Application of ML324 in preparation of medicine for treating and / or preventing heart diseases

Abstract

The invention provides application of ML324 in preparation of drugs for treating and / or preventing heart diseases. The invention shows that the ML324 can prevent and / or treat heart diseases related to myocardial cell death, obviously inhibit activity decline of myocardial cells induced by a cell death inducer Erastin and H2O2, and improve the cell survival rate; generation of active oxygen in cells is reduced, and death modes such as myocardial cell apoptosis are reduced; the compound can be used for preventing, treating and improving heart function damage, myocardial fibrosis and heart organic damage caused by heart diseases, has a good application prospect in the aspect of developing medicines for resisting the heart diseases, and provides a new medicine research and development direction for preventing, treating and improving the heart diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of ML324 in the preparation of drugs for the treatment and / or prevention of heart disease. Background Technology

[0002] Myocardial infarction (MI) is one of the most common cardiovascular diseases and the most serious clinical manifestation of coronary artery disease. During MI, cardiomyocytes die due to ischemia, a process accompanied by the generation of a large number of free radicals, primarily reactive oxygen species (ROS). These free radicals further cause cell damage and inflammatory responses, thus exacerbating myocardial injury. Cardiomyocytes, as the main building blocks of the heart, account for approximately 75% of the total heart volume and are the most severely damaged cell group in MI. Cardiomyocyte death is considered a major cause of cardiac dysfunction. Because cardiomyocytes are terminally differentiated cells with extremely limited regenerative capacity, excessive death of cardiomyocytes increases the burden on surviving cells, further aggravating the condition and leading to more severe heart disease. In conclusion, reducing cardiomyocyte death is crucial for the treatment and prognosis of related heart diseases.

[0003] Epigenetic modification is a common and important regulatory mechanism that can regulate gene expression without altering the DNA sequence. This primarily includes DNA methylation, RNA methylation, histone methylation, acetylation, and non-coding RNA regulation. Numerous studies have shown that cell death is regulated by epigenetic modifications. Epigenetics connects genetics with environmental factors, explaining genetic phenomena that are difficult to explain using traditional genetics. Furthermore, due to its reversibility, drug intervention targeting epigenetic regulators is more likely to correct abnormal gene expression than genetic alterations. In cardiomyocytes, epigenetic and post-translational modifications maintain homeostasis by regulating the expression levels of genes related to cardiomyocyte death, determining cardiomyocyte sensitivity, and thus participating in the occurrence and development of cardiovascular diseases. Therefore, exploring the progression of heart disease driven by cardiomyocyte death from the perspective of epigenetic regulation will help to more accurately identify its regulatory targets and provide new ideas for developing novel drugs to prevent heart disease caused by cardiomyocyte death.

[0004] ML324 (CAS:1222800-79-4) is a selective jumonji histone demethylase (JMJD2) inhibitor with an IC50 of 920 nM against JMJD2E. Compared to previously reported JMJD2 inhibitors, ML324 exhibits good cell permeability, providing an opportunity for broader research on the JMJD2 protein. Previous studies have indicated that ML324 demonstrates good antiviral activity against herpes simplex virus and human cytomegalovirus by inhibiting the expression of early viral genes. Specifically, ML324 effectively inhibits the formation of herpes simplex virus plaques and blocks the reactivation of herpes simplex virus-1 in a mouse ganglion explant model of latent infection. In addition, ML324 can reduce the production of interleukin-6 stimulated by lipopolysaccharide, suggesting that it has a certain anti-inflammatory effect, but its role in cardiomyocyte death and heart disease has not yet been explored. Summary of the Invention

[0005] In view of this, to solve the above-mentioned technical problems, this invention proposes the application of ML324 in the preparation of drugs for the treatment and / or prevention of heart disease. This invention demonstrates that ML324 can prevent and / or treat heart diseases related to cardiomyocyte death, improve cardiac function impairment, myocardial fibrosis, and organic heart damage caused by heart disease, and has promising application prospects in the development of drugs for treating heart disease, providing a new direction for drug research and development for the prevention, treatment, and improvement of heart disease.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] The use of ML324 in the preparation of drugs for the treatment and / or prevention of heart disease.

[0008] The structural formula of ML324 is:

[0009]

[0010] In some preferred embodiments of the present invention for use in the preparation of medicaments for the treatment and / or prevention of heart disease, the heart disease is at least one of myocardial infarction, heart failure, arrhythmia, cardiomyopathy, myocarditis, and myocardial ischemia-reperfusion injury.

