WO2022141805A1

WO2022141805A1 - Application of crotonic acid salt, crotonic acid salt preparation and method for preparing same

Info

Publication number: WO2022141805A1
Application number: PCT/CN2021/080588
Authority: WO
Inventors: 段金柱; 蔡文倩; 曾锤; 许达才; 林应炯; 赵悦; 汪庆文; 董文艳
Original assignee: 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心）
Priority date: 2020-12-31
Filing date: 2021-03-12
Publication date: 2022-07-07
Also published as: CN112691097B; CN112691097A

Abstract

An application of a crotonic acid salt in the preparation of a drug for treating a heart disease. A crotonic acid salt preparation and a method for preparing same. The method is characterized by comprising: mixing a crotonic acid solution with an alkaline substance and adjusting the pH to 6.8-7.4, so as to form a crotonic acid salt preparation.

Description

The application of crotonate, its preparation and preparation method of preparation

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of the Chinese Patent Application No. 202011634530.3 and entitled "Application of Crotonate, Its Formulation and Its Preparation Method" filed with the China Patent Office on December 31, 2020, the entire content of which is approved by Reference is incorporated in this application.

technical field

The present application relates to the field of pharmaceutical application and preparation, in particular, to the application of crotonate, its preparation and its preparation method.

Background technique

At present, the number of patients with hypertension, coronary heart disease and diabetes in my country is increasing day by day, and the problem of social aging is becoming more and more serious, which makes the prevalence of heart disease in my country continue to rise.

In 2014, the Cardiovascular Branch of the Chinese Medical Association issued the "Guidelines for the Diagnosis and Treatment of Heart Failure in China 2014". The "Golden Triangle" has become the basic treatment plan for various acute and chronic heart diseases. In recent years, with the extensive application of the Golden Triangle in the treatment of heart disease, the mortality rate has been greatly reduced, but its mortality rate is still comparable to that of cancer, with a 5-year mortality rate close to 50%, and it is still one of the major diseases threatening human health. Therefore, it is necessary to develop a new drug with protective effect on acute and chronic cardiac injury.

Cardiac arrest is the sudden stop of the heart's pumping function, usually resulting in sudden death. Sudden cardiac death is the most common cause of death in both developed and developing countries. The most commonly used drug for cardiac arrest is epinephrine, which increases the rate of recovery of spontaneous circulation and increases the chance of transporting the patient to the hospital for further rescue. But there are no other vasoactive drugs that improve survival more than epinephrine. Therefore, there is a need to find a new drug that can be used in cardiac arrest to improve the recovery rate of spontaneous circulation.

SUMMARY OF THE INVENTION

In view of the above situation, the present application provides the application of crotonate, its preparation and its preparation method.

The present application provides the application of a crotonate in the preparation of a medicine for treating heart disease.

Wherein, the crotonate used is sodium crotonate. It can be understood that in addition to sodium crotonate, other crotonates, such as potassium crotonate, can also be used.

And crotonate can treat heart disease, mainly because it can repair damaged heart, and it mainly increases the level of crotonylation modification of mitochondrial and skeletal proteins in cardiomyocytes, and increases the remodeling and arrangement of mitochondria, skeletal proteins and cardiomyocytes. The integrated connection between the two can reduce cardiac fibrosis, regulate cardiomyocyte autophagy and inhibit cardiomyocyte death, therefore, in some embodiments, crotonate can be used to prepare at least one of inhibiting cardiac fibrosis and cardiomyocyte death An inhibitor of the condition, or an improver for increasing the level of crotonylation modification of mitochondrial and skeletal proteins of cardiomyocytes. Alternatively, crotonates can also be used in drugs that increase the overall level of crotonylation.

In some embodiments, crotonate is also used as a rescue drug for cardiac arrest and/or sudden cardiac death; in particular, it can enhance myocardial cell viability, increase myocardial contractility, regulate heart rate stabilization, and facilitate restoration of spontaneous heart rate. Thus, in some embodiments, crotonate is used as an improver to increase myocardial contractility and spontaneous circulation, or as a modulator of heart rate stabilization and/or restoration of spontaneous heart rate, or to increase skeletal protein remodeling, mitochondrial stability, and Cardiomyocyte connectivity improver.

