated in this embodiment.

[0055] In some optional examples of the present invention, the solvent is water, the auxiliary solvent is polyethylene glycol 300, the emulsifier is Tween 80, the stabilizer is β-cyclodextrin, and the solubilizer is glycerol.

[0056] That is to say, the preparation for treating cardiovascular diseases according to an embodiment of the present invention mainly includes trichostatin A, N-suberylanilide hydroxamic acid, water (i.e., water for injection or pure water), polyethylene glycol 300, Tween 80, β-cyclodextrin and glycerol. The liquid preparation prepared by containing the above ingredients can have higher stability.

[0057] According to one embodiment of the present invention, the preparation comprises, by weight:

[0058] 10 to 30 parts of the trichostatin A, 10 to 30 parts of the N-suberylanilide hydroxamic acid, 275 to 550 parts of the auxiliary solvent, 250 to 500 parts of the emulsifier, 5 to 30 parts of the stabilizer, 5 to 30 parts of the solubilizer, and the solvent.

[0059] In this embodiment, the liquid preparation is prepared by using the above-mentioned components in fractions by mass, which can ensure the stability of the preparation and the encapsulation rate of the preparation.

[0060] In some specific embodiments of the present invention, the preparation comprises, by weight:

[0061] 10 to 20 parts of the trichostatin A, 20 to 30 parts of the N-suberylanilide hydroxamic acid, 275 to 550 parts of the auxiliary solvent, 250 to 500 parts of the emulsifier, 5 to 15 parts of the stabilizer, 5 to 15 parts of the solubilizer, and the solvent.

[0062] In other words, in the preparation of the present invention, if the mass content of the stabilizer and the solubilizer is greater than 15 parts, it will affect the stability of the drug, and the side effects of the drug will increase. Therefore, this embodiment selects 5 to 15 parts of stabilizer and 5 to 15 parts of solubilizer, which can ensure the stability of the drug and avoid excessive side effects of the drug.

[0063] According to one embodiment of the present invention, the preparation comprises, by mass, 10 to 20 parts of the trichostatin A, 20 to 30 parts of the N-suberylanilide hydroxamic acid, 275 to 550 parts of the auxiliary solvent, 250 to 500 parts of the emulsifier, 15 parts of the stabilizer, 15 parts of the solubilizer, and the solvent.

[0064] In this example, the stabilizer and the solubilizer have the same mass fraction, which can further improve the stability of the preparation, further ensure the encapsulation rate of the preparation, and avoid excessive side effects of the drug.

[0065] In some optional examples of the present invention, in every 5 ml of the preparation, the content of the trichostatin A is 10 mg~30 mg, the content of the N-suberylanilide hydroxamic acid is 10 mg~30 mg, the content of the auxiliary solvent is 275 mg~550 mg, the content of the emulsifier is 250 mg~500 mg, the stabilizer is 15 mg~30 mg, the solubilizer is 15 mg~30 mg, and the balance is the solvent.

[0066] Specifically, the content of trichostatin A can be 10mg, 15mg, 20mg, 25mg, 30mg, etc., the content of N-suberoylanilide hydroxamic acid can be 10mg, 15mg, 20mg, 25mg, 30mg, etc., the content of auxiliary solvent is 275mg, 350mg, 400mg, 450mg, 500mg, 550mg, etc., the content of emulsifier is 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, etc., the content of stabilizer can be 10mg, 15mg, 20mg, 25mg, 30mg, etc., the content of solubilizer can be 10mg, 15mg, 20mg, 25mg, 30mg, etc. The preparation of liquid phase preparation using the above mass fractions of each component can ensure the stability of the preparation and the encapsulation rate of the preparation.

[0067] In some specific embodiments of the present invention, in every 5 ml of the preparation, the content of the trichostatin A is 10 mg~30 mg, the content of the N-suberylanilide hydroxamic acid is 10 mg~30 mg, the content of the auxiliary solvent is 275 mg~550 mg, the content of the emulsifier is 250 mg~500 mg, the content of the stabilizer is 5 mg~15 mg, the content of the solubilizer is 5 mg~15 mg, and the balance is the solvent.