[0011] In some preferred embodiments of the present invention for use in the preparation of medicaments for the treatment and / or prevention of heart disease, the ML324 can improve cardiac function impairment, myocardial fibrosis and organic damage to the heart caused by heart disease.

[0012] In some preferred embodiments of the present invention for use in the preparation of medicaments for the treatment and / or prevention of heart disease, the heart disease is a heart disease associated with myocardial cell death.

[0013] In some preferred embodiments of the present invention, the ML324 is used in the preparation of medicaments for the treatment and / or prevention of heart disease, wherein the ML324 treats and / or prevents heart disease by inhibiting myocardial cell death.

[0014] In some preferred embodiments of the present invention for use in the preparation of medicaments for the treatment and / or prevention of heart disease, the ML324 inhibits cardiomyocyte death by suppressing the production and excessive accumulation of reactive oxygen species within cardiomyocytes.

[0015] In some preferred embodiments of the present invention for use in the preparation of medicaments for the treatment and / or prevention of heart disease, the medicament comprises ML324 as an active ingredient and a pharmaceutically acceptable carrier.

[0016] Some examples suitable as carriers include: lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methylcellulose, methylparaben and propylparaben, talc, magnesium stearate and liquid paraffin, one or more of these.

[0017] The method for preparing the active ingredient into a drug in this invention can be prepared using methods known to those skilled in the art. For example, the active ingredient can be diluted or encapsulated in a carrier so that it can be released immediately, slowly, or with a delayed release after being administered to a subject.

[0018] In some preferred embodiments of the present invention for use in the preparation of medicaments for the treatment and / or prevention of heart disease, the dosage form of the medicament is one of tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, tinctures, lotions, and films.

[0019] In some preferred embodiments of the present invention for the preparation of medicaments for the treatment and / or prevention of heart disease, the medicament is administered via one or more of the following routes: oral, injection, implantation, and topical application.

[0020] Compared with existing technologies, the application of ML324 described in this invention in the preparation of drugs for treating and / or preventing heart disease has the following advantages:

[0021] This invention provides a novel use for ML324: its application in drugs for the treatment and / or prevention of heart disease. In this invention, ML324 can inhibit cardiomyocyte death, reduce the production and accumulation of reactive oxygen species (ROS), alleviate cardiomyocyte damage, reduce ROS-induced apoptosis, and improve cardiac function, myocardial fibrosis, and organic cardiac damage. This provides a new drug for the treatment and / or prevention of heart diseases related to cardiomyocyte death and has promising application prospects. Attached Figure Description

[0022] Figure 1 The results of the experiment in Example 1 showing that ML324 reduces Erastin-induced cardiomyocyte death are as follows: Figure 1 A represents the measured cell viability result; Figure 1 B represents the results of the cell live / dead staining experiment;

[0023] Figure 2 The experimental results of ML324 reducing Erastin-induced reactive oxygen species accumulation in Example 1 are as follows: Figure 2 A represents the results of intracellular reactive oxygen species (ROS) levels measured using flow cytometry. Figure 2 B represents the results of analyzing intracellular reactive oxygen species levels in each treatment group using FlowJo software; Figure 2 C represents the results of detecting intracellular reactive oxygen species levels using a fluorescence microscope; Figure 2 D represents the results of analyzing intracellular reactive oxygen species levels in each treatment group using ImageJ software;

[0024] Figure 3 The results of the experiment in Example 2 on reducing H2O2-induced cardiomyocyte death by ML324 are as follows: Figure 3 A represents the measured cell viability result; Figure 3 B represents the results of the cell live / dead staining experiment;

[0025] Figure 4 The experimental results of ML324 reducing H2O2-induced reactive oxygen species accumulation in Example 2 are as follows: Figure 4 A represents the results of intracellular reactive oxygen species (ROS) levels measured using flow cytometry. Figure 4 B represents the results of analyzing intracellular reactive oxygen species levels in each treatment group using FlowJo software; Figure 4 C represents the results of detecting intracellular reactive oxygen species levels using a fluorescence microscope; Figure 4 D represents the results of analyzing intracellular reactive oxygen species levels in each treatment group using ImageJ software;

[0026] Figure 5 The results of the experiment in Example 2 on reducing H2O2-induced apoptosis by ML324 are as follows: Figure 5 A represents the results of intracellular apoptosis levels determined using flow cytometry. Figure 5 B represents the proportion of apoptotic positive cells in each treatment group analyzed using FlowJo software;