The above-mentioned heart diseases include cardiac arrest, heart damage, arrhythmia, myocarditis, congenital heart disease and rheumatic heart disease. It should be noted that, although only some heart diseases are listed in the examples of the present application, other heart diseases that can be treated by crotonate are also within the protection scope of the present application.

Optionally, the inventors found that different concentrations of crotonate have different effects. In animal models, the effective therapeutic concentration of crotonate is: 0.22mg/kg/d～5.5mg/kg/d or 275mg /kg/d～825mg/kg/d, specifically, when the effective therapeutic concentration of the crotonate is 0.22mg/kg/d～5.5mg/kg/d, it can be used to repair heart damage; when it is effective When the concentration is 275mg/kg/d ~ 825mg/kg/d, it can be used for first aid in cardiac arrest.

Alternatively, the crotonate formulation is administered by intraperitoneal injection.

The present application also provides a preparation method of a crotonate preparation for treating heart disease, comprising: mixing a crotonic acid solution with an alkaline substance and adjusting the pH to 6.8-7.4 to form the crotonate preparation.

Optionally, the preparation of the crotonic acid solution includes: mixing the crotonic acid crystals with an organic solvent, and then ultrasonically crushing them, so that the crotonic acid crystals are dissolved to form the crotonic acid solution;

Optionally, the alkaline substance is hydroxide; it can be sodium hydroxide;

Optionally, the alkaline substance is carbonate, bicarbonate or alkoxide; it can be sodium carbonate, sodium bicarbonate or sodium alkoxide.

Optionally, the organic solvent is DMSO.

Optionally, the crotonate formulation is stored at -25 to -18°C.

The present application also provides a crotonate preparation for the treatment of heart disease, which is prepared by the preparation method of the crotonate preparation described in the foregoing embodiments.

The present application also provides a crotonate preparation for treating heart disease, which is prepared by the preparation method of the crotonate preparation described in the foregoing embodiment, and the crotonate preparation is a liquid preparation.

The present application also provides a crotonate preparation for the treatment of heart disease, which is prepared by the preparation method of the crotonate preparation described in the foregoing embodiment, and the crotonate preparation is a solid preparation.

The present application also provides a crotonate preparation for the treatment of heart disease, which is prepared by the preparation method of the crotonate preparation described in the foregoing embodiment, and the crotonate preparation is a capsule preparation.

The present application also provides the use of a crotonate in the treatment of heart disease.

The present application also provides a method of cardiac disease comprising: administering to a subject in need thereof a therapeutically effective amount of a crotonate.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present application more clearly, the following drawings will briefly introduce the drawings that need to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the organelle localization map of the sites with significant changes in the crotonylation modification level provided by Experimental Example 1 of this application;

Fig. 2 is the schematic diagram of the protein and its site whose crotonylation modification level is significantly changed to mitochondria provided by Experimental Example 1 of this application;

Fig. 3 is the schematic diagram of the protein and the site of which the crotonylation modification level of the backbone protein is significantly changed provided by the first experimental example of the application;

FIG. 4 is a graph of the results of a large increase in crotonylation modification after I/R surgery observed by immunoelectron microscopy provided by Experimental Example 1 of the present application;

Figure 5 is a graph of changes in apoptosis and autophagy in cardiomyocytes under the condition that mitochondrial crotonylation modification is increased, provided in Experimental Example 1 of the present application;

Figure 6 is a graph of changes in cardiomyocyte apoptosis affected by the crotonylation modification of skeleton protein provided in Experimental Example 1 of the application;

Figure 7 is a graph of changes in cardiomyocyte autophagy affected by crotonylation modification of skeletal proteins provided in Experimental Example 1 of the present application;

FIG. 8 is a graph showing the effect of overexpression of crotonylation modification on specific sites of mitochondrial proteins and specific sites of skeletal proteins provided in Experimental Example 2 of this application on the viability of cardiomyocytes after injury;