[0068] In this example, the use of the above-mentioned masses of components to prepare a liquid preparation can further improve the stability of the preparation, while further ensuring the encapsulation rate of the preparation and avoiding excessive side effects of the drug.

[0069] According to one embodiment of the present invention, the sum of the mass fractions of the N-suberoylanilide hydroxamic acid and the trichostatin A is 20 to 40 parts.

[0070] That is to say, if the content of N-suberylanilide hydroxamic acid and trichostatin A is too small, the therapeutic effect of the preparation will be weak, and if the content of N-suberylanilide hydroxamic acid and trichostatin A is too large, some significant side effects will be caused. Therefore, in this embodiment, the sum of the mass fractions of N-suberylanilide hydroxamic acid and the trichostatin A is 20 to 40 parts. For example, in every 5 ml of the preparation, the sum of the mass of trichostatin A and N-suberylanilide hydroxamic acid is 20 mg to 40 mg. Within this content range, the preparation can show a more significant therapeutic effect on heart failure, and there will be no significant toxicity and no significant side effects.

[0071] In some specific embodiments of the present invention, in every 5 ml of the preparation, the content of trichostatin A is 20 mg, the content of N-suberylanilide hydroxamic acid is 20 mg, the content of the auxiliary solvent is 550 mg, the content of the emulsifier is 500 mg, the content of the stabilizer is 15 mg, the content of the solubilizer is 15 mg, and the balance is the solvent.

[0072] In this example, the use of the above-mentioned masses of components to prepare the liquid preparation can further improve the stability of the preparation and further ensure the encapsulation rate of the preparation.

[0073] In some optional examples of the present invention, the preparation is configured as an injection.

[0074] That is to say, the preparation can be injected into patients as an injection. Injection as an injection can effectively increase the local effective concentration of trichostatin A and N-suberylanilide hydroxamic acid in the cardiovascular system, reduce or avoid the side effects of the preparation on other parts of the body, and at the same time improve the therapeutic effect of the preparation on cardiovascular diseases.

[0075] According to some embodiments of the present invention, trichostatin A accounts for 25%-75% of the active ingredients of the preparations of the embodiments of the present invention. Taking 5 ml of the preparation as an example, the effective dose is about 40 mg. The individual mass concentrations of the remaining auxiliary solvents, solubilizers and stabilizers are about 2.5%-5%. Significant liver and kidney toxicity can be seen in the portion exceeding 10%.

[0076] Optionally, the concentration of trichostatin A in the active ingredient of the embodiment of the present invention is less than 50% of the active ingredient concentration without causing significant toxicity to myocardial cells, but the concentration exceeding 50% can reduce cell activity relative to the control group. It can be seen from the different prescriptions that the dosage form of polyethylene glycol 300 with a mass concentration of 5%, Tween 80 with a mass concentration of 5%, and β-cyclodextrin 3mg / ml has good stability and solubility.

[0077] The embodiments of the present invention also provide the use of the preparation described in any of the above embodiments in the preparation of a drug for treating cardiovascular diseases. Since the preparation for treating cardiovascular diseases according to the embodiments of the present invention has the above technical effects, the use of the preparation described in the embodiments of the present invention in the preparation of a drug for treating cardiovascular diseases also has corresponding technical effects, which will not be described in detail in this embodiment.

[0078] An embodiment of the present invention further provides a method for preparing the preparation described in any of the above embodiments, and the preparation method comprises: steps S1 to S3.

[0079] S1, dissolving the active ingredient in a solvent to obtain a first mixed solution;

[0080] S2, adding a solubilizing agent to the first mixed solution and shear mixing to obtain a second mixed solution;

[0081] S3. Homogenize the second mixed solution to obtain the preparation.

