Application of BRD4770 in preparation of medicine for treating and / or preventing DOX-induced cardiomyopathy

Abstract

The invention provides an application of BRD4770 in preparation of a medicine for treating and / or preventing DOX-induced cardiomyopathy. The BRD4770 can significantly reduce the level of active oxygen in cells and the generation of lipid peroxidation, inhibits DOX-induced myocardial cell ferroptosis and apoptosis, provides a new potential drug for prevention and / or treatment of cardiotoxicity induced when DOX is used as a clinical chemotherapy drug, and does not affect the chemotherapy effect of DOX on tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the use of BRD4770 in the preparation of a drug for treating and / or preventing DOX-induced cardiomyopathy. Background Art

[0002] Doxorubicin (DOX) belongs to anthracycline antitumor drugs, with a broad anti-tumor spectrum, and is used to treat hematological malignancies (such as leukemia) and solid tumors (such as breast cancer). The pharmacological effect of doxorubicin in anti-tumor is to intercalate with DNA, thereby blocking tumor cell proliferation and inducing apoptosis. It has a good killing effect on tumor cells in various growth cycles, and can significantly prolong the survival period of patients when used alone or in combination with other drugs. However, the clinical application of DOX is limited due to numerous side effects, and the most prominent one is cardiotoxicity, including various types of arrhythmias, pericarditis and myocarditis, acute hypertension, congestive heart failure and even death. In practical observations and clinical studies, about 30% of patients are affected by doxorubicin-induced cardiomyopathy (DIC). Therefore, the mechanism of doxorubicin cardiotoxicity has always been a hot topic in tumor treatment. Previous studies have shown that traditional drugs for treating heart failure can no longer reverse the damage caused by DOX-induced cardiomyopathy. Therefore, the prevention and treatment of cardiotoxic side effects induced by chemotherapeutic drugs represented by doxorubicin are still the main obstacles in the use of chemotherapeutic drugs. Developing highly efficient and safe cardioprotective agents has important research significance for improving the quality of life and survival level of cancer chemotherapy patients.

[0003] Epigenetic modifications (such as DNA modifications and histone modifications) change gene expression through various mechanisms, and then regulate phenotypes. During the process of DOX-induced cardiomyopathy, many epigenetic modifications change, and these changes can be used as molecular markers for cancer prognosis and potential molecular targets for preventing DOX-induced cardiomyopathy.

[0004] Increasingly more studies have shown that epigenetic modifications are involved in the occurrence and development of DOX-induced cardiomyopathy, but the specific mechanism is still unclear. Epigenetics refers to the reversible and heritable changes in gene expression without changes in the nucleotide sequence of genes. Epigenetic modifications mainly include DNA methylation, histone modification, non-coding RNA regulation, and chromatin remodeling, etc. Epigenetics links genetics and environmental factors, can explain genetic phenomena that cannot be explained in genetics, and at the same time, compared with genetic changes, drugs are more likely to correct abnormal gene expression by affecting such regulatory factors. During the process of DOX producing cardiotoxicity, the generation of oxidative stress and metabolic changes in cells are accompanied, and these changes further cause changes in epigenetic modifications. Changes in epigenetic modifications can be used as molecular markers for cancer prognosis and can also be used as potential molecular targets to reduce DOX-induced cardiotoxicity in cancer patients.

[0005] DNA methylation, histone modification, and non-coding RNA expression play roles in DOX-induced cardiotoxicity, and these modifications can relieve DOX-induced cardiotoxicity by inhibiting the cardiomyocyte apoptosis pathway. For example, epigenetic modification enzymes such as histone arginine transferase PRMT4 and RNA methyltransferase METIL14 can promote the occurrence of DOX-induced ferroptosis in cardiomyocytes by inhibiting the Nrf2 / GPX4 24 and KCNQ1OT1-miR-7-5p-TFRC 25 pathway, and inhibiting the above pathways can effectively inhibit ferroptosis and prevent DOX-induced cardiotoxicity. Although the causal relationship between epigenetic modifications and DOX-induced cardiomyopathy has not been fully explored, it is generally believed that epigenetic modifications are involved in the progression of DOX-induced cardiomyopathy. Therefore, exploring the process of DOX-induced cardiomyopathy from the perspective of epigenetic regulation will more directly and accurately explore its regulatory targets and provide new ideas for the development of drugs to prevent DOX-induced cardiomyopathy.