Fig. 9 is a graph showing the effect of adding crotonylation to a specific site of the skeleton protein provided by Experimental Example 2 of this application on the remodeling and arrangement of myocardial cytoskeleton protein after injury;

Figure 10 is a graph of the result that sodium crotonate provided by experimental example 2 of the application increases the viability of neonatal rat cardiomyocytes and thereby inhibits the decrease in myocardial viability induced by ISO;

Fig. 11 is the result diagram that sodium crotonate reduces ISO-induced cardiomyocyte apoptosis provided by Experimental Example 2 of the application;

Fig. 12 is the result diagram that the sodium crotonate that the application experiment example 2 provides improves mitochondrial stability;

Figure 13 is an echocardiogram of mice after 7 days and 14 days of I/R surgery and its result analysis diagram provided by Experimental Example 3 of the application;

Figure 14 provides the results of Masson staining and TUNEL staining of mouse hearts 15 days after I/R operation provided in Experimental Example 3 of the application;

Figure 15 provides the results of heart rate monitoring after injecting epinephrine and high-concentration NaCr respectively, provided in Experimental Example 4 of the present application.

Detailed ways

To make the purposes, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The examples of the present application provide a crotonate preparation, which is a liquid preparation, and the preparation is stable, can be stored at -25 to -18° C. for 6 months, the properties are still stable, and the therapeutic effect is still outstanding. Therefore, the crotonate Salt formulations can be used for industrial mass production.

The embodiment of the present application provides a preparation method of a crotonate preparation, comprising:

The crotonic acid crystals are mixed with an organic solvent and then sonicated to dissolve the crotonic acid crystals to form the crotonic acid solution.

The solvent used in the examples of this application is DMSO (dimethyl sulfoxide), but it can be understood that other solvents that can dissolve crotonic acid and will not affect the physiological and biochemical activities can also be used. within the scope of protection.

The use of ultrasonic crushing of crotonic acid crystals is conducive to the dissolution of crotonic acid, is conducive to the stability of the formed crotonic acid solution, and ensures that the crotonic acid is fully dissolved. At the same time, the use of crotonic acid crystals can reduce impurities in the crotonic acid solution and improve the therapeutic effect of the subsequently formed crotonate.

At this time, the pH of the crotonic acid solution is 2-3, and then the above-mentioned crotonic acid solution is mixed with an alkaline substance, and the crotonic acid and the alkaline substance undergo a neutralization reaction to form a crotonic acid salt. And after the crotonic acid solution is mixed with the alkaline substance, the pH is adjusted to 6.8-7.4, thereby ensuring the formation of crotonate and reducing the residue of crotonic acid and alkaline substances, thereby ensuring the therapeutic effect of the crotonic acid preparation.

Optionally, the alkaline substance is hydroxide, which can be sodium hydroxide. It should be noted that, in addition to sodium hydroxide, other hydroxides capable of forming salts, such as potassium hydroxide, can also be used as the hydroxide. Similarly, the alkaline substance is not limited to hydroxide, and other alkaline substances that can form crotonate, such as carbonate, bicarbonate or alkoxide, for example, sodium carbonate, sodium bicarbonate or sodium alkoxide can be used Wait.

Then, the volume was reconstituted to form crotonate liquid formulations of different concentrations. The crotonate liquid is stored at -25 to -18° C., so as to ensure that the crotonate preparation still has good physiological and biochemical properties even after being stored for a long time.

The examples of the present application also provide new applications of crotonate, specifically, crotonate can be used to treat heart disease, such as cardiac arrest, heart injury, arrhythmia, myocarditis, congenital heart disease and rheumatic heart disease disease or other heart disease.

Specifically, crotonate increases the level of crotonylation modification on mitochondrial and skeletal proteins of cardiomyocytes, thereby reducing cardiac fibrosis, inhibiting cardiomyocyte autophagy, and cardiomyocyte death, thereby repairing damaged hearts. At the same time, it can improve myocardial cell viability, increase myocardial contractility, regulate the stability of heart rate, and help restore voluntary heart rate, so that it can be used as a rescue drug for cardiac arrest and/or sudden cardiac death.

