Application of T-5224 in preparation of medicine for preventing and treating doxorubicin-induced cardiotoxicity

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and relates to application of T-5224 in preparation of a medicine for preventing and treating doxorubicin-induced cardiotoxicity. According to the application, the pharmacological action of the T-5224 is researched, the application and the action mechanism of the T-5224 in the aspect of preventing and treating the doxorubicin-induced cardiotoxicity are found and verified, the effect of the T-5224 in the aspect of improving the doxorubicin-induced cardiotoxicity is remarkable, and a solid pharmacodynamic basis is provided for new clinical research and application of the medicine.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the development of new uses of known drugs, specifically the use of T-5224 for preparing drugs for preventing and treating doxorubicin-induced cardiotoxicity. Background Art

[0002] Doxorubicin is an anthracycline antibiotic widely used in cancer chemotherapy, but its cardiotoxic side effects are the main clinical limiting factor. Doxorubicin-induced cardiotoxicity is manifested by decreased left ventricular ejection fraction, increased serum myocardial enzymes, including creatine kinase (CK), creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), increased myocardial damage and increased cardiac fibrosis.

[0003] AP-1 is mainly composed of heterodimers of c-Jun and c-Fos proteins in mammals. It is actually one of the most critical transcription factors for regulating cell growth and differentiation, and is involved in important biological processes such as cell proliferation and differentiation. AP-1 can act on the binding site on the promoter of the target gene, namely the phorbol ester (12-O-Tetradecanoylphorbol-13-Acetate, TPA) response component, to regulate the transcription of the target gene. T-5224 is a selective AP-1 inhibitor with anti-inflammatory activity. It can specifically inhibit the DNA binding activity of c-Fos / c-Jun without affecting the binding activity of other transcription factors. Studies have shown that T-5224 can inhibit the upregulation of MMPs transcription induced by IL-1β.

[0004] Sarcolipin's sarcoplasmic lipoprotein SLN, SLN is a small molecule sarcoplasmic reticulum protein. It is mainly composed of 31 amino acid residues and is a highly conserved protein that is expressed in both the myocardial and skeletal muscles of mammals. In muscle cells, SLN is mainly localized in the sarcoplasmic reticulum, where it interacts with the calcium pump (SERCA) in the sarcoplasmic reticulum. In cardiomyocytes, this interaction is important for regulating the contraction and relaxation functions of the myocardium. For example, during myocardial relaxation, the sarcoplasmic reticulum needs to take in calcium ions, and SLN can regulate the activity of the calcium pump, thereby affecting the calcium ion uptake rate. In some heart diseases such as heart failure, the expression level or function of SLN may change. Studies have found that the expression of SLN in cardiomyocytes of patients with heart failure increases, which may further affect the calcium homeostasis of cardiomyocytes and aggravate myocardial dysfunction. Summary of the invention

[0005] In view of the defects in the prior art, the present invention aims to provide the use of T-5224 in preventing doxorubicin-induced cardiotoxicity and its specific mechanism of action.

[0006] One aspect of the present invention is to provide a use of T-5224 or a pharmaceutically acceptable salt or derivative thereof in the preparation of a medicament for treating and / or preventing cardiac injury.

[0007] Furthermore, the heart damage is heart failure, arrhythmia, and myocardial disease;

[0008] Furthermore, the cardiac injury is specifically cardiac injury with elevated troponin;

[0009] Furthermore, the heart damage is heart damage induced by anti-tumor drugs. Preferably, the anti-tumor drugs are doxorubicin, daunorubicin, epirubicin, and mitoxantrone; most preferably, the drug is doxorubicin.

[0010] Furthermore, the medicine also includes a pharmaceutically acceptable carrier.

[0011] The second aspect of the present invention provides a use of T-5224 or a pharmaceutically acceptable salt or derivative thereof in the preparation of a medicament for treating a disease with high expression of sarcoplasmic reticulum adiponectin (SLN).

[0012] Furthermore, the diseases are Duchenne muscular dystrophy (DMD) and heart failure.