[0006] BRD4770 is a methyltransferase inhibitor, initially reported to be synthesized in 2012, and can inhibit H3K9me2 and H3K9me3 catalyzed by G9a (also known as euchromatic histone methyltransferase 2 [EHMT2]) in pancreatic cancer PANC-1 cells. There is currently no report on the use of BRD4770 for the prevention and treatment of DOX-induced cardiomyopathy. Summary of the Invention

[0007] In view of this, in order to overcome the problem of inducing cardiotoxic side effects existing in chemotherapy drugs represented by doxorubicin, the present invention provides the use of BRD4770 in the preparation of a drug for treating and / or preventing doxorubicin-induced cardiomyopathy, providing a new potential drug for preventing and / or treating the cardiotoxicity induced by doxorubicin when used as a clinical chemotherapy drug, and without affecting the chemotherapy effect of doxorubicin on tumors.

[0008] To achieve the above object, the technical solution of the present invention is realized as follows:

[0009] On the one hand, the present invention provides the use of BRD4770 in the preparation of a drug for treating and / or preventing doxorubicin-induced cardiomyopathy.

[0010] The structural formula of BRD4770 is:

[0011]

[0012] Further, the doxorubicin-induced cardiomyopathy is the cardiotoxic side effects induced when doxorubicin is used as a chemotherapy drug for tumor chemotherapy.

[0013] Further, the doxorubicin-induced cardiomyopathy is at least one of arrhythmia, pericarditis, myocarditis, acute hypertension, and congestive heart failure.

[0014] Further, the BRD4770 relieves doxorubicin-induced cardiac function injury, cardiac atrophy, and cardiac fibrosis, improves the cardiac antioxidant level, and reduces the accumulation of lipid peroxides.

[0015] Further, the BRD4770 inhibits doxorubicin-induced cardiomyocyte death.

[0016] Further, the BRD4770 inhibits doxorubicin-induced cardiomyocyte death by inhibiting the production of reactive oxygen species and the accumulation of lipid peroxidation in cardiomyocytes caused by doxorubicin.

[0017] Further, the drug includes BRD4770 as an active ingredient and a pharmaceutically acceptable carrier.

[0018] Further, the dosage form of the drug is one of tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, medicinal wines, tinctures, extracts, and films.

[0019] Further, the administration route of the drug is one of oral administration, injection, implantation, and external use.

[0020] On the other hand, the present invention provides the use of the combination of BRD4770 and doxorubicin in the preparation of an antitumor drug, and the BRD4770 does not affect the antitumor effect of doxorubicin.

[0021] BRD4770 is involved in DOX-induced cardiomyocyte injury. Compared with other epigenetic regulation compounds, BRD4770 can completely reverse Erastin-induced cardiomyocyte ferroptosis and rescue DOX-induced cardiomyocyte ferroptosis and apoptosis. BRD4770 is a specific inhibitor of histone methyltransferase EHMT2 except BIX-01294, and it can participate in epigenetic regulation by inhibiting H3K9 monomethylation and dimethylation. In addition, BRD4770 has certain anti-tumor ability. BRD4770 inhibits the production of reactive oxygen species and lipid peroxidation in cardiomyocytes by affecting the change of histone methylation, thereby reducing ferroptosis and apoptosis, and thus preventing and treating DOX-induced cardiotoxicity.

[0022] Compared with the prior art, the application of BRD4770 described in the present invention in the preparation of drugs for treating and / or preventing DOX-induced cardiomyopathy has the following advantages:

[0023] (1) Through cell experiments and animal experiments, the present invention explores from two dimensions in vivo and in vitro and finds that BRD4770 can effectively inhibit the production of reactive oxygen species and the accumulation of lipid peroxidation in cardiomyocytes caused by DOX, and further inhibit DOX-induced cardiomyocyte death, playing a role in treating and / or preventing DOX-induced cardiomyopathy, thereby confirming that BRD4770 can be used as a potential drug for preventing or treating DOX-induced cardiomyopathy.