[0082] In some optional examples of the present invention, the step of dissolving the active ingredient in a solvent to obtain a first mixed solution includes: step S11 and step S12.

[0083] S11, mixing the active ingredient with a solvent, a co-solvent, an emulsifier and a stabilizer to obtain a mixture;

[0084] S12, heating the mixture, and performing ultrasonic treatment on the mixture to obtain a first mixed solution.

[0085] Specifically, in step S11, the required amounts of trichostatin A and N-suberoylanilide hydroxamic acid are first weighed, and then Tween 80, polyethylene glycol 300, β-cyclodextrin and water are added to obtain a mixture consisting of the various components.

[0086] In step S12, the mixture is heated to a set temperature (eg, about 50°C), and the heated mixture is ultrasonically treated for 15s*5 cycles until the API is completely dissolved, thereby obtaining a first mixed solution.

[0087] In step S2, glycerol is added to the first mixed solution, and then mixed uniformly at a shearing speed of 5000 rpm by a shear mixing setting to obtain a second mixed solution.

[0088] In step S3, the second mixed liquid can be homogenized by high-pressure homogenization equipment at a pressure of about 700 bar, and the process can be repeated for multiple times. For example, the desired preparation can be obtained after 6 cycles.

[0089] According to some embodiments of the invention, the ampoule containing the preparation is packaged and then sterilized at a temperature of 115° C. for about 30 minutes, thereby obtaining the final product.

[0090] The technical solution of the present invention will be further described below through specific embodiments.

[0091] Example 1

[0092] 1. Prescription A

[0093]

[0094] Prepare 5 ml.

[0095] 2. Preparation method

[0096] Weigh the prescribed amount of trichostatin A and N-suberylanilide hydroxamic acid, add Tween 80, polyethylene glycol 300 and β-cyclodextrin, add about 4 ml of water, heat to 50°C, and ultrasonicate for 15s*5 cycles until the API is completely dissolved. Then add glycerol and mix evenly at 5000rpm shear, homogenize at 700bar pressure, cycle 6 times, put into ampoules and seal. Sterilize at 115°C for 30 minutes to obtain the final product.

[0097] Example 2

[0098] 1. Prescription B

[0099]

[0100] Prepare 5 ml.

[0101] 2. Preparation method

[0102] Weigh the prescribed amount of trichostatin A and N-suberylanilide hydroxamic acid, add Tween 80, polyethylene glycol 300 and β-cyclodextrin, add about 4 ml of water, heat to 50°C, and ultrasonicate for 15s*5 cycles until the API is completely dissolved. Then add glycerol and mix evenly at 5000rpm shear, homogenize at 700bar pressure, cycle 6 times, put into ampoules and seal. Sterilize at 115°C for 30 minutes to obtain the final product.

[0103] Example 3

[0104] 1. Prescription C

[0105]

[0106] Prepare 5 ml.

[0107] 2. Preparation method

[0108] Weigh the prescribed amount of trichostatin A and N-suberylanilide hydroxamic acid, add Tween 80, polyethylene glycol 300 and β-cyclodextrin, add about 4 ml of water, heat to 50°C, and ultrasonicate for 15s*5 cycles until the API is completely dissolved. Then add glycerol and mix evenly at 5000rpm shear, homogenize at 700bar pressure, cycle 6 times, put into ampoules and seal. Sterilize at 115°C for 30 minutes to obtain the final product.

[0109] Example 4

[0110] 1. Prescription D

[0111]

[0112] Prepare 5 ml.

[0113] 2. Preparation method

[0114] Weigh the prescribed amount of trichostatin A and N-suberylanilide hydroxamic acid, add Tween 80, polyethylene glycol 300 and β-cyclodextrin, add about 4 ml of water, heat to 50°C, and ultrasonicate for 15s*5 cycles until the API is completely dissolved. Then add glycerol and mix evenly at 5000rpm shear, homogenize at 700bar pressure, cycle 6 times, put into ampoules and seal. Sterilize at 115°C for 30 minutes to obtain the final product.