[0027] Figure 6 The experimental results of ML324 reducing cardiac function damage caused by myocardial infarction in Example 3 are as follows: Figure 6 A is a schematic diagram of mouse surgical and drug treatment grouping; Figure 6B is a schematic diagram of the surgical induction of myocardial infarction in mice, drug treatment, and echocardiography protocol; Figure 6 C is a representative schematic diagram of cardiac ultrasound in mice of each treatment group 28 days after surgery; Figure 6 D is a statistical graph of left ventricular ejection fraction in mice; Figure 6 E is a statistical graph of the shortening rate of the short axis of the left ventricle in mice;

[0028] Figure 7 The results of Example 3 show that ML324 reduces myocardial fibrosis and cardiac damage induced by myocardial infarction in mice: where, Figure 7 A shows the preparation of paraffin sections of heart samples from mice in each treatment group 28 days after myocardial infarction surgery, with representative H&E images. Figure 7 B shows paraffin sections of heart samples from mice in each treatment group 28 days after surgery, with representative Masson staining images. Figure 7 C represents the quantitative statistical analysis of fibrosis area in mouse heart sections using Masson staining results obtained from ImageJ software. Figure 7 D represents the quantitative statistical analysis of infarct area in mouse heart sections using ImageJ software based on masson staining results. Figure 7 E represents the quantitative analysis of Myh7 mRNA levels in the heart tissues of mice in each treatment group using qRT-PCR. Detailed Implementation

[0029] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0030] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0031] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0032] Example 1

[0033] ML324 reduces Erastin-induced cardiomyocyte death.

[0034] 1. The main experiments involved in this embodiment are as follows:

[0035] (1) ML324 reduces the sensitivity of cardiomyocytes to Erastin;

[0036] (2) ML324 reduces Erastin-induced reactive oxygen species production.

[0037] 2. The specific experimental procedure is as follows:

[0038] (1) ML324 reduces the sensitivity of cardiomyocytes to Erastin.

[0039] The experimental steps are as follows:

[0040] 1) H9C2 cardiomyocytes were pretreated with DMSO and ML324 (1, 2, 5, 10, 20 μM) for 2 hours respectively;

[0041] 2) After treating cells from step 1) with Erastin (3 μM) for 16 hours, cell viability was measured (see results below). Figure 1 A);

[0042] 3) H9C2 cells were pretreated with DMSO and ML324 (5 μM) for 2 hours respectively;

[0043] 4) After treating the cells in 3) with Erastin (3 μM) for 16 hours, observe the cell status under a microscope;

[0044] 5) Add Calcein-AM / PI detection working solution to the cells from step 4) to perform cell viability staining experiments, and observe the staining effect of the cells under a fluorescence microscope (see results). Figure 1 B, where green fluorescence (Calcein-AM) represents live cells and red fluorescence (PI) represents dead cells.

[0045] Depend on Figure 1 A and Figure 1 As shown in B, ML324 can effectively reduce Erastin-induced cardiomyocyte death.

[0046] (2) ML324 reduces Erastin-induced reactive oxygen species production.

[0047] The experimental steps are as follows:

[0048] 1) H9C2 cells were pretreated with DMSO and ML324 (5 μM) for 2 hours;

[0049] 2) After treating cells from step 1) with Erastin (3 μM) for 16 hours, intracellular reactive oxygen species (ROS) levels were measured using flow cytometry (results are shown in [link to results]). Figure 2 A);

[0050] 3) FlowJo software was used to analyze the intracellular reactive oxygen species levels in each treatment group (results are shown in...). Figure 2 B);

[0051] 4) H9C2 cells were pretreated with DMSO and ML324 (5 μM) for 2 hours;

[0052] 5) After treating cells from step 4) with Erastin (3 μM) for 16 hours, the intracellular reactive oxygen species (ROS) level was measured using fluorescence microscopy (see results below). Figure 2 C, where the intensity of green fluorescence represents the level of reactive oxygen species in the cell;

[0053] 6) ImageJ software was used to analyze the intracellular reactive oxygen species levels in each treatment group (results are shown in...). Figure 2 D).

[0054] Depend on Figure 2 As shown in A to 2D, ML324 can significantly reduce Erastin-induced reactive oxygen species production.

[0055] In summary, the above experimental results demonstrate that ML324 can resist cell death induced by Erastin stimulation, significantly reduce the sensitivity of cardiomyocytes to Erastin, reduce the increase in reactive oxygen species levels and excessive accumulation of intracellular reactive oxygen species caused by Erastin, and significantly inhibit Erastin-induced cardiomyocyte death.

[0056] Example 2

[0057] ML324 reduces H2O2-induced cardiomyocyte death.

[0058] 1. The main experiments involved in this embodiment are as follows:

[0059] (1) ML324 reduces H2O2-induced cardiomyocyte death;

[0060] (2) ML324 reduces H2O2-induced accumulation of reactive oxygen species;

[0061] (3) ML324 reduces H2O2-induced apoptosis.