[0024] (2) While playing a role in treating and / or preventing DOX-induced cardiomyopathy, BRD4770 described in the present invention does not affect the chemotherapy effect of DOX on tumors, and even has a certain anti-tumor effect. Description of the Drawings

[0025] Figure 1 It is a flow chart for compound screening;

[0026] Figure 2 It is the process of screening out the small molecule compound BRD4770: Among them, Figure 2 A is the Venn diagram of compound screening after treatment with compound DOX and Erastin; Figure 2 B is Figure 2 The list of 8 compounds in the intersection in A; Figure 2 C is the effect of BG45 and BRD4770 on DOX-induced H9C2 cell injury; Figure 2 D is the effect of BG45 and BRD4770 on Erastin-induced H9C2 cell injury;

[0027] Figure 3 It is the experimental result of BRD4770 inhibiting DOX-induced cardiomyocytes: Among them, Figure 3A is the statistical analysis of the activity of H9C2 cells detected by the CCK-8 method after pretreatment of H9C2 cells with BRD4770 or DMSO for 2 hours, followed by addition of DOX or DMSO and continued treatment for 24 hours; Figure 3 B is a representative diagram of the morphological changes of H9C2 cells after pretreatment of H9C2 cells with BRD4770 or DMSO for 2 hours, followed by addition of DOX or DMSO and continued treatment for 24 hours;

[0028] Figure 4 are the experimental results of BRD4770 inhibiting the accumulation of reactive oxygen species in cardiomyocytes induced by DOX: Among them, Figure 4 A is the detection of the ROS level of H9C2 cells by flow cytometry after staining with DCFH-DA probe after pretreatment of H9C2 cells with BRD4770 or DMSO for 2 hours, followed by addition of DOX or DMSO and continued treatment for 24 hours; Figure 4 B is Figure 4 the statistical analysis of A; Figure 4 C is the detection of the ROS level of H9C2 cells by fluorescence microscopy after staining with DCFH-DA probe after pretreatment of H9C2 cells with BRD4770 or DMSO for 2 hours, followed by addition of DOX or DMSO and continued treatment for 24 hours; Figure 4 D is Figure 4 the statistical analysis of C;

[0029] Figure 5 are the experimental results of BRD4770 inhibiting lipid peroxidation in cardiomyocytes induced by DOX: Among them, Figure 5 A is the detection of the lipid peroxidation level in H9C2 cells by fluorescence microscopy after staining with BODIPY after pretreatment of H9C2 cells with BRD4770 or DMSO for 2 hours, followed by addition of DOX or DMSO and continued treatment for 24 hours; Figure 5 B is for Figure 5 the quantitative statistical analysis of Ox-BODIPY in A; Figure 5 C is the statistical analysis of the MDA level in H9C2 cells after pretreatment of H9C2 cells with BRD4770 or DMSO for 2 hours, followed by addition of DOX or DMSO and continued treatment for 24 hours;

[0030] Figure 6 are the experimental results of BRD4770 reducing cardiomyocyte apoptosis induced by DOX: Among them, Figure 6 A is the effect of BRD4770 and different inhibitors on the activity of H9C2 cells treated with DOX; Figure 6 B is the effect of BRD4770 on the protein level of Cleaved-Caspase 3 in H9C2 cells induced by DOX; Figure 6 C is Figure 6 the statistical analysis of B; Figure 6D shows the effect of BRD4770 on apoptosis of DOX-treated H9C2 cells detected by flow cytometry; Figure 6 E is Figure 6 Statistical analysis of D;