[0115] Example 5

[0116] 1. Prescription E

[0117]

[0118] Prepare 5 ml.

[0119] 2. Preparation method

[0120] Weigh the prescribed amount of trichostatin A and N-suberylanilide hydroxamic acid, add Tween 80, polyethylene glycol 300 and β-cyclodextrin, add about 4 ml of water, heat to 50°C, and ultrasonicate for 15s*5 cycles until the API is completely dissolved. Then add glycerol and mix evenly at 5000rpm shear, homogenize at 700bar pressure, cycle 6 times, put into ampoules and seal. Sterilize at 115°C for 30 minutes to obtain the final product.

[0121] The drug toxicity and stability of the above formulation were verified in primary neonatal rat cardiomyocytes. Figure 1 .

[0122] Specifically, the CCT8 method was used to detect the toxicity of different prescriptions on primary neonatal rat cardiomyocytes, and the OD value at 450nm was detected and compared with the control group. It can be seen that prescriptions A / B / C had no significant toxicity to cardiomyocytes, while prescriptions D / E had significant differential drug toxicity to cardiomyocytes.

[0123] It can be seen that when the mass content of trichostatin A is less than or equal to the mass content of N-suberylanilide hydroxamic acid, the significant toxicity of the preparation can be avoided, which is beneficial for the treatment of cardiovascular diseases.

[0124] The stability tests of different formulations are shown in Table 1 below.

[0125] Table 1: Stability test of preparations

[0126]

[0127] As shown in Table 1, the drug solubility of prescription A is poor, and precipitation and insolubility occur after one week. Prescription E precipitates after 16 weeks and cannot be completely dissolved after heating. Prescriptions B / C / D can still maintain good stability.

[0128] Furthermore, the encapsulation efficiency was determined by ultracentrifugation to separate the oil phase and the aqueous phase, and the contents of trichostatin A and N-suberylanilide hydroxamic acid in the aqueous phase were measured and calculated. The contents of trichostatin A and N-suberylanilide hydroxamic acid were analyzed by reversed-phase high performance liquid chromatography, and the chromatographic conditions were as follows: a C18 column (250 mm × 4.6 mm, particle size 5 μm) was selected, the mobile phase was acetonitrile-0.1% phosphoric acid water (volume ratio 30:70), and the pH was adjusted to 3.0 using triethylamine; the detection wavelength was set to 241 nm, and the flow rate was 1.0 ml / min. The encapsulation efficiencies of formulations B and C were both maintained above 90%, with good stability.

[0129] The technical solution of the present invention will be further illustrated by specific experimental examples below.

[0130] Experimental example

[0131] Establishment of animal model

[0132] The wild-type C57BL / 6N mice used were purchased from Charles River Laboratories (USA). Since HFpEF patients have gender differences and the incidence rate in males is significantly higher than that in females, male mice were selected as experimental modeling subjects. Experimental mouse rearing conditions: lighting time is 6:00-18:00, humidity is maintained at 40%-50%, and temperature is maintained at 65-75 degrees Fahrenheit. 7-8 week old C57BL / 6N male experimental mice were selected and given a mixed diet of HFpEF (heart failure group): 60kcal% high-fat diet (D12492, Research Dist Inc. HFD) + L-Name0.5g / L (SigmaAldrich); the control group was given a regular diet (#2916, Teklad).

[0133] Drug treatment method

[0134] Subcutaneous injection was used as follows: the back of the mouse's neck or the base of the hind limbs was selected, and the surface skin of the mouse was wiped and disinfected with 75% alcohol. The local skin was lifted up in a triangular shape with the thumb and index finger of the left hand, and a certain amount of TS001 was drawn up with a 1ml syringe. In this experimental example, prescription C was used and injected into the subcutaneous tissue of the triangular area according to the standard of 50ulvolume / 10gbodyweight. The syringe needle was pressed with two fingers to prevent the liquid from seeping out, and rubbing was used to promote the dispersion of the liquid into the subcutaneous tissue.