[0062] 2. The specific experimental procedure is as follows:

[0063] (1) ML324 reduces H2O2-induced cardiomyocyte death

[0064] The experimental steps are as follows:

[0065] 1) H9C2 cardiomyocytes were pretreated with DMSO and ML324 (5 μM) for 2 hours;

[0066] 2) After treating the cells in 1) with H2O2 (200 μM) for 24 hours, cell viability was measured (see results). Figure 3 A);

[0067] 3) H9C2 cells were pretreated with DMSO and ML324 (5 μM) for 2 hours respectively;

[0068] 4) After treating the cells in 3) with H2O2 (200 μM) for 24 hours, observe the cell status under a microscope;

[0069] 5) Add Calcein AM / PI detection working solution to the cells from step 4) to perform a live-cell staining experiment. Observe the staining effect of the cells under a fluorescence microscope (see results). Figure 3 B, where green fluorescence (Calcein-AM) represents live cells and red fluorescence (PI) represents dead cells.

[0070] Depend on Figure 3 As shown in A and 3B, ML324 significantly reduces H2O2-induced cardiomyocyte death.

[0071] (2) ML324 reduces H2O2-induced reactive oxygen species accumulation.

[0072] 1) H9C2 cardiomyocytes were pretreated with DMSO and ML324 (5 μM) for 2 hours;

[0073] 2) After treating the cells from step 1) with H2O2 (200 μM) for 24 hours, the cells were harvested, and the intracellular reactive oxygen species (ROS) levels were measured using flow cytometry (see results). Figure 4 A);

[0074] 3) FlowJo software was used to analyze the intracellular reactive oxygen species levels in each treatment group (results are shown in...). Figure 4 B);

[0075] 4) H9C2 cells were pretreated with DMSO and ML324 (5 μM) for 2 hours;

[0076] 5) After treating the cells from step 4) with H2O2 (200 μM) for 24 hours, the intracellular reactive oxygen species (ROS) level was measured using a fluorescence microscope (see results). Figure 4 C, where the intensity of green fluorescence represents the level of reactive oxygen species in the cell;

[0077] 6) ImageJ software was used to analyze the intracellular reactive oxygen species levels in each treatment group (results are shown in...). Figure 4 D).

[0078] Depend on Figure 4 From A to 4D, it can be seen that ML324 significantly reduces H2O2-induced reactive oxygen species accumulation.

[0079] (3) ML324 reduces H2O2-induced apoptosis

[0080] 1) H9C2 cardiomyocytes were pretreated with DMSO and ML324 (5 μM) for 2 hours;

[0081] 2) After treating the cells from step 1) with H2O2 (200 μM) for 24 hours, the cells were harvested, and the intracellular apoptosis level was determined by flow cytometry (see results). Figure 5 A);

[0082] 3) FlowJo software was used to analyze the proportion of apoptosis-positive cells in each treatment group (results are shown in [link to results]). Figure 5 B);

[0083] Depend on Figure 5 As shown in A and 5B, ML324 effectively reduces H2O2-induced apoptosis.

[0084] In summary, the above experimental results demonstrate that ML324 can effectively alleviate H2O2-induced cardiomyocyte damage by reducing the excessive accumulation of reactive oxygen species, thus protecting cardiomyocytes from the effects of H2O2-induced myocardial damage. At the same time, it can significantly inhibit H2O2-induced apoptosis, thereby effectively reducing H2O2-induced cardiomyocyte death.

[0085] Example 3

[0086] ML324 treatment reduces cardiac damage following myocardial infarction in mice.

[0087] 1. The main experiments involved in this embodiment are as follows:

[0088] (1) ML324 reduces cardiac function damage caused by myocardial infarction;

[0089] (2) ML324 reduces myocardial fibrosis and cardiac damage in mice caused by myocardial infarction.

[0090] 2. The specific experimental procedure is as follows:

[0091] (1) ML324 reduces cardiac function damage caused by myocardial infarction.

[0092] The experimental steps are as follows:

[0093] 1) Eight-week-old male mice were randomly divided into four groups and pretreated with ML324 (1.74 mg / kg / d, intraperitoneal injection) one day in advance.

[0094] 2) Surgical modeling was performed on the mice in 1) (see details of mouse surgery and drug treatment grouping). Figure 6 A);

[0095] 3) Mice that underwent surgery as described in 2) were treated daily with ML324, and their cardiac function was assessed on days 3, 7, 14, and 28 (see details of treatment and echocardiography protocol). Figure 6 B. See echocardiography results. Figure 6 C);

[0096] 4) Statistical analysis of left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) in mice in step 3) (see results). Figure 6 D-6E);

[0097] Depend on Figure 6 From A to 6E, it can be seen that ML324 pretreatment in mice can improve cardiac function after myocardial infarction.