[0031] Figure 7 The experimental results of the effect of BRD4770 on DOX-induced cardiomyopathy are as follows: Among them, Figure 7 A is the experimental flow chart of the effect of BRD4770 on DOX-induced cardiomyopathy; Figure 7 B is the representative diagram of the effect of BRD4770 on DOX-induced cardiac function detected by echocardiography; Figure 7 C shows the effect of BRD4770 on left ventricular ejection fraction (EF%) and fractional shortening (FS%) induced by DOX; Figure 7 D shows the effect of BRD4770 on DOX-induced cardiac atrophy; Figure 7 E is the detection of the effect of BRD4770 on DOX-induced cardiac atrophy and cardiac fibrosis by heart photography and section staining; Figure 7 F shows the effect of BRD4770 on the level of DOX-induced cardiac fibrosis; Figure 7 G shows the effect of BRD4770 on the cardiac injury marker Nppa produced by DOX; Figure 7 H shows the effect of BRD4770 on the cardiac injury marker Nppb produced by DOX; Figure 7 I shows the effect of BRD4770 on the cardiac injury marker Myh7 produced by DOX; Figure 7 J shows the effect of BRD4770 on the antioxidant level of the heart; Figure 7 K shows the effect of BRD4770 on the lipid peroxidation product MDA induced by DOX in the heart; Figure 7 L is the detection of the transcriptional levels of the ferroptosis marker genes Ptgs2 and Hmox1, confirming the protective effect of BRD4770 on DOX-induced ferroptosis of cardiomyocytes;

[0032] Figure 8 The experimental results of the effect of BRD4770 on the antitumor effect of DOX are as follows: Among them, Figure 8 A shows the antitumor effect of BRD4770 and its effect on the antitumor activity of DOX itself in murine breast cancer cells 4T1, human breast cancer cells MCF-7 and MDA-MB-231; Figure 8 B is the experimental flow chart of the effect of BRD4770 on the antitumor effect of DOX; Figure 8 C shows the effect of BRD4770 on the weight loss of mice caused by DOX treatment; Figure 8 D is the detection of the effect of BRD4770 on the cardiac function of mice after DOX treatment by echocardiography; Figure 8E is the effect of BRD4770 on the left ventricular ejection fraction (EF%) after DOX treatment; Figure 8 F is the effect of BRD4770 on the fractional shortening (FS%) after DOX treatment; Figure 8 G is the effect of BRD4770 on cardiac fibrosis after DOX treatment detected by slice staining; Figure 8 H is the effect of BRD4770 on cardiac fibrosis after DOX treatment; Figure 8 I is the effect of BRD4770 on the anti-tumor effect of DOX, confirming that BRD4770 does not affect the anti-tumor effect of DOX itself. Specific Embodiments

[0033] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following embodiments are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0034] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings.

[0035] I. Process of High-Throughput Screening of BRD4770 as a Treatment for DOX-Induced Cardiomyopathy

[0036] 1. The main implementation processes involved in this embodiment are as follows:

[0037] (1) Screen small molecule compounds from the epigenetic small molecule compound library that can prevent DOX-induced cardiomyocyte damage;

[0038] (2) Treat H9C2 cardiomyocytes with the small molecule compounds screened in (1), and conduct an activity screening experiment for anti-DOX-induced cardiomyocyte damage. Detect the changes in cardiomyocyte activity by CCK-8;

[0039] (3) Verify the effectiveness of the high-throughput screening drugs by CCK-8 experiment.

[0040] 2. The main experimental parts involved in this embodiment are as follows:

[0041] (1) Screen out small molecule compounds that can prevent DOX-induced cardiomyocyte damage

[0042] The experimental steps are as follows:

[0043] Using 773 small molecule compounds from the epigenetic small molecule compound library provided by TaoLab (Catalog No.: L1200), the compounds in the library can regulate more than a dozen epigenetic related targets such as HDACs, SIRTs, HATs, and HMTs. Since some compounds in the compound library are inhibitors synthesized by ChemDiv, which are suitable for biological screening and are difficult to purchase in the market, and their biological safety has not been proven, these drugs are not considered in the candidate drug screening for the time being.