[0135] Detection Methods

[0136] Mouse heart ultrasound examination

[0137] Ultrasound detection was performed using the VisualSonics Vevo 2100 system and MS400 probe (VisualSonics Inc). Chest hair was removed 24 h before detection to expose the entire chest skin. The ultrasound coupling agent was heated to 37°C during detection to avoid stimulation of the mice. The left ventricular short axis was selected from the parasternal short axis section in the awake state to measure FS, EF, LVID and other indicators under M-mode conditions; then the mice were anesthetized (maintaining oxygen flow of 2.0 L / min, isoflurane 1.5%-2.0%), maintaining body temperature at 38.0°C and heart rate at 400-500 / min. The left ventricular long axis motion image was obtained under B-Mode conditions on the parasternal long axis section; the mitral valve blood flow spectrum was obtained under Pulse-wave Mode conditions at the maximum opening of the mitral valve on the apical four-chamber view; and the tissue Doppler spectrum was obtained under Tissue-Doppler conditions at the junction of the mitral valve ring and the ventricular septum on the apical four-chamber view. VevoLab5.5.0 (VisualSonics) was used for subsequent ultrasound analysis, and at least 5 cycles of systolic and diastolic function were selected to take the average value.

[0138] Mouse glucose tolerance test

[0139] This experiment uses Advanced Glucose Meter (CVS) blood glucose meter for glucose tolerance test. On the day of the experiment, the experimental mice were starved for 6 hours, the feeding food was removed and the residual food in the cage was cleared, and only drinking water was given. Glucose solution (2.5g Glucose / 10ml PBS) was prepared and 8ulglucose / g bodyweight was injected into the abdomen. The tail of the mouse was cut off by about 0.2cm, and the blood glucose concentration was tested at 0min, 15min, 30min, 45min, 1h, and 2h.

[0140] Mouse mitochondrial isolation

[0141] Prepare mitochondrial buffer as follows:

[0142]

[0143] The experimental mice were killed after 3 minutes of isoflurane anesthesia in the gas anesthesia chamber. The mouse chest was disinfected with 70% alcohol. The chest was opened with surgical scissors to expose the heart. The atrium and aorta connection were lifted with curved forceps. The heart tissue was cut, the atrial tissue and blood vessels were separated and discarded, cut into 3mm diameter small pieces and placed in mitochondrial buffer, and transferred to a 5ml centrifuge tube. The tissue block was broken by homogenizer and homogenized for 15s and placed on ice. It was centrifuged at 4℃ and 2000rpm for 5min, and the tissue fragments were filtered and discarded to obtain the filtrate containing mitochondria. The filtrate was centrifuged at 4℃ and 10000rpm for 10min to obtain mitochondrial precipitation, and resuspended with 50ul mitochondrial buffer to obtain active mitochondria.

[0144] Mitochondrial function assay

[0145] Prepare oxidative phosphorylation buffer as follows:

[0146]

[0147] After obtaining the mitochondria as described above, add the above-mentioned oxidative phosphorylation buffer, add saponin (50ng / ml), and permeabilize for 5min; use a microsyringe to place a fluorescent oxygen sensor (OceanOptics, NeofoxHIOXY) and add different substrates, palmitic acid or pyruvic acid, to measure the mitochondrial oxygen consumption rate; the oxygen consumption rate is based on respiratory state II, state III is the maximum oxygen consumption rate supplemented by ADP; state IV is the oxygen consumption rate that is not related to ADP after ADP is depleted. Calculate OCR, measure each sample 3 times with each substrate, and take the average value.