[0013] Furthermore, the heart failure is heart damage induced by anti-tumor drugs. Preferably, the anti-tumor drugs are doxorubicin, daunorubicin, epirubicin, and mitoxantrone; most preferably, the drug is doxorubicin.

[0014] The third aspect of the present invention provides an application of T-5224 or a pharmaceutically acceptable salt or derivative thereof in the preparation of a preparation for inhibiting the overexpression of sarcoplasmic reticulum adiponectin (SLN), wherein the application is an in vitro cytological application, preferably, the cell is a cardiomyocyte cell line, and most preferably, the cell is AC16.

[0015] Furthermore, the T-5224 or a pharmaceutically acceptable salt or derivative thereof inhibits the activity of the transcription factor AP-1, thereby reducing its ability to bind to the SLN promoter and inhibiting the translation expression of SLN. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The effects of doxorubicin-induced cardiotoxicity on the heart in the animal experiment of the present invention, the animal experiment grouping and drug administration conditions, among which, A, drug administration time of mice; B, doxorubicin-induced cardiotoxicity modeling, and comparison of survival curves of the control group and the model group; C, echocardiograms of the hearts of the two groups of mice; D, statistics of left ventricular ejection fraction and shortening fraction of the hearts of the two groups of mice; E, Sirius red staining of cardiac pathological sections of the two groups of mice; F, expression levels of serum myocardial enzymes in the two groups of mice; G, transcription levels of cardiac fibrosis-related genes in the two groups of mice; H, transcription levels of cardiac injury-related genes in the two groups of mice; I, transcription levels of AP-1 family-related genes in the hearts of the two groups of mice; J, expression levels of AP-1 family-related proteins in the hearts of the two groups of mice; K, statistics of AP-1 family-related proteins in the hearts of the two groups of mice; L, transcription levels of troponin-related genes;

[0017] Figure 2 The animal experiment of the present invention confirms that T-5224 has the effect of preventing the onset of doxorubicin-induced cardiotoxicity, and the animal experiment grouping and drug administration conditions; wherein, A, drug administration time of mice; B, doxorubicin-induced cardiotoxicity modeling, and AP-1 inhibitor T-5224 were used at the same time, and the survival curves of the model group and the treatment group were compared; C, echocardiograms of the hearts of the two groups of mice; D, statistics of left ventricular ejection fraction and shortening fraction of the hearts of the two groups of mice; E, Sirius red staining of heart pathological sections of the two groups of mice; F, expression levels of serum myocardial enzymes in the two groups of mice; G, transcription levels of cardiac fibrosis-related genes in the two groups of mice; H, transcription levels of cardiac injury-related genes in the two groups of mice; I, transcription levels of AP-1 family-related genes in the hearts of the two groups of mice; J, expression levels of AP-1 family-related proteins in the hearts of the two groups of mice; K, statistics of AP-1 family-related protein expression levels in the hearts of the two groups of mice; L, transcription levels of troponin-related genes in the hearts of the two groups of mice;

[0018] Figure 3 The transcriptional regulation of SLN by AP-1 described in the present invention: A. Statistics of differentially expressed genes by volcano plots of transcriptomics screening of two groups of mice; B. Prediction of AP-1 binding sites in mammalian SLN promoter sequences and their corresponding mutant forms; C. ChIP-qPCR analysis of the interaction between AP-1 and two AP-1 binding sites in the SLN promoter region under normal conditions or under doxorubicin treatment conditions in human cardiomyocytes AC16; D. Transfection of SLN promoter luciferase reporter plasmids containing wild-type (WT) or mutant (Mut) AP-1 binding sites into human cardiomyocytes AC16 to detect their luciferase reporter activity; E. Expression of transcriptional levels of ANP and BNP in human cardiomyocyte cell line AC16 after treatment with SLN siRNA and 500 nM doxorubicin for 24 hours, respectively; DETAILED DESCRIPTION

[0019] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0020] Example 1 Phenotypes associated with the development of doxorubicin-induced cardiotoxicity