[0044] (2) Activity screening experiment for anti-DOX-induced cardiomyocyte injury

[0045] The experimental steps are as follows:

[0046] 1) Seed 5000 rat cardiomyocytes H9C2 per well into a 96-well plate and culture in a cell incubator for 24 hours;

[0047] 2) Add 1 μL of the small molecule compound in the 5 mM epigenetic library to the cells in 1), and after 2 hours, add the inducer DOX (1 μM) or Erastin (2.5 μM) to induce the injury model;

[0048] 3) After culturing the cells in 2) for another 24 hours, add CCK-8 (10%), and after incubating for 2 hours, measure the absorbance value (OD value) at 450 nm using a microplate reader; (Steps 1)-3) are shown in Figure 1 the figure);

[0049] 4) Calculate the values of log 2 (inhibitor + DOX group / DOX group) and log2 (inhibitor + Erastin group / Erastin group) using the OD values;

[0050] 5) Screen for compounds with log 2 (foldchange) > 0, and compare the effective compounds obtained from the two groups of screening and take the intersection to screen for potential compounds with preventive and protective effects on DOX-induced cardiomyocyte injury; (Select the top 25 compounds in terms of numerical order for both the DOX group and the Erastin group, and take the intersection of the two groups of drugs through a bioinformatics website. Finally, 8 compounds are screened (see Figure 2 A-B) which can simultaneously protect against Erastin-induced ferroptosis of cardiomyocytes and DOX-induced myocardial injury. The screened compounds and their corresponding targets are sorted according to the degree of alleviating DOX myocardial injury ( Figure 2 B)).

[0051] (3) Verification of the effectiveness of high-throughput screening drugs by CCK-8 experiment

[0052] The experimental steps are as follows:

[0053] 1) Seed rat cardiomyocytes H9C2 into 96-well plates;

[0054] 2) The next day, add drugs to the cells in 1) according to the experimental groups: control (DMSO) group, DOX group, inhibitor + DOX group;

[0055] 3) After 24 hours, detect the OD value by a multifunctional microplate reader to verify and screen the effectiveness of the compound against DOX-induced cardiomyopathy;

[0056] 4) Compare the final results and select the compounds that are effective at the same time for later experiments. The results are shown in Figure 2 C-D (DOX treatment can significantly inhibit cell proliferation activity. After the action of BRD4770 and BG45, both can protect DOX-induced cardiomyocyte damage, but the protective effect of BRD4770 is more significant; similarly, Erastin treatment can significantly inhibit cell proliferation activity. After the action of BRD4770 and BG45, both can protect Erastin-induced cardiomyocyte ferroptosis, but the protective effect of BRD4770 is more significant).

[0057] Summary: In the above experimental process of this example, by screening the small molecule library with known activities of TargetMol, compounds with preventive and protective effects on DOX-induced cardiomyocyte damage were screened out. Again, the top 25 small molecule compounds with high docking scores were selected. The intersection of the two groups of drugs was taken through a bioinformatics website. The screened compounds and their corresponding targets were sorted according to the degree of alleviating DOX myocardial damage. Finally, 8 compounds, BRD4770 and BG45, were screened out, which can protect both Erastin-induced cardiomyocyte ferroptosis and DOX-induced myocardial damage at the same time. The screened compounds and their corresponding targets were sorted according to the degree of alleviating DOX myocardial damage. The two small molecule compounds BRD4770 and BG45 obtained by the final screening were respectively applied to cardiomyocytes damaged by DOX and Erastin induction, and the CCK-8 experiment was used to screen more effective compounds for preventing and treating DOX-induced myocardial damage. DOX or Erastin treatment can significantly inhibit cell proliferation activity. After the action of BRD4770 and BG45, both can protect DOX or Erastin-induced cardiomyocyte damage and ferroptosis, but the protective effect of BRD4770 (Cat. No.: T1923) is more significant.

[0058] II. BRD4770 significantly inhibits DOX-induced cardiomyocyte damage

[0059] The main experimental parts involved in this example are as follows:

[0060] 1. BRD4770 significantly inhibits DOX-induced cardiomyocyte death

[0061] 1) Pretreat H9C2 cells with BRD4770 (5 μM) or DMSO for 2 hours, and then add DOX (1 μM) or DMSO and continue to treat for 24 hours;

[0062] 2) Detect the activity of H9C2 cells in 1) by CCK-8 method. Compared with the control group, DOX significantly inhibited cell activity, and BRD4770 treatment could rescue the decrease in cardiomyocyte activity after DOX treatment. The results are shown in Figure 3 A;

[0063] 3) Observe the morphological changes of H9C2 cells in 1) under a microscope. After DOX treatment, the number of cells decreased, and some cells shrank, became round and floated in the cell culture medium. BRD4770 treatment reduced DOX-induced cardiomyocyte death. The results are shown in Figure 3 B.