[0148] HE staining:

[0149] The experimental mice were killed after being anesthetized with isoflurane for 3 minutes in an anesthesia chamber. The mouse chest was disinfected with 70% alcohol, and the chest was opened with surgical scissors to expose the heart. The left atrial appendage was cut off, and PBS was perfused and flushed in the heart using a 10 ml syringe. The connection between the atrium and the aorta was lifted with curved forceps, and the heart tissue was cut. It was placed in pre-cooled PBS for washing, and a 3 mm-5 mm thick tissue block in the middle part of the ventricle was cut and fixed overnight in a 4% paraformaldehyde shaker.

[0150] Prepare alcohol solutions of varying concentrations, dehydrate the tissue from low to high concentrations, place in xylene to make it transparent, and then embed in paraffin. Cut into 5um-8um paraffin sections, flatten with hot water and stick to the slide, and dry in a 45℃ constant temperature oven. Then wash the sections in xylene I for 10min-xylene II for 10min-anhydrous ethanol I for 5min-anhydrous ethanol II for 5min-95% alcohol for 5min-80% alcohol for 5min-70% alcohol for 5min-distilled water to deparaffinize the sections.

[0151] Prepare hematoxylin stain (0.5g hematoxylin powder + 24g ammonium alum / 70ml distilled water, 31g NaIO, 5ml water, 30ml glycerol, 2ml acetic acid) and eosin stain (0.5g water-soluble eosin stain / 100ml distilled water) and then stain. The steps are as follows:

[0152] 1) Place the slices in a hematoxylin aqueous solution and stain for 5-10 minutes;

[0153] 2) Color separation in acid water and ammonia water for 5s-10s;

[0154] 3) Rinse with running water for 1 hour and then put into distilled water for 5 minutes;

[0155] 4) Dehydrate in 70% and 90% alcohol for 10 minutes each;

[0156] 5) Place in alcohol eosin staining solution for 2-3 minutes. Dehydrate with anhydrous ethanol, make it transparent with xylene, seal with resin, and observe under a microscope.

[0157] Masson staining

[0158] Obtain the aforementioned paraffin sections and prepare the following reagents:

[0159]

[0160] Masson trichrome staining was performed as follows:

[0161] 1) Dewax paraffin sections as described above;

[0162] 2) Rinse with distilled water for 5 minutes;

[0163] 3) Use Regaud's hematoxylin staining solution for nuclear staining for 5min-10min;

[0164] 4) Wash with water to observe the degree of nuclear staining. If it is overstained, hydrochloric acid and alcohol can be used for differentiation;

[0165] 5) Stain with Masson Ponceau acid red solution for 5-10 minutes;

[0166] 6) Soak in 2% glacial acetic acid aqueous solution for 3 minutes;

[0167] 7) Soak in 2% glacial acetic acid solution for 3 minutes;

[0168] 8) Soak in 1% phosphomolybdic acid aqueous solution for 3-5 minutes;

[0169] 9) Use aniline blue or light green liquid to dye for 5 minutes;

[0170] 10) Soak in 0.2% glacial acetic acid aqueous solution for 2 minutes;

[0171] 11) Dehydrate with 95% alcohol-anhydrous ethanol, make transparent with xylene, seal with resin, and observe under a microscope.

[0172] PSR (Sirius Red) staining

[0173] Prepare Sirius Red-Piric Acid Solution (0.5% Sirius Red 10ml / picric acid saturated aqueous solution 100ml) and Lapis Lazuli Dye Solution (Lapis Lazuli Blue B 1.25g + Iron Alum 1.25g / distilled water 250ml, boil to dissolve, filter after cooling, add 30ml glycerol and 0.5ml concentrated sulfuric acid in turn)

[0174] The steps of PSR staining are as follows:

[0175] 1) Dewax the paraffin sections as described above;

[0176] 2) Use lapis lazuli dye solution to dye for 5min-10min;

[0177] 3) Rinse with distilled water 3 times;

[0178] 4) Use Sirius red-picric acid solution to stain for 15min-30min;

[0179] 5) Perform anhydrous ethanol differentiation and dehydration;

[0180] 6) Xylene is transparent, add a drop of resin to seal the slide and observe under a microscope.

