Application of drunkanin A in prevention or treatment of adriamycin cardiomyopathy

Abstract

The invention relates to an application of drunkanin A in prevention or treatment of adriamycin cardiomyopathy. The invention discloses a novel medical application of drunkanin A, namely the drunkanin A can be used as a medicine for preventing and treating doxorubicin cardiomyopathy, and the compound can improve the heart function of doxorubicin mice, reduce the oxidative stress level in the doxorubicin mice, relieve myocardial cell death caused by doxorubicin and relieve cardiac organ injury. Through further research, the drunkanin A is found to play a role through a DUSP-1 target spot, and the DUSP-1 expression level reduction caused by adriamycin can be improved, so that the treatment of adriamycin cardiomyopathy is realized. More importantly, doxorubicin cardiomyopathy is common complications generated when doxorubicin is used for treating tumors, although the existing unique clinical medicine can relieve the complications, the existing unique clinical medicine has the risk of promoting generation and development of some tumors, and the medicine disclosed by the invention has the effects of inhibiting tumors and treating cardiomyopathy, so that the medicine has a good application prospect. The medicine is expected to be a powerful alternative for treating complications in the tumor treatment process of adriamycin.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of withaferin A in preventing or treating adriamycin cardiomyopathy. Background Art

[0002] Doxorubicin cardiomyopathy is one of the most serious and most common complications in patients with cancer chemotherapy. It is a dose-dependent and cumulative myocardial toxicity of doxorubicin, and can further develop into irreversible congestive heart failure and dilated cardiomyopathy and eventually lead to death. More importantly, it has been found clinically that the use of doxorubicin can cause liver and kidney damage. Doxorubicin (DOX) belongs to the anthracycline family and is a first-line chemotherapy drug for the clinical treatment of various tumors with the characteristics of good chemotherapy effect, wide application range, low resistance, and first choice for tumor recurrence. However, serious complications have severely limited its clinical application and many cancer patients cannot continue to receive efficient chemotherapy, resulting in easy tumor recurrence and severe shortening of survival. At present, there are no effective prevention and treatment measures for multi-organ damage caused by doxorubicin. Therefore, it is urgent to find new treatment measures to prevent and treat multi-organ damage caused by doxorubicin.

[0003] So far, there is no specific treatment for doxorubicin-induced cardiomyopathy in clinical practice. Currently, the main preventive treatment is dexrazoxane. Dexrazoxane is a derivative of ethylenediaminetetraacetic acid and is the only cardioprotective agent for cancer patients receiving anthracycline chemotherapy. A large number of clinical studies have shown that the use of dexrazoxane before the administration of anthracycline chemotherapy drugs can significantly reduce the heart damage caused by doxorubicin. However, it should be noted that dexrazoxane may increase the incidence of certain malignant tumors.

[0004] Therefore, although the current clinical use of dexrazoxane preventive treatment has been the core strategy to save doxorubicin-induced cardiomyopathy, there is a risk of increasing the incidence of malignant tumors. In view of this, it is particularly urgent to continue preclinical basic research to provide effective and specific prevention and treatment plans. Summary of the invention

[0005] In order to solve the above technical problems, the present invention discloses a new use of Withaferin A, which can be used as a powerful alternative drug for preventing heart damage caused by tumor treatment drugs including doxorubicin. The compound can improve the cardiac function of doxorubicin mice, reduce the level of oxidative stress in doxorubicin mice and alleviate the damage of the heart organ.

[0006] The invention discloses application of withaferin A or its derivatives in preparing a drug for preventing or treating adriamycin cardiomyopathy.