[0064] The above results indicate that BRD4770 can rescue DOX-induced cardiomyocyte death.

[0065] 2. BRD4770 inhibits the accumulation of reactive oxygen species in cardiomyocytes induced by DOX

[0066] 1) Pretreat H9C2 cells with BRD4770 (5 μM) or DMSO for 2 hours, and then add DOX (1 μM) or DMSO and continue to treat for 24 hours;

[0067] 2) Stain the H9C2 cells in 1) with DCFH-DA-FITC probe and detect the intracellular reactive oxygen species level by flow cytometry. After DOX treatment, the intracellular reactive oxygen species level in cardiomyocytes increased significantly compared with the control group. BRD4770 treatment significantly inhibited the increase in intracellular reactive oxygen species caused by DOX treatment. The results are shown in Figure 4 A - B;

[0068] 3) Stain the H9C2 cells in 1) with DCFH-DA-FITC probe and detect the intracellular reactive oxygen species level of H9C2 cells by fluorescence microscopy. The results are similar to those of flow cytometry. The results are shown in Figure 4 C - D.

[0069] The above results indicate that BRD4770 inhibits the accumulation of reactive oxygen species in cardiomyocytes caused by DOX treatment.

[0070] 3. BRD4770 significantly inhibits lipid peroxidation in cardiomyocytes induced by DOX

[0071] 1) Pretreat H9C2 cells with BRD4770 (5 μM) or DMSO for 2 hours, and then add DOX (1 μM) or DMSO and continue to treat for 24 hours;

[0072] 2) After staining the H9C2 cells in 1) with BODIPY, the level of lipid peroxidation in H9C2 cells was detected by fluorescence microscopy. Treatment with DOX increased the accumulation of lipid peroxides (Ox-BODIPY) in cells, while treatment with BRD4770 significantly reduced the level of lipid peroxidation in cells induced by DOX. The results are shown in Figures 5A-B;

[0073] 3) The level of malondialdehyde (MDA) in the H9C2 cells in 1) was detected. After treatment with DOX, the level of malondialdehyde increased significantly, while treatment with BRD4770 significantly reduced the level of malondialdehyde in cells induced by DOX. The results are shown in Figure 5C.

[0074] The above results indicate that BRD4770 can significantly reduce the occurrence of intracellular lipid peroxidation induced by DOX treatment.

[0075] 4. BRD4770 significantly reduces DOX-induced cardiomyocyte apoptosis

[0076] 1) The rat cardiomyocytes H9C2 were seeded into 96-well plates;

[0077] 2) The next day, the ferroptosis inhibitor Fer-1, apoptosis inhibitor z-VAD-FMK, and antioxidant Trolox were added according to the grouping;

[0078] 3) After 2 hours, a DOX-induced injury model was added;

[0079] 4) The effects of BRD4770 and different programmed death inhibitors on DOX-induced cardiomyocyte injury were detected and compared by CCK-8 assay. Compared with Ferrostatin-1 (ferroptosis inhibitor), Deferoxamine (iron chelator), and Z-VAD-FMK (apoptosis inhibitor), BRD4770 more significantly rescued DOX-induced cardiomyocyte injury. The results are shown in Figure 6A; This result suggests that in addition to alleviating DOX-induced ferroptosis, BRD4770 can also protect DOX-induced cardiomyocyte injury through other pathways;

[0080] 5) The H9C2 cells were seeded in 6-cm dishes;

[0081] 6) The next day, drugs were added, and the cells were grouped. The drug addition treatment was the same as in 2);

[0082] 7) The proteins of the treated cells were harvested, and the expression of the apoptotic marker protein cleaved Caspase-3 in the cells was detected by immunoblotting. Treatment with DOX significantly increased the protein level of Cleaved-Caspase3, indicating that DOX induced apoptosis of cardiomyocytes. Treatment with BRD4770 significantly inhibited the increase in the protein level of Cleaved-Caspase3 caused by DOX, suggesting that BRD4770 prevented DOX-induced apoptosis of cardiomyocytes. The results are shown in Figures 6B-C.