[0181] Western Blot detection of protein expression:

[0182] Prepare 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide) separation gel: Taking 10ml separation gel as an example, add 4.07ml of distilled water, 30% Acr-Bis (acrylamide-methylene bisacrylamide) 3.33ml, 4XTris-HCl-SDS (trishydroxymethylaminomethane-sodium dodecyl sulfate) separation gel buffer (PH8.8) 2.5ml, 10% APS (ammonium persulfate) 100ul, TEMED (tetramethylethylenediamine) 4ul into the test tube in sequence, and vortex to mix.

[0183] Prepare 5% SDS-PAGE (sodium dodecyl sulfate polyacrylamide) stacking gel: Taking 4 ml stacking gel as an example, add 2.28 ml of distilled water, 0.68 ml of 30% Acr-Bis (acrylamide-methylene bisacrylamide), 1.0 ml of 4XTris-HCl-SDS (trishydroxymethylaminomethane-sodium dodecyl sulfate) stacking gel buffer (PH6.8), 40 ul of 10% APS (ammonium persulfate), and 4 ul of TEMED (tetramethylethylenediamine) into the test tube in sequence, and vortex to mix.

[0184] Clean and dry the glass plate, test the airtightness, first add 5 ml of separation gel and let it stand at room temperature for 30 minutes, then add 2 ml of concentrated gel and insert a 1.5 mm comb, let it stand at room temperature for 20 minutes, and the gel preparation is completed. If it is not used temporarily, add distilled water at 4°C to keep it moist for 3-5 days for use.

[0185] Protein loading and gel running

[0186] Prepare 5X electrophoresis buffer as follows:

[0187]

[0188] Prepare 5X transfer buffer as follows:

[0189]

[0190] Fix the sample loading glass plate, add electrophoresis solution to cover the upper edge of the gel, discard the comb, add 10ul-20ul of protein sample to each well, perform electrophoresis at 90V constant voltage for 30min, wait for the protein marker to separate, adjust the voltage to 120V constant voltage for about 60min, and perform electrotransfer.

[0191] Cut a PVDF (polyvinylidene fluoride) membrane of appropriate size, activate it with methanol for 30 seconds, place it on the electrophoresis protein gel, cover it with filter paper, clamp it into the transfer tank, and pour in the pre-cooled transfer solution. Place the transfer tank in an ice box and transfer the membrane at a constant current for 120 minutes. The specific current and time depend on the size of the protein.

[0192] Obtain the successfully transferred protein membrane, block it with 5% BSA (skim milk) at room temperature for 1 hour; wash it with TBST once, add the primary antibody diluent (1:1000, 5% BSA), incubate the primary antibody at 4°C overnight; wash it with TBST for 5 minutes * 3 times; add the secondary antibody diluent (1:5000, 5% BSA), incubate it at room temperature for 1 hour-2 hours; wash it with TBST for 5 minutes * 3 times, use ECLkit (EfficientChemiluminescencekit) for fluorescence luminescence to determine the strength of protein signals, and use SPSS for statistical analysis.

[0193] RNA reverse transcription and real-time quantitative PCR

[0194] TAKARAkit was used for RNA reverse transcription, and the 20ul reaction system was as follows:

[0195]

[0196] The reaction conditions were as follows: 37°C, 15 min reverse transcription reaction; 85°C, 5 s reverse transcriptase inactivation; 4°C, end of reaction, add RNaseFreeH2O to dilute cDNA 10 times, and store at -20°C.

[0197] The Biorad realtime-qPCR kit was used for qPCR, and the 20ul reaction system was as follows:

[0198]

[0199] The reaction conditions were as follows: pre-step, Step 1: 95°C 30 sec; cycle step, number of cycles: 40; Step 1: 95°C 3 sec; Step 2: 60°C 30 sec; melting step. The CT value of the amplification curve and the melting curve were observed to judge the qPCR results, and SPSS was used for statistics.