Specifically, different concentrations of crotonate have different effects. In animal models, when the effective therapeutic concentration of the crotonate is 0.22mg/kg/d to 5.5mg/kg/d, its effect is to repair Heart injury; when the effective therapeutic concentration of crotonate is 275mg/kg/time to 825mg/kg/time, it can be used for emergency cardiac arrest.

And the crotonate provided in the examples of the present application is sodium crotonate (Sodium Crotonate, NaCr). Of course, it can be understood that in addition to sodium crotonate, other crotonates, such as potassium crotonate, can also be used.

The features and properties of the present application will be described in further detail below with reference to the embodiments.

The embodiment of the present application provides a preparation method of a sodium crotonate solution, comprising:

Weigh 8.6 g of crotonic acid crystals, add 4 mL of DMSO solution, and dissolve all crotonic acid crystals with the help of ultrasonic waves. At this time, the pH value of the crotonic acid solution is about 2.0 to 3.0. Adjust the pH value to 7.0 with NaOH solution, add Ultrapure water, titrated to 40ml to obtain 2.5mol/L sodium crotonate solution, subpackage and freeze at -20℃, and then take it out when needed.

Experimental Example 1: Proteomic Sequencing and Verification

Step 1: The chest of 6 C57 mice was opened under inhalation anesthesia respectively. Among them, the left anterior descending coronary artery was ligated 1 mm below the left atrium in 4 mice, and the ligation was released after 30 minutes. The electrocardiogram was connected to confirm the successful modeling of myocardial ischemia-reperfusion injury; the other 2 mice only underwent thoracotomy without ligation, that is, sham surgery was performed to establish a sham model (Sham). Postoperatively, they were given disinfection, analgesia, liquid food, and heat preservation. Left atrial tissue was collected 3 days and 5 days after the operation, respectively, for protein mass spectrometry sequencing.

Step 2: Analyze the proteomic data

Step 3: Analyze and validate proteomic results

(1) As shown in Figure 1: After I/R surgery, the proteomic results showed that about 60%-70% of the sites with significant changes in crotonylation modification levels were concentrated in mitochondria (dark gray) and skeletal proteins ( gray), and other organelles (light gray) only account for 30%-40%.

(2) The sites with significant changes in the level of crotonylation modification and their specific proteins are shown in Figures 2 and 3. The inventors found that changing the crotonylation modification at a specific site may have the potential to affect the survival of cardiomyocytes.

(3) To observe how the crotonylation of mitochondrial and skeletal proteins of mouse cardiomyocytes changed after I/R injury by immunoelectron microscopy. The results are shown in Figure 4. Compared with the sham group, the crotonylation modification of mitochondrial and skeletal proteins of mouse cardiomyocytes after I/R surgery was greatly increased (each black dot is a crotonylation modification signal) .

(4) Isolate mouse cardiomyocytes, and by immunofluorescence detection, it was found that cardiomyocyte apoptosis (cleaved caspase3) and autophagy (LC3B II) changed in the case of increased crotonylation of mitochondrial proteins and cytoskeletal proteins (Fig. 5-7).

Experimental Example 2: Cell Experiment Verification

(1) Regulation of crotonylation at specific sites

Step 1: Euthanize 10 rat suckling mice 2-3 days after birth, take out the heart, digest with trypsin and collagenase, and obtain cardiomyocytes.

Step 2: Overexpress the target gene (WT) or the target gene mutated at a specific site (KR will make the site no longer acylated, and KQ will simulate the site and add a croton) in neonatal mouse cardiomyocytes by adenovirus. acyl group), and then use 100uM isoproterenol (ISO) to induce cardiomyocyte apoptosis. After 24h, the morphology of cytoskeleton protein was detected, and the apoptosis signal was detected after 48h—TUNEL (terminal-deoxynucleotidyltransferase mediated nick end labeling), 72h Cell viability was then detected.