[0021] 1. Establishment of doxorubicin-induced cardiotoxicity model in mice

[0022] C57BL / 6J mice were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0023] All animals were housed in pathogen-free facilities with access to standard mouse chow and water and a 12-h light / 12-h dark cycle under controlled temperature conditions. All animal experiments were performed in accordance with the Guide for the Use and Care of Laboratory Animals and approved by the Ethics Committee of China Agricultural University. Forty male 8-week-old mice were randomly divided into two groups. The model group was given intraperitoneal injections of doxorubicin (3 mg / kg, Figure 1 A), cumulative dose of 24 mg / kg, modeling doxorubicin-induced cardiotoxicity. (See Figure 1 B), the heart was removed and preserved in tissue fixative on the day of death during the experiment. For the rest, cardiac ultrasound was performed the next day after the last administration, and the corresponding cardiac indicators were recorded (see Figure 1 C and D), the eyeballs were removed and blood was collected. The blood was centrifuged at 3500 rpm for 5 minutes to obtain serum for serum myocardial enzyme detection (see Figure 1 F), the heart was removed and stored in tissue fixative for Sirius red staining (see Figure 1 E), extract mRNA and protein from the heart to detect the transcriptional levels of fibrosis-related genes (see Figure 1 G), and detect the transcriptional levels of genes related to cardiac injury (see Figure 1 H), and the transcriptional levels of AP-1 family-related genes and SLN in the heart were detected (see Figure 1 I), and the levels of AP-1 family-related genes and SLN protein in the heart were detected (see Figure 1 J and K), and the transcription levels of troponin-related genes in the heart were detected (see Figure 1 Middle L).

[0024] Animal experiments have found that after doxorubicin-induced cardiotoxicity, the mortality rate of mice increased significantly compared with the control group, and the left ventricular ejection fraction and shortening fraction of the model group decreased, indicating that the heart's pumping function was impaired. Figure 1 The results shown in F and H show that compared with the control group, after adriamycin induction, the expression of serum myocardial enzymes AST, ALT, CK, CK-MB, LDH, and LDH-1 were upregulated, and the transcription levels of cardiac injury-related genes were upregulated, indicating that the degree of myocardial injury increased. Figure 1 As shown in E and 1G, compared with the control group, the area of ​​cardiac collagen fibers increased significantly after adriamycin induction. Figure 1 As shown in IK, after adriamycin induction, the expression levels of AP-1 family and SLN in mouse hearts increased significantly. Figure 1 As shown in L, after doxorubicin induction, the transcription levels of troponin-related genes in mouse hearts were upregulated.

[0025] Example 2 Protective Effect of T-5224 on Adriamycin-Induced Cardiotoxicity

[0026] Forty male mice aged 8 weeks were randomly divided into two groups. The model group was given intraperitoneal injection of doxorubicin (3 mg / kg, Figure 2 A), cumulative dose of 24 mg / kg, modeling doxorubicin-induced cardiotoxicity. On the basis of doxorubicin-induced cardiotoxicity, the treatment group was given T-5224 intraperitoneal injection (5 mg / kg / d) from the first day until the end of doxorubicin induction. During the experiment, survival was monitored every day and the survival curve was recorded (see Figure 2 B), the heart was removed and preserved in tissue fixative on the day of death during the experiment. For the rest, cardiac ultrasound was performed the next day after the last administration, and the corresponding cardiac indicators were recorded (see Figure 2 C and D), the eyeballs were removed and blood was collected. The blood was centrifuged at 3500 rpm for 5 minutes to obtain serum for serum myocardial enzyme detection (see Figure 2 F), the heart was removed and stored in tissue fixative for Sirius red staining (see Figure 2 E), extract mRNA and protein from the heart to detect the transcriptional levels of fibrosis-related genes (see Figure 2 G) and detect the transcriptional levels of genes related to cardiac injury (see Figure 2 H), and the transcriptional levels of AP-1 family-related genes and SLN in the heart were detected (see Figure 2 I), and the levels of AP-1 family-related genes and SLN protein in the heart were detected (see Figure 2 J and K), and the transcription levels of troponin-related genes in the heart were detected (see Figure 2 Middle L).