[0098] (2) ML324 reduces myocardial fibrosis and cardiac damage induced by myocardial infarction in mice.

[0099] 1) Prepare paraffin sections of the heart from mice 28 days after myocardial infarction in step 3) of (1);

[0100] 2) The paraffin sections from 1) were stained with hematoxylin and eosin (H&E) (see results). Figure 7 A);

[0101] 3) Masson staining was performed on the paraffin sections from 1) (see results). Figure 7 B);

[0102] 4) ImageJ software was used to quantify the fibrosis area in mouse heart sections based on Masson staining results (see results below). Figure 7 C);

[0103] 5) ImageJ software was used to quantify the infarct area in mouse heart sections based on Masson staining results (see results). Figure 7 D);

[0104] 6) qRT-PCR quantitative analysis of Myh7 mRNA expression levels in the heart tissues of mice in each treatment group (results are shown in...). Figure 7 E).

[0105] Depend on Figure 7 From A to 7D, it can be seen that ML324 can effectively reduce myocardial fibrosis and heart damage in mice caused by myocardial infarction.

[0106] In summary, the above experimental results demonstrate that ML324 treatment can improve cardiac function after myocardial infarction, alleviate myocardial cell damage and myocardial fibrosis induced by myocardial infarction, and play a protective role in myocardial injury induced by myocardial infarction.

[0107] This invention primarily employs laser confocal microscopy, qRT-PCR, Western blotting, and molecular biology techniques to investigate the therapeutic and / or preventative effects of the small molecule compound ML324 on cardiomyocyte death-related heart diseases, thereby advancing its clinical application in the treatment of these diseases. The Erastin-induced H9C2 cardiomyocyte death model was used. Cell viability was detected by CCK-8 assay, and reactive oxygen species (ROS) levels were detected using a kit. Microscopic imaging was also used to clarify the role of ML324 in Erastin-induced cardiomyocyte death. In the H2O2-induced cardiomyocyte oxidative stress model, cell viability was detected by CCK-8 assay, and changes in intracellular ROS levels and the number of apoptotic positive cells were detected by kit and flow cytometry to clarify the effect of ML324 on H2O2-induced cardiomyocyte damage. The effect of ML324 on cardiomyocyte death was explored in vivo. A mouse myocardial infarction (MI) model was constructed and treated with ML324 (1.74 mg / kg / d). Changes in left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were detected by echocardiography. The effect of ML324 on mouse myocardial infarction was evaluated by qRT-PCR, Western blotting, hematoxylin and eosin (H&E) staining, and Masson staining. The results showed that ML324 effectively reduced Erastin and H2O2-induced cardiomyocyte death and intracellular reactive oxygen species accumulation, decreased H2O2-induced cardiomyocyte apoptosis, and alleviated cardiac dysfunction, myocardial fibrosis, and organic cardiac damage caused by cardiomyocyte death. In conclusion, ML324 provides a novel potential drug for the clinical prevention and / or treatment of cardiac diseases related to cardiomyocyte death.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of ML324 in the preparation of drugs for the treatment and / or prevention of heart disease.

2. The application according to claim 1, characterized in that: The heart disease is at least one of myocardial infarction, heart failure, arrhythmia, cardiomyopathy, myocarditis, or myocardial ischemia-reperfusion injury.

3. The application according to claim 2, characterized in that: The ML324 can improve cardiac function impairment, myocardial fibrosis, and organic heart damage caused by heart disease.

4. The application according to claim 1, characterized in that: The heart disease mentioned refers to heart diseases related to the death of myocardial cells.

5. The application according to claim 4, characterized in that: The ML324 treats and / or prevents heart disease by inhibiting cardiomyocyte death.

6. The application according to claim 5, characterized in that: The ML324 inhibits cardiomyocyte death by suppressing the production and excessive accumulation of reactive oxygen species within cardiomyocytes.

7. The application according to any one of claims 1 to 6, characterized in that: The drug comprises ML324 as the active ingredient and a pharmaceutically acceptable carrier.

8. The application according to any one of claims 1 to 6, characterized in that: The dosage form of the drug is one of the following: tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, tinctures, lotions, and films.

9. The application according to any one of claims 1 to 6, characterized in that: The drug can be administered via one or more of the following routes: oral, injection, implantation, or topical application.

Images