(1) The inventors selected some proteins and sites for verification, and found that regulating the crotonylation modification of specific sites of these proteins can affect the viability of cardiomyocytes. As shown in Figure 8, increasing the crotonylation modification of the K200 site of the mitochondrial protein IDH3a and the K72 site of ATPIF1, as well as the K29&30 of the cytoskeletal protein TPM1 and the K138 of Tnnc1 can resist the reduction of cell viability induced by high concentrations of ISO, indicating that the increase in the Crotonylation at specific sites in mitochondrial proteins can enhance cardiomyocyte viability and may serve as a therapeutic target for repairing cardiac injury.

(2) As shown in Figure 9, increasing the crotonylation modification of K29&30 of TPM1 can significantly change the arrangement of the skeleton protein and make it remodeled, which may enhance its ability to resist damage, indicating that the increase of specific sites on the skeleton protein The crotonylation modification of the dots can not only improve cell viability, but also change the arrangement of cytoskeletal proteins, and can also be used as a therapeutic target for repairing cardiac injury (K for lysine, WT for wild type, KR for reduced crotonylation, KQ stands for enhanced crotonylation, PBS stands for phosphate buffer saline, and GFP stands for Green fluorescent protein.

(2), increase the overall crotonylation modification

Step 1 is the same as step 1 in (1) of experimental example 2; step 2, pre-treated neonatal rat cardiomyocytes with 5mM sodium crotonate solution for 24h, and then used 100uM isoproterenol (ISO) to induce cardiomyocyte apoptosis, Mitochondrial membrane potential was detected after 24h, and cell viability was detected after 72h.

(1) As shown in Figure 12, the mitochondrial membrane potential was detected. The stronger the fluorescence intensity, the more stable the mitochondrial membrane potential was, indicating that sodium crotonate could improve mitochondrial stability.

(2) As shown in Figures 10 and 11, 5mM sodium crotonate solution can improve cell viability, and can resist ISO-induced decrease in cell viability and reduce ISO-induced cardiomyocyte apoptosis, indicating that sodium crotonate can protect cardiomyocytes.

Conclusion: In cell experiments, the inventors found that increasing the crotonylation modification of specific protein sites can protect cardiomyocytes. In addition, increasing the overall crotonylation modification level can also protect cardiomyocytes. All in all, within a certain range of Increased crotonylation can protect cardiomyocytes.

Experimental Example 3: Animal Experiments

Step 1: 40 C57 mice were opened under inhalation anesthesia, and the left anterior descending coronary artery was ligated 1 mm below the left atrium. After 45 minutes, the ligation was released. After confirming the recanalization of the blood vessels, the chest was closed. Intraoperative ECG connections were made to confirm the successful modeling of myocardial ischemia-reperfusion injury. Postoperative disinfection, analgesia, liquid food, and heat preservation were given.

Step 2 (pre-experiment of single concentration administration): 0.22 or 5.5 mg/kg sodium crotonate solution (sodium crotonate dissolved in 10% DMSO) was injected intraperitoneally 2 hours immediately after the operation, followed by intraperitoneal injection of 0.22 or 5.5 for the next 5 days mg/kg/day sodium crotonate solution, (per mouse) for a total of 6 days of injection. Mice in the control group were injected with an equivalent volume of 10% DMSO solution for 5 consecutive days.

Step 3 (optimized dosing mode, mixed concentration administration): 2 hours after the operation, 5.5 mg/kg sodium crotonate solution (sodium crotonate dissolved in 10% DMSO) was injected intraperitoneally immediately, and then 5.5 mg/kg intraperitoneal injection was administered for the next 4 days. kg/day sodium crotonate solution, followed by intraperitoneal injection of 0.22 mg/kg/day sodium crotonate solution for the next 7 days (per mouse) for a total of 12 days. Mice in the control group were injected with an equivalent volume of 10% DMSO solution for 12 consecutive days.

(1) On the 7th and 14th days after the operation, the cardiac function of the mice was monitored by echocardiography.