[0007] Adriamycin accumulates in myocardial cells and causes severe myocardial toxicity, which is the result of the participation of many factors, including: oxidative stress, autophagy, mitochondrial damage, DNA damage and other important factors. Adriamycin interacts with DNA by inserting and inhibiting the biosynthesis of macromolecules. This action inhibits topoisomerase II that unwinds DNA supercoils. After topoisomerase II unwinds the DNA chain for replication, adriamycin stabilizes topoisomerase II to prevent the DNA double helix from combining again, thereby stopping the replication process and inhibiting the synthesis of nucleic acids. The applicant has found through a large number of experiments that the use of withaferin A pretreatment in the adriamycin-induced cardiomyopathy model can significantly improve cardiac function and myocardial cell damage, indicating that withaferin A may be used for the treatment of adriamycin-induced cardiomyopathy. At the same time, in vitro experimental results show that withaferin A improves myocardial cell damage caused by adriamycin in cultured primary cardiomyocytes and can induce the expression of DUSP-1. Based on this, we imagine that withaferin A may provide a protective effect on adriamycin-induced myocardial toxicity by inducing the expression of DUSP-1.

[0008] Furthermore, the use comprises the step of administering withaferin A before administering doxorubicin.

[0009] Furthermore, the use also includes the step of administering withaferin A after administering doxorubicin.

[0010] Furthermore, the dosage form of the drug includes capsules, tablets, powders, injections or oral preparations.

[0011] Furthermore, the administration method includes intravenous administration or intraperitoneal administration.

[0012] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0013] Furthermore, the auxiliary materials include one or more of fillers, excipients, stabilizers, diluents, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, flavoring agents, preservatives, dispersants, film formers, plasticizers, pore formers, opacifiers, retardants, and solvents.

[0014] Furthermore, the medicine consists of 0.1-100% of withaferin A and 99.9-0% of auxiliary materials.

[0015] The second object of the present invention is to provide the use of withaferin A or its derivatives in the preparation of drugs for preventing or treating cardiomyopathy caused by tumor therapeutic drugs.

[0016] Furthermore, the tumor treatment drug specifically refers to a drug that may cause adverse reactions including cardiomyopathy during tumor treatment, including but not limited to doxorubicin.

[0017] The third object of the present invention is to provide a method for inhibiting doxorubicin toxicity of cardiomyocytes in vitro, comprising the step of adding withaferin A or its derivatives to the cardiomyocyte culture system.

[0018] Furthermore, the concentration of withaferin A is preferably 50-200 nM. Of course, those skilled in the art will appreciate that it can be adjusted as needed during actual use.

[0019] The fourth object of the present invention is to provide a drug for preventing or treating adriamycin-induced cardiomyopathy, comprising withaferin A or its derivatives.

[0020] By means of the above scheme, the present invention has at least the following advantages:

[0021] The present invention discloses a new use of withaferin A, which can reduce organ damage caused by doxorubicin, and provides a direction for the development of new drugs for the prevention of doxorubicin cardiomyopathy. A large number of animal experiments have verified that withaferin A has the effects of improving cardiac function in doxorubicin mice, reducing the level of oxidative stress in doxorubicin mice, and reducing myocardial cell damage. Secondly, withaferin A is easy to obtain, breaking through the limitations of other treatment options.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0024] Figure 1 To investigate the role of withaferin A in doxorubicin-induced cardiomyocyte death and oxidative stress;

[0025] Figure 2 The effect of different concentrations of withaferin A on doxorubicin-induced cardiomyocyte apoptosis;

[0026] Figure 3 To investigate the role of withaferin A in doxorubicin-induced cardiac dysfunction;

[0027] Figure 4 The role of withaferin A in myocardial tissue injury induced by adriamycin;

[0028] Figure 5 Withaferin A induces the expression of DUSP-1 in cardiomyocytes;

[0029] Figure 6 The role of DUSP-1 in doxorubicin-induced cardiomyocyte death and oxidative stress;

[0030] Figure 7 The role of DUSP-1 in doxorubicin-induced cardiac dysfunction and myocardial tissue damage;

[0031] Figure 8 To investigate the role of withaferin A and dexrazoxane in adriamycin-induced cardiomyocyte injury;

[0032] Fig. 9 The role of withaferin A in tumor cells.