[0083] 8) The treated glass slides were placed in a 12-well plate, and H9C2 cells were seeded on the glass slides.

[0084] 9) The next day, drugs were added, and the cells were grouped. The drug treatment was the same as in 2).

[0085] 10) The treated cells were stained with the Tunel kit, and the stained glass slides were photographed using a Zeiss confocal microscope. Compared with the control group, the proportion of AnnexinV-positive cells in the DOX treatment group was significantly increased. Treatment with BRD4770 significantly inhibited the increase in the proportion of AnnexinV-positive cells. The results are shown in Figures 6D-E.

[0086] The above results indicate that BRD4770 can alleviate DOX-induced cardiomyocyte injury by inhibiting apoptosis.

[0087] Summary: The above results show that BRD4770 can significantly inhibit DOX-induced cardiomyocyte death, intracellular reactive oxygen species accumulation and lipid peroxidation, and can alleviate DOX-induced cardiomyocyte injury by inhibiting apoptosis.

[0088] III. BRD4770 effectively alleviates DOX-induced heart injury in vivo

[0089] The main experimental parts involved in this example are as follows:

[0090] 1) DOX (5 mg / kg) was intraperitoneally injected once a week for three consecutive weeks to establish a chronic DOX injury model. BRD4770 (1 mg / kg) was pretreated 2 days before DOX stimulation and administered until the end of DOX induction.

[0091] 2) The cardiac function of the mice was detected by echocardiography, and the effect on the cardiac structure of the mice was further judged by section staining. The results are shown in Figure 7A.

[0092] 3) The echocardiography data showed that BRD4770 could significantly alleviate the decrease in left ventricular ejection fraction (EF%) and fractional shortening (FS%) induced by DOX, that is, alleviate DOX-induced cardiac function injury. The results are shown in Figures 7B-C.

[0093] 4) Heart photography and section staining confirmed that BRD4770 could effectively alleviate the cardiac atrophy and elevated levels of cardiac fibrosis caused by DOX, and the results are shown in 7D-F;

[0094] 5) Detection of cardiac injury markers Nppa, Nppb, and Myh7 confirmed that BRD4770 could effectively alleviate the upregulation of injury markers caused by DOX, and the results are shown in Figure 7 G-I;

[0095] 6) BRD4770 could effectively increase the antioxidant level of the heart, reduce the accumulation of lipid peroxides, and detection of the ferroptosis marker genes Ptgs2 and Hmox1 at the transcriptional level also confirmed the protection of BRD4770 against DOX-induced cardiomyocyte ferroptosis, and the results are shown in Figure 7 J-L.

[0096] Summary: The above results indicate that BRD4770 can effectively alleviate DOX-induced cardiac injury in vivo.

[0097] IV. Effect of BRD4770 on the antitumor effect of DOX

[0098] Since the only drug currently approved by the FDA for the treatment of DOX-induced cardiotoxicity, dexrazoxane, has the problem of reducing the efficacy of antitumor drugs, in this study, we explored the effect of BRD4770 on the antitumor effect of DOX in vivo and in vitro.

[0099] The main experimental parts involved in this example are as follows:

[0100] 1) Detection of the antitumor effect of BRD4770 in murine 4T1, human MCF-7, and MDA-MB-231 cells found that BRD4770 could exert a certain antitumor effect without affecting the antitumor activity of DOX itself, and the results are shown in Figure 8 A;

[0101] 2) Establish a breast cancer model by orthotopic injection of murine 4T1 cells;

[0102] 3) After the tumor reached an appropriate volume, inject BRD4770 (1 mg / kg) for pretreatment for 2 days;

[0103] 4) After treating the mice in 3) with DOX (1.5 mg / kg) for 10 days, take samples, and the results are shown in Figure 8 B;

[0104] 5) Detect the body weight of the mice in 4) and found that BRD4770 could significantly alleviate the decrease in body weight of the mice caused by DOX treatment, and the results are shown in Figure 8 C;

