CN111067884A

CN111067884A - Pharmaceutical composition and application thereof

Info

Publication number: CN111067884A
Application number: CN202010025091.XA
Authority: CN
Inventors: 高恶斌; 刘坤; 叶鹏林; 朱杨杰
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2020-04-28

Abstract

The invention relates to the field of biological medicine, in particular to a pharmaceutical composition and application thereof. The pharmaceutical composition provided by the invention remarkably reduces the myocardial cell damage caused by adriamycin in the SD suckling mouse myocardial cell injury test, and improves the cell activity; after detecting the activity of mitochondrial ATP of the cardiac muscle cells after the combined application, the combination of caffeic acid and adriamycin is found to enhance Na in the cardiac muscle cells⁺‑K⁺-ATPase and Ca²⁺-ATPase activity. The caffeic acid and adriamycin can be used for preventing and treating myocardial damage caused by adriamycin. Enhancing activity of myocardial cell after injury, reducing injury degree and apoptosis rate of myocardial cell, protecting myocardial cell mitochondria, reducing adverse side effect of adriamycin, and enlargingThe clinical application range of the adriamycin.

Description

Pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a pharmaceutical composition and application thereof.

Background

Adriamycin (DOX), also known as doxorubicin, is an anthracycline tumor antibiotic, can inhibit the synthesis of DNA and RNA, has a wide antitumor spectrum, and has killing effect on tumor cells of various growth cycles. It is mainly suitable for treating solid tumors such as acute leukemia, malignant lymphoma, breast cancer, etc. However, the clinical application process, which produces severe damage to cardiac muscle cells, can seriously affect the physical health of patients and even threaten the life safety of patients. Therefore, how to develop new drugs for inhibiting doxorubicin cardiotoxicity has been an important issue in medicine.

Caffeic Acid (CA) is a natural phenolic compound, widely exists in plant bodies, can be safely used in cosmetics, has wide antibacterial and antiviral activities, is mainly a medicine for stopping bleeding and increasing leucocytes, and has the functions of contracting and reinforcing capillaries, improving blood coagulation factors and increasing leucocytes and thrombocytes. In recent years, there have been reports in the literature that caffeic acid may be used for ameliorating symptoms such as oxidative stress, inflammatory reaction, and viral infection. However, at present, whether caffeic acid has a protective effect on adriamycin myocardial damage or not and a specific mechanism thereof have no literature report.

Disclosure of Invention

In order to solve the above technical problems, the present invention aims to provide a combination drug which can better alleviate drug-induced myocardial damage caused by adriamycin. The test result of the invention shows that the combined application of the caffeic acid and the adriamycin can obviously relieve the myocardial damage caused by the adriamycin, thereby reducing the adriamycin myocardial cytotoxicity, and the combined application of the caffeic acid and the adriamycin can be used for preventing and treating the myocardial cell damage caused by the adriamycin and expanding the clinical application range of the adriamycin.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a pharmaceutical composition comprising caffeic acid and adriamycin.

In some embodiments of the invention, the molar ratio of caffeic acid to doxorubicin is 0.25-3: 1.

In some embodiments of the invention, the molar ratio of caffeic acid to doxorubicin is 1.5: 1.

The invention also provides application of the pharmaceutical composition in preparation of drugs for treating drug-induced cardiomyopathy. Further, the drug-induced cardiomyopathy is cardiomyopathy caused by adriamycin.

The invention also provides a medicament for treating drug-induced cardiomyopathy, which comprises the medicinal composition.

Further, the molar ratio of caffeic acid to adriamycin in the medicine is 0.25-3: 1;

1. further preferably, the molar ratio of caffeic acid to adriamycin in the medicament is 0.25-3: 1. Further, the drug-induced cardiomyopathy is a myocardial injury disease caused by adriamycin.

The invention also provides application of the composition in preparing a medicament for preventing and treating myocardial cell damage caused by adriamycin.

The pharmaceutical composition provided by the invention remarkably reduces the myocardial cell damage caused by adriamycin in the SD suckling mouse myocardial cell injury test, and improves the cell activity; after detecting the ATP enzyme activity of mitochondria of the cardiac muscle cells after the medicine is taken, the combination of caffeic acid and adriamycin is found to enhance Na in the cardiac muscle cells⁺- K⁺-ATPase and Ca²⁺-ATPase activity.