[0033] The meanings of the p values ​​in the above figures are as follows: Figure 1 :*p<0.05vs Vehicle+Saline, vs Vehicle + DOX; Figure 2 : *p<0.05 vs Sham, vs DOX; Figure 3 :*p<0.05vs Vehicle+Saline, vs Vehicle+DOX; Figure 4 :*p<0.05vs Vehicle+Saline, vs Vehicle+DOX; Figure 6 :*p<0.05vsAd-gal orNC+Saline, vs Ad-gal or NC+DOX; Figure 7 :*p<0.05vsAAV9-cTNT-EGFP+Saline, vs AAV9-cTNT-EGFP+DOX; Figure 8 :*p<0.05vs Vehicle+Saline, vs Vehicle+DOX, vs Dexrazoxane+DOX; Fig. 9 : *p<0.05vsSaline. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0035] The materials and methods involved in the present invention are as follows:

[0036] (1) Preparation of 0.5 mg / mL Withaferin A working solution

[0037] Taking 1mL working solution as an example, take 100μL of 5mg / mL withaferin A (dissolved in DMSO) stock solution and add it to 400μL PEG300 and mix well; then add 50μL Tween-80 to the above system and mix well; then continue to add physiological saline to make up to 1mL.

[0038] (2) Construction of doxorubicin-induced primary cardiomyocyte injury model and doxorubicin-induced cardiomyopathy model in mice

[0039] (1) Isolation and culture of primary cardiomyocytes: Wild-type newborn mice (C57BL / 6) within 24 hours of birth were decapitated and their hearts were removed and cut into small pieces. Roche Liberase digestive enzyme was added and incubated and blown to obtain a single cardiomyocyte suspension. The suspension was centrifuged at 200 g for 5 minutes, the supernatant was removed, and the cardiomyocytes were resuspended and allowed to adhere to the culture dish for 2 hours. The non-adherent cells were gently aspirated to obtain primary cardiomyocytes and seeded into 24-well plates (about 3-5×10 5 Cells / well) were cultured overnight for subsequent experiments.

[0040] (2) The isolated primary cardiomyocytes were pretreated with withaferin A (100 nM) for 1 hour and stimulated with doxorubicin (1 μM) for 24 hours before use in subsequent experiments.

[0041] (3) Male SPF wild-type mice (C57BL / 6, weighing about 25 g) aged 8-10 weeks were randomly divided into four groups: control group, withaferin A group, dexrazoxane group, doxorubicin group, doxorubicin + withaferin A group, and doxorubicin + dexrazoxane group (the number of mice in each group was greater than 6).

[0042] (4) Each group of mice was intraperitoneally injected with withaferin A (2 mg / kg) or solvent control, and 1 hour later, they were intraperitoneally injected with doxorubicin (15 mg / kg, dissolved in saline) or saline to establish the doxorubicin myocardial toxicity model. On the 3rd day, withaferin A (2 mg / kg) or solvent control was intraperitoneally injected again. Five days later, the cardiac function of the mice was analyzed and myocardial tissue and serum were collected for subsequent experiments.

[0043] (5) After 5 days, the cardiac function of mice in each group was detected using an ultra-high-resolution cardiac ultrasound device. After euthanasia, peripheral serum and heart tissue samples of all mice were collected, some of which were fixed and some were placed in a -80°C refrigerator for analysis. The representative biochemical parameters and injury indicators of the hearts of doxorubicin-treated mice were detected. The specific steps are as follows:

[0044] A. Ultrasound evaluation of cardiac function: The tested mice were placed in an anesthesia chamber, and the isoflurane concentration was adjusted to 2% for anesthesia. The anesthetized mice were taken out and fixed in a supine position on a 37°C constant temperature operating table. The four paws were fixed to the stage and in good contact with the wafer. The chest hair was removed with depilatory cream. The Vevo 2100 high-resolution small animal ultrasound imaging system of Visual Sonics, Canada was used. The frequency of the linear array probe was 40MHz, and the plane and spatial resolution were: 40μm axially and 80μm laterally. The probe was placed on the left chest of the mouse, and the short-axis papillary muscle section was recorded with 2D ultrasound, and the left ventricular condition and tissue Doppler imaging were recorded using M-mode ultrasound at the papillary muscle level. The left ventricular end-systolic inner diameter (LVESD) and left ventricular end-diastolic inner diameter (LVEDD) were measured continuously for three cardiac cycles, and the fractional shortening (FS%) was calculated according to the following formula: FS% = [(LVEDD-LVESD) / LVEDD] × 100%, which was used to evaluate the cardiac contractile function of mice.

[0045] B. Serological testing: The peripheral serum of mice was collected and the indicators of myocardial cell injury, cTnI and LDH, were detected using commercial kits.

[0046] C. Detection of reactive oxygen species: Grind mouse heart tissue or collect cultured and processed primary cardiomyocytes, add horseradish peroxidase and Amplex Red to detect the amount of tissue reactive oxygen species produced. The final form of reactive oxygen species in tissue cells is hydrogen peroxide, and the product of hydrogen peroxide catalyzed by horseradish peroxidase can be labeled by the fluorescent signal carried by Amplex Red. The strength of the fluorescent signal represents the amount of tissue reactive oxygen species produced.

[0047] D. Detection of malondialdehyde (MDA): This detection method is based on the colorimetric reaction of MDA and thiobarbituric acid (TBA) to produce a red product. MDA is a natural product produced by lipid oxidation in organisms. When oxidative stress occurs in body cells, lipid oxidation also occurs. Some fatty acids are decomposed into a series of complex compounds after oxidation, including MDA. The level of lipid oxidation in the body can be reflected by detecting the content of MDA, so the determination of MDA is widely used as an indicator of lipid oxidation. In an acidic environment at a higher temperature, MDA can react with TBA to form a red MDA-TBA adduct, which has a maximum absorption light at 535nm, so it can be detected by colorimetry. The specific reaction principle is shown in the following formula:

[0048]

[0049] E. Cell apoptosis detection: The activity of caspase-3 in cardiomyocytes was analyzed using a caspase-3 activity detection kit; the content of DNA fragmentation in cardiomyocytes was analyzed using a DNA fragmentation ELISA detection kit from Roche.

[0050] F. Cell necrosis detection: CCK8 kit was used to analyze cardiomyocyte viability; LDH activity in culture supernatant and serum was analyzed using LDH activity detection kit.

[0051] G. Histopathological analysis: The pathological analysis of mouse heart tissue was performed using the hematoxylin-eosin (H&E) staining method. After the mice were euthanized, the heart tissue was collected and added with an appropriate volume of 10% formalin tissue fixative. After standing at room temperature for 24 hours, it was dehydrated with alcohol and embedded in paraffin. Paraffin sections with a thickness of 6 μm were cut using a paraffin slicer, and hematoxylin-eosin staining was performed after dewaxing, hydration, nucleus and cytoplasm staining, and decolorization. The pathological changes of the heart tissue structure were then observed under a microscope.

[0052] Example 1 Effect of Withaferin A on Doxorubicin-induced Cardiomyocyte Death and Oxidative Stress

[0053] Primary cardiomyocytes were isolated and cultured, pretreated with withaferin A (100 nM) for 1 hour, and treated with doxorubicin (1 μM) for 24 hours, and cardiomyocyte cell death and oxidative stress were detected. Figure 1 As shown in AB, withaferin A improved myocardial cell apoptosis induced by doxorubicin; at the same time, withaferin A improved myocardial cell necrosis induced by doxorubicin ( Figure 1CD). Further analysis revealed that withaferin A improved the production of reactive oxygen species and oxidative stress in cardiomyocytes induced by doxorubicin ( Figure 1 EF).