[0105] 6) Detect the cardiac function of the mice in 4), and it was found that the addition of BRD4770 alleviated the decline in cardiac function of the mice after DOX treatment. The results are shown in Figure 8 D-F;

[0106] 7) Detect the cardiac fibrosis of the mice in 4), and it was found that the addition of BRD4770 alleviated the increase in cardiac fibrosis of the mice after DOX treatment. The results are shown in Figure 8 G-H;

[0107] 8) Detect the anti-tumor effect of the mice during the DOX treatment involving BRD770, and it was found that the anti-tumor effect of DOX itself was not affected during this process. The results are shown in Figure 8 I.

[0108] Summary: The above results indicate that BRD4770 has a certain anti-tumor effect. While improving the DOX-induced cardiac toxicity in mice, it does not affect the chemotherapeutic effect of DOX on tumors.

[0109] This invention mainly uses laser confocal microscopy, q-RT PCR technology, immunoblotting technology (Western Blot) and molecular biology technology to study the effect and mechanism of the small molecule compound BRD4770 in preventing DOX-induced cardiac toxicity, and to promote its clinical application in the treatment of DOX-induced cardiomyopathy. In the early stage, an epigenetic compound library was used to screen drugs that can simultaneously prevent erastin-induced ferroptosis of cardiomyocytes and DOX-induced damage of cardiomyocytes; the CCK-8 experiment was used to detect the effect of BRD4770 on DOX-induced damage of cardiomyocytes. At the same time, combined with microscope photography, the morphological changes of cells after the action of BRD4770 were characterized, and its effect on cell proliferation activity was characterized; cell experiments were used to detect the anti-tumor effect of BRD4770 in breast cancer cells treated with DOX. A breast cancer model was constructed by in-situ injection of murine 4T1 cells, and BRD4770 was injected to participate in DOX treatment. The changes in body weight, cardiac function and fibrosis degree of the mice were detected to characterize its effect on the anti-tumor effect of DOX. The results show that BRD4770 can effectively inhibit the production of reactive oxygen species in cardiomyocytes, thereby reducing ferroptosis and apoptosis, thus preventing and treating DOX-induced cardiac toxicity. At the same time, it was detected that BRD4770 has a certain anti-tumor effect. While improving the DOX-induced cardiac toxicity in mice, it does not affect the chemotherapeutic effect of DOX on tumors. In summary, BRD4770 can be used as a new potential drug for clinically treating or preventing DOX-induced cardiomyopathy.

[0110] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of BRD4770 in the preparation of a drug for treating and / or preventing DOX-induced cardiomyopathy.

2. The use according to claim 1, characterized in that: The DOX-induced cardiomyopathy is a cardiac toxic side effect induced when DOX is used as a drug for tumor chemotherapy.

3. The use according to claim 1, characterized in that: The DOX-induced cardiomyopathy is at least one of arrhythmia, pericarditis, myocarditis, acute hypertension, and congestive heart failure.

4. The use according to claim 1, characterized in that: The BRD4770 alleviates DOX-induced cardiac function damage, cardiac atrophy, and cardiac fibrosis, improves cardiac antioxidant levels, and reduces lipid peroxide accumulation.

5. The use according to claim 1, characterized in that: The BRD4770 inhibits DOX-induced cardiomyocyte death.

6. The use according to claim 5, characterized in that: The BRD4770 inhibits DOX-induced cardiomyocyte cell death by inhibiting the generation of reactive oxygen species and accumulation of lipid peroxidation in cardiomyocytes caused by DOX.

7. The use according to any one of claims 1 to 6, characterized in that: The drug comprises BRD4770 as an active ingredient and a pharmaceutically acceptable carrier.

8. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the medicine is one of tablets, capsules, pills, suppositories, aerosols, granules, powders, injections, syrups, wine preparations, tinctures, dew preparations, and membrane preparations.

9. The use according to any one of claims 1 to 6, characterized in that: The administration route of the drug is one of oral administration, injection, implantation and external application.

10. The application of BRD4770 and DOX in the preparation of drugs for treating tumors, wherein BRD4770 does not affect the anti-tumor effect of DOX.

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