[0200] It should be noted that the content of active ingredients used in each experimental group and control group is the same, which will not be repeated in this example.

[0201] Experimental data such as Figures 2 to 10 As shown in the figure, it can be seen from the experimental data that after treatment with the preparation for treating cardiovascular diseases according to the embodiment of the present invention, the impaired glucose tolerance of HF mice was improved, the cardiac diastolic function was significantly improved, and the mitochondrial morphology and function were significantly improved. This therapeutic effect is closely related to the inhibition of myocardial ceramide synthesis by the preparation for treating cardiovascular diseases according to the embodiment of the present invention.

[0202] In summary, the preparation for treating cardiovascular diseases according to the embodiment of the present invention can have a more significant therapeutic effect on heart failure, especially heart failure with preserved ejection fraction, through its unique dual active ingredients, trichostatin A and N-suberylanilide hydroxamic acid. The preparation can not only effectively alleviate cardiac diastolic dysfunction in heart failure, but also treat and optimize insulin resistance in HFpEF; in addition, it can also significantly reduce the level of ceramide in cardiac tissue, thereby improving mitochondrial functional damage; in addition, the preparation can also treat and improve mitochondrial edema and structural disorder in heart failure, increase the activity of enzyme complexes, and improve cardiac fibrosis associated with heart failure.

[0203] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that the above embodiments may be modified without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A preparation for treating heart failure, characterized in that: The preparation contains the following active ingredients: Trichostatin A; and N-suberylanilide hydroxamic acid, wherein the mass content of the trichostatin A is less than or equal to the mass content of the N-suberylanilide hydroxamic acid; The preparation comprises, by weight: 10 to 20 parts of the trichostatin A, 20 to 30 parts of the N-suberoylanilide hydroxamic acid, 275 to 550 parts of an auxiliary solvent, 250 to 500 parts of an emulsifier, 5 to 15 parts of a stabilizer, 5 to 15 parts of a solubilizer, and a solvent.

2. The preparation according to claim 1, characterized in that The solvent is water, the auxiliary solvent is polyethylene glycol 300, the emulsifier is Tween 80, the stabilizer is β-cyclodextrin, and the solubilizer is glycerol.

3. The preparation according to claim 1, characterized in that The preparation comprises, by weight: 10 to 20 parts of the trichostatin A, 20 to 30 parts of the N-suberylanilide hydroxamic acid, 275 to 550 parts of the auxiliary solvent, 250 to 500 parts of the emulsifier, 15 parts of the stabilizer, 15 parts of the solubilizer, and the solvent.

4. The preparation according to any one of claims 1 to 3, characterized in that The sum of the mass parts of the N-suberoylanilide hydroxamic acid and the trichostatin A is 40 parts.

5. The preparation according to claim 4, characterized in that In every 5 ml of the preparation, the content of trichostatin A is 20 mg, the content of N-suberylanilide hydroxamic acid is 20 mg, the content of the auxiliary solvent is 550 mg, the content of the emulsifier is 500 mg, the content of the stabilizer is 15 mg, the content of the solubilizer is 15 mg, and the balance is the solvent.

6. The preparation according to claim 1, characterized in that The preparation is configured as an injection.

7. Use of the preparation according to any one of claims 1 to 6 in the preparation of a medicament for treating heart failure.

8. A method for preparing the preparation according to any one of claims 1 to 6, characterized in that: The preparation method comprises: dissolving the active ingredient in a solvent to obtain a first mixed solution; adding a solubilizing agent to the first mixed solution and shear mixing the mixture to obtain a second mixed solution; The second mixed solution is homogenized to obtain the preparation.

9. The preparation method according to claim 8, characterized in that: The step of dissolving the active ingredient in a solvent to obtain a first mixed solution comprises: mixing the active ingredient with a solvent, a co-solvent, an emulsifier and a stabilizer to obtain a mixture; The mixture is heated and subjected to ultrasonic treatment to obtain a first mixed solution.

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