The test results are shown in Figure 13. As shown in A in Figure 13, both 0.22 mg/kg and 5.5 mg/kg of sodium crotonate can improve the cardiac function of mice after I/R, and the ejection fraction was 7 and 14 days after the operation. have improved. After optimizing the way of administration (step 3), sodium crotonate significantly improved the cardiac function of mice after I/R, the results are shown in B (left) in Figure 13, and the line with arrows (left) White arrows), the height between the peaks and troughs represents the systolic and diastolic capacity of the mouse heart, and the greater the distance, the stronger the systolic capacity.

Taking ejection fraction as an example, ejection fraction refers to the percentage of stroke volume in ventricular end-diastolic volume (ie, cardiac preload), which is one of the important indicators for judging the type of heart failure. Ventricular ejection fraction refers to the ratio of ventricular stroke volume to ventricular end-diastolic volume. The calculation formula is: EF=(EDV-ES)×100%/EDV, where EF is ejection fraction; EDV is ventricular end-diastolic volume. volume; ES is the ventricular end-systolic volume. Ejection fraction is a volume ratio indicator that reflects the ejection function of the ventricle in terms of volume. As shown in B (right) in Figure 13, after injection of sodium crotonate, the ejection fraction of mice increased, and the cardiac pumping ability was enhanced, which was manifested as an increase in cardiac function.

(2) The mouse hearts were harvested after surgery, embedded in paraffin, sliced, and subjected to Masson staining and TUNEL staining to calculate the fibrosis area and the number of apoptotic cells.

The test results are shown in Figure 14. According to A in Figure 14, it can be seen that sodium crotonate can significantly reduce postoperative cardiac fibrosis in mice, and according to B in Figure 14, it can be seen that sodium crotonate can significantly reduce myocardial cell apoptosis.

Conclusion: From the above analysis, it can be seen that sodium crotonate can repair the damaged heart, and then it can be used to treat heart damage.

Experimental Example 4: Comparison of the effects of sodium crotonate and isoproterenol on heart rate

Monitoring ECG: 8 C57 mice were connected to ECG under mild anesthesia. After the mice were stable, NaCr (experimental group) or isoproterenol (control group) were injected into the abdominal cavity respectively to observe the effect of NaCr on heart rate.

The test results are shown in Figure 15. The left figure in Figure 15 shows the effect on heart rate after a one-time injection of 275mg/kg of NaCr, and the middle figure in Figure 15 shows two injections of NaCr, one 275mg /kg, once 550mg/kg, the effect on heart rate after a total injection of 825mg/kg, the right panel in Figure 15 shows the effect of isoproterenol injection on heart rate. As shown in the figure, injection of sodium crotonate can increase the heart rate of mice; in addition, although the injection of isoproterenol can also increase the heart rate of mice, but this increase quickly calms down, in contrast, sodium crotonate can be more durable promotes an increase in heart rate.

Conclusion: High concentration of sodium crotonate can increase the heart rate for a long time, and has the potential to treat cardiac arrest.

In this application, the crotonate preparation of the liquid preparation is used as an example to illustrate, but it is not limited to this, and can also be made into a solid preparation or a capsule preparation according to actual needs, and the solid preparation or capsule preparation can also achieve the above effects.

To sum up, the present application has the following beneficial effects: the examples of the present application provide a new application of crotonate, which can be used to treat heart disease, especially cardiac arrest and acute and chronic heart damage. Specifically, it can reduce cardiac fibrosis, regulate cardiomyocyte autophagy and cardiomyocyte death by increasing the level of crotonylation of mitochondrial proteins and skeletal proteins in cardiomyocytes, and then repair cardiac damage. At the same time, crotonate can also enhance myocardial cell viability, increase myocardial contractility, regulate heart rate stability, and help restore spontaneous heart rate, which can then be used for cardiac arrest.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Industrial Applicability

The new application of crotonate provided by the present application can not only have a protective effect on acute and chronic heart damage, and then repair the heart damage, but also can be used for emergency cardiac arrest, and then can prevent sudden cardiac death.