[0054] Example 2 Effects of different concentrations of withaferin A on adriamycin-induced cardiomyocyte apoptosis

[0055] In order to analyze the role of different concentrations of withaferin A in doxorubicin-induced cardiomyocyte apoptosis, primary cardiomyocytes were isolated and cultured, and pretreated with 50nM, 100nM, and 200nM withaferin A for 1 hour. After stimulation with doxorubicin (1μM) for 24 hours, caspase-3 activity and DNA fragmentation in cardiomyocytes were detected using commercial kits. Figure 2 As shown in AB, withaferin A dose-dependently inhibited the caspase-3 activity and DNA fragmentation of cardiomyocytes induced by doxorubicin, that is, it improved the apoptosis of cardiomyocytes induced by doxorubicin.

[0056] Example 3 Effect of Withaferin A on Doxorubicin-induced Cardiac Dysfunction

[0057] To study the role of withaferin A in doxorubicin-induced cardiac dysfunction, an adriamycin cardiomyopathy model was established. Withaferin A (2 mg / kg) was intraperitoneally administered to mice 1 hour before and 3 days after doxorubicin administration, and the cardiac function of the mice was analyzed 5 days later. Figure 3 As shown in AC, withaferin A improved the decrease in cardiac ejection fraction and shortening fraction induced by doxorubicin, indicating that it can improve the cardiac function disorder caused by doxorubicin.

[0058] Example 4 Effect of Withaferin A on Myocardial Tissue Injury Induced by Adriamycin

[0059] To analyze the role of withaferin A in myocardial tissue damage and oxidative stress induced by doxorubicin, an adriamycin cardiomyopathy model was established. Withaferin A (2 mg / kg) was intraperitoneally administered to mice 1 hour before and 3 days after doxorubicin administration, and serum and myocardial tissue were collected 5 days later for detection. Figure 4 As shown in A, withaferin A improves myocardial tissue damage caused by doxorubicin, and withaferin A improves the increase of Troponin I, a specific indicator of myocardial cell damage caused by doxorubicin ( Figure 4 B), and also improve doxorubicin-induced cardiomyocyte apoptosis and reactive oxygen species production ( Figure 4 CD).

[0060] Example 5 Withaferin A induces the expression of DUSP-1 in cardiomyocytes

[0061] In order to study the target of withaferin A, primary cardiomyocytes were isolated and cultured, and treated with 50nM, 100nM and 200nM withaferin A for 24 hours, and the protein expression level of DUSP-1 was detected. Figure 5 As shown, withaferin A significantly upregulated the protein level of DUSP-1.

[0062] Example 6 Role of DUSP-1 in doxorubicin-induced cardiomyocyte death and oxidative stress

[0063] In order to study the role of DUSP-1 in doxorubicin-induced cardiomyocyte apoptosis and oxidative stress, primary cardiomyocytes were isolated and cultured, infected with adenovirus carrying the DUSP-1 gene for 24 hours, and then stimulated with doxorubicin (1 μM) for 24 hours. The caspase-3 activity and DNA fragmentation in cardiomyocytes were detected using commercial kits. Overexpression of DUSP-1 significantly reduced doxorubicin-induced cardiomyocyte apoptosis ( Figure 6 AB). However, knockdown of DUSP-1 by cholesterol-modified DUSP-1 siRNA exacerbated doxorubicin-induced cardiomyocyte apoptosis ( Figure 6 CD). At the same time, overexpression of DUSP-1 significantly improved the production of reactive oxygen species in cardiomyocytes induced by doxorubicin ( Figure 6 E), while knockdown of DUSP-1 exacerbated the production of reactive oxygen species ( Figure 6 F).