[0027] Animal experiments have found that after 14 days of intervention with a dose of 5 mg / kg / day (AP-linhibitor) of T-5224, the survival of patients with doxorubicin-induced cardiotoxicity was significantly improved, reaching 100%; compared with the model group, the left ventricular ejection fraction of the T-5224 intervention group increased, indicating that heart function was significantly improved. Figure 2 The results shown in F and H show that compared with the model group, after T-5224 intervention, the expression of serum myocardial enzymes AST, ALT, CK, CK-MB, LDH, and LDH-1 were downregulated, and the transcription level of cardiac injury-related genes was downregulated, indicating that the degree of myocardial injury was reduced. Figure 2 As shown in E and G, compared with the model group, the area of ​​cardiac collagen fibers was also reduced after intervention with 5 mg / kg / day of T-5224. Figure 2 As shown in IK, after T5224 intervention, the expression levels of AP-1 family and SLN in mouse hearts decreased. Figure 2 As shown in L, after T5224 intervention, the transcription levels of troponin-related genes in mouse hearts were downregulated.

[0028] Example 3: Transcriptional regulation of SLN by AP-1

[0029] Targeting the human cardiomyocyte cell line AC16, the SLN promoter-luciferase reporter plasmid containing wild-type (WT) or mutant (Mut) AP-1 binding sites, and SLN siRNA were synthesized from Miaoling Biotechnology Co., Ltd.

[0030] Transcriptomics screening of differentially expressed genes was performed on the hearts of mice in the control group and the model group, such as Figure 3 As shown in A, SLN has the highest differential expression fold; Figure 3 As shown in Figure B, the mammalian SLN promoter sequence was analyzed and two AP-1 binding sites and their corresponding mutant forms were predicted; in human cardiomyocytes AC16, ChIP-qPCR analysis of the interaction between AP-1 and the two AP-1 binding sites in the SLN promoter region under normal conditions or adriamycin treatment conditions was performed, and the SLN promoter luciferase reporter plasmid containing wild-type (WT) or mutant (Mut) AP-1 binding sites was transfected into human cardiomyocytes AC16, as shown in Figure 2B. Figure 3 As shown in C and D, the binding ability of AP-1 to the AP-1 binding site on the SLN promoter was enhanced after doxorubicin induction; after 24 hours of treatment with SLN siRNA and doxorubicin in the human cardiomyocyte cell line AC16, as shown in Figure 3 As shown in E, after doxorubicin treatment, the transcription levels of ANP and BNP were downregulated after SLN reduction, indicating that reducing SLN levels effectively alleviated the damage of cardiomyocytes.

[0031] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. Use of T-5224 or a pharmaceutically acceptable salt or derivative thereof in the preparation of a medicament for treating and / or preventing cardiac damage.

2. The use according to claim 1, characterized in that The heart damage is heart failure, arrhythmia, and myocardial disease.

3. The use according to claim 1, characterized in that The cardiac injury is specifically cardiac injury with elevated troponin.

4. The use according to claim 1, characterized in that The heart damage is heart damage induced by anti-tumor drugs, and the anti-tumor drugs are adriamycin, daunorubicin, epirubicin and mitoxantrone.

5. The use according to claim 4, characterized in that The medicine also includes a pharmaceutically acceptable carrier.

6. Use of T-5224 or a pharmaceutically acceptable salt or derivative thereof in the preparation of a drug for treating a disease with high expression of sarcoplasmic reticulum adiponectin (SLN).

7. The use according to claim 6, characterized in that The diseases are Duchenne muscular dystrophy (DMD) and heart failure.

8. The use according to claim 7, characterized in that The heart failure is heart damage induced by anti-tumor drugs, and the anti-tumor drugs are adriamycin, daunorubicin, epirubicin and mitoxantrone.

9. A use of T-5224 or a pharmaceutically acceptable salt or derivative thereof in the preparation of a preparation for inhibiting the overexpression of sarcoplasmic reticulum adiponectin (SLN), wherein the use is an in vitro cytological application, and the cells are a cardiomyocyte cell line.

10. The use according to claim 9, characterized in that The T-5224 or a pharmaceutically acceptable salt or derivative thereof inhibits the translation expression of SLN by inhibiting the binding ability of the transcription factor AP-1 to the SLN promoter.

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