Claims

The application of a crotonate in the preparation of a medicine for treating heart disease.
The application according to claim 1, wherein the crotonate is sodium crotonate.
The application according to claim 1, wherein the medicine is a medicine for repairing damaged heart;

Preferably, the drug is a drug for reducing cardiac fibrosis;

Preferably, the drug is a drug that regulates cardiomyocyte autophagy and/or inhibits cardiomyocyte death;

More preferably, the drug is a rescue drug for cardiac arrest and/or sudden cardiac death;

Preferably, the drug is a drug for enhancing myocardial cell viability;

Preferably, the drug is a drug that increases myocardial contractility;

Preferably, the medicine is a medicine for regulating and stabilizing heart rate;

Preferably, the drug is a drug for restoring spontaneous heart rate.
The use according to any one of claims 1 to 3, wherein the drug is a drug that increases the level of crotonylation modification on mitochondrial proteins and skeletal proteins of cardiomyocytes or improves the overall level of crotonylation modification .
The application according to any one of claims 1-3, wherein the heart disease includes cardiac arrest, cardiac injury, arrhythmia, myocarditis, congenital heart disease and rheumatic heart disease.
The application according to claim 1, characterized in that, in an animal model, when the effective therapeutic concentration of the crotonate is: 0.22 mg/kg/d to 5.5 mg/kg/d, the crotonate is administered with When the effective therapeutic concentration of the crotonate is: 275mg/kg/d～825mg/kg/d, the crotonate is used for preparing the emergency medicine for cardiac arrest.
The application according to any one of claims 1 to 6, characterized in that,

In animal models, the crotonate formulation is administered by intraperitoneal injection.
The use of a crotonate as an inhibitor for inhibiting at least one of the following (1)-(2);

(1) Cardiac fibrosis; (2) Cardiomyocyte death.
Application of a crotonate as an improving agent for improving at least one of the following (1)-(6);

(1) Myocardial contractility; (2) Autonomic circulation; (3) The level of crotonylation of mitochondrial proteins and cytoskeletal proteins in cardiomyocytes; (4) Remodeling and arrangement of skeletal proteins; (5) Mitochondrial stability; (6) Myocardium cell connection.
Use of a crotonate as a regulator for regulating heart rate stabilization and/or restoring voluntary heart rate.
A method for preparing a crotonate preparation for treating heart disease, characterized in that it comprises: mixing a crotonic acid solution with an alkaline substance and adjusting the pH to 6.8-7.4 to form a crotonate preparation;

Preferably, the preparation of the crotonic acid solution comprises: mixing crotonic acid crystals with an organic solvent, and then ultrasonically crushing, so that the crotonic acid crystals are dissolved to form the crotonic acid solution;

Preferably, the crotonate preparation is stored at -25°C to -18°C, and the stability is good at this temperature, and the acylation level of cardiomyocytes can still be increased after being stored for half a year before use.
The preparation method of the crotonate preparation for the treatment of heart disease according to claim 11, is characterized in that,

The alkaline substance is hydroxide; it can be sodium hydroxide or potassium hydroxide.
The preparation method of the crotonate preparation for the treatment of heart disease according to claim 11, is characterized in that,

The alkaline substance is carbonate, bicarbonate or alkoxide; it can be sodium carbonate, sodium bicarbonate or sodium alkoxide.
The method for preparing a crotonate preparation for treating heart disease according to any one of claims 11 to 13, wherein the organic solvent is DMSO.
A crotonate preparation for treating heart disease, characterized in that it is prepared by the method for preparing a crotonate preparation for treating heart disease according to claim 11 .
A crotonate preparation for the treatment of heart disease, characterized in that, it is prepared by the preparation method of the crotonate preparation for the treatment of heart disease according to claim 11, and the crotonate preparation is a liquid preparation .
A crotonate preparation for the treatment of heart disease, characterized in that, it is prepared by the preparation method of the crotonate preparation for the treatment of heart disease according to claim 11, and the crotonate preparation is a solid preparation .
A crotonate preparation for the treatment of heart disease, characterized in that, it is prepared by the preparation method of the crotonate preparation for the treatment of heart disease according to claim 11, and the crotonate preparation is a capsule preparation .
Use of a crotonate in the treatment of heart disease.
A method of treating heart disease comprising: administering to a subject in need thereof a therapeutically effective amount of a crotonate.