[0064] Example 7 The role of DUSP-1 in doxorubicin-induced cardiac dysfunction and myocardial tissue damage

[0065] In order to study the role of DUSP-1 in doxorubicin-induced cardiomyopathy, mice were given adeno-associated virus AAV9-cTNT-m-dusp1-EGFP carrying the cTNT promoter and DUSP-1 gene through the tail vein to achieve overexpression of DUSP-1 in cardiomyocytes. Four weeks later, doxorubicin (15 mg / kg) was given intraperitoneally, and the cardiac function and myocardial tissue damage indicators of the mice were detected after 5 days. Figure 7 As shown in AB, overexpression of DUSP-1 improved the reduction of cardiac ejection fraction and shortening fraction caused by doxorubicin, indicating that it can improve the cardiac dysfunction caused by doxorubicin. Further studies found that overexpression of DUSP-1 reduced the level of Troponin I, a specific marker of myocardial cell injury, and the level of LDH, a marker of myocardial cell injury.

[0066] Example 8 Effects of Withaferin A and Dexrazoxane on Adriamycin-induced Myocardial Cell Injury

[0067] In order to study the difference in the protective effects of withaferin A and dexrazoxane on doxorubicin-induced cardiomyocyte injury, primary cardiomyocytes were isolated and cultured, pretreated with withaferin A (100 nM) or dexrazoxane (10 μM) for 1 hour, and treated with doxorubicin (1 μM) for 24 hours, and the levels of cardiomyocyte apoptosis and oxidative stress were analyzed. Figure 8 As shown in AB, withaferin A or dexrazoxane can improve the apoptosis of cardiomyocytes induced by doxorubicin, and the degree of improvement of withaferin A is significantly better than that of dexrazoxane. Further analysis found that withaferin A or dexrazoxane can improve the level of oxidative stress of cardiomyocytes induced by doxorubicin, and the degree of improvement of withaferin A is also significantly better than that of dexrazoxane ( Figure 8 CD).

[0068] Example 9 Effects of Withaferin A in Tumor Cells

[0069] Literature reports that dexrazoxane increases the risk of malignant tumors. In order to study the effect of withaferin A on tumor cells, tumor cell line A549 was cultured and treated with withaferin A (100 nM) for 24 hours, and tumor cell apoptosis indicators were analyzed. Fig. 9 As shown, withaferin A significantly upregulated the activity of caspase-3 in tumor cells ( Fig. 9 A) and DNA fragmentation ( Fig. 9 B), namely, promoting apoptosis of tumor cells, indicating that withaferin A has more advantages than dexrazoxane in treating adriamycin-induced cardiomyopathy in tumor patients.

[0070] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Use of withaferin A or its derivatives in the preparation of drugs for the prevention or treatment of adriamycin cardiomyopathy.

2. The use according to claim 1, characterized in that: The use comprises the step of administering withaferin A before administering doxorubicin.

3. The use according to claim 1 or 2, characterized in that: The use further comprises the step of administering withaferin A after the administration of doxorubicin.

4. The use according to claim 1, characterized in that: The drug is available in capsules, tablets, powders, injections or oral preparations.

5. The use according to claim 1, characterized in that: The administration routes include intravenous administration or intraperitoneal administration.

6. The use according to claim 1, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that: Contains at least one of the following characteristics: (1) The excipients include one or more of fillers, excipients, stabilizers, diluents, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, colorants, sweeteners, flavoring agents, preservatives, dispersants, film formers, plasticizers, pore formers, opacifiers, retardants, and solvents; (2) The drug is composed of 0.1-100% withaferin A and 99.9-0% excipients.

8. Use of withaferin A or its derivatives in the preparation of drugs for preventing or treating tumor complications, characterized in that: The tumor complication is cardiomyopathy caused by tumor treatment drugs.

9. A method for inhibiting adriamycin toxicity in cardiomyocytes in vitro, characterized in that: The method comprises the steps of adding withaferin A or its derivatives into the cardiomyocyte culture system.

10. A drug for preventing or treating adriamycin cardiomyopathy, characterized in that: The medicine contains withaferin A or its derivatives.

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