Compared with the prior art, the invention has the beneficial effects that:

a series of test results prove that the combined use of the caffeic acid and the adriamycin can obviously improve the activity of the myocardial cells and reduce the myocardial cell injury and apoptosis caused by the adriamycin. The combined application of caffeic acid and adriamycin is found to enhance Na in the myocardial cells through the detection of cell mitochondrial ATPase activity⁺- K⁺-ATPase and Ca²⁺-ATPase activity to protect cardiomyocyte mitochondria. Based on the research results, the combined use of caffeic acid and adriamycin can prevent and treat myocardial damage caused by adriamycin. The method has the advantages of enhancing the activity of the damaged myocardial cells, reducing the damage degree and the apoptosis rate of the myocardial cells, protecting the mitochondria of the myocardial cells, reducing the side effect of the adriamycin and expanding the clinical application range of the adriamycin.

Drawings

FIG. 1 is a graph comparing the results of myocardial cell injury experiments in various groups;

FIG. 2 is a comparison of OD values of nucleosome contents in lysates of various groups of myocardial cells;

FIG. 3 shows the respective groups Na in example 4⁺- K⁺-a comparison of ATPase activity;

FIG. 4 shows groups of Ca in example 4²⁺Comparison of ATPase activity.

Detailed Description

The invention discloses a pharmaceutical composition and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The pharmaceutical composition and the raw materials and reagents used in the application thereof provided by the invention are all available in the market. The SD suckling mouse is provided by the experimental animal center of Jiangsu university; doxorubicin, caffeic acid, pancreatin were purchased from Sigma; 10% fetal bovine serum was purchased from Hyclone; DMEM medium was purchased from Gibico.

Example 1: effect of caffeic acid on Activity of Adriamycin-damaged cardiomyocytes

Taking SD suckling mice on one day of birth, taking the heart tip part after being sterilized and killed, and obtaining the myocardial cells by conventional digestion of pancreatin. Suspending the cardiomyocytes in DMEM medium containing 10% fetal calf serum at 37 deg.C with 5% CO₂The resulting cardiomyocytes were purified after 2h of culture. According to 10⁵one/mL of the cells was inoculated into a 96-well plate and cultured for another 48 h. The purified cardiomyocytes were randomly divided into 8 groups, which were a normal control group, an adriamycin group (DOX), a caffeic acid group (CA), and 5 groups of adriamycin + caffeic acid mixed groups (DOX + CA) with different concentrations. Dissolving adriamycin with normal saline to obtain a final concentration of 10 μ M; caffeic acid was dissolved in preheated double distilled water to a final concentration of 30 μ M and then diluted to the desired concentration in each group. Each set of detailed information is as follows:

① Control group (Control group) 200. mu.L DMEM

② doxorubicin group (1. mu.M) 180. mu.L DMEM + 20. mu.L DOX

③ caffeic acid (0.25 μ M) + doxorubicin group (1 μ M) 160 μ L DMEM +20 μ L CA +20 μ L DOX

④ caffeic acid (0.5 μ M) + doxorubicin group (1 μ M) 160 μ L DMEM +20 μ L CA +20 μ L DOX

⑤ caffeic acid (1 μ M) + doxorubicin group (1 μ M) 160 μ L DMEM +20 μ L CA +20 μ L DOX

⑥ caffeic acid (1.5 μ M) + doxorubicin group (1 μ M) 160 μ L DMEM +20 μ L CA +20 μ L DOX

⑦ caffeic acid (3 μ M) + doxorubicin group (1 μ M) 160 μ L DMEM +20 μ L CA +20 μ L DOX

⑧ caffeic acid (3. mu.M) group 180. mu.L DMEM + 20. mu.L CA.

The myocardial cells of the above groups were incubated at 37 ℃ with 5% CO₂After 24h incubation in the incubator 20. mu.L MTT (5mg/mL) was added per well. Note: MTT is unfrozen before use, and MTT is sensitive to light and can be operated under the condition of no light as far as possible. After 3-4h of incubation, the incubation was terminated and the culture medium in the wells was carefully aspirated. Add 150. mu.L of dimethyl sulfoxide (DMSO) per well and incubate at 37 ℃ for 10 min in an incubator or shake the shaker for 10 min at low speed. Then detecting OD by using a microplate reader_490nmThe absorbance (A) value of each well was measured, and the cell viability was measured.

The results of the experiments are shown in Table 1, and the cell viability of the doxorubicin group is lower than that of the Control group (C:)P<0.05), the cell activity of the adriamycin and caffeic acid mixed group is higher than that of the adriamycin group, and the adriamycin and caffeic acid mixed group has certain dose dependence; the cell viability of the single caffeic acid group is similar to that of the Control group, and therefore, the myocardial cell viability is remarkably improved after the caffeic acid and the adriamycin are combined.

TABLE 1 statistics of cardiomyocyte viability

Grouping	Cell survival rate (%)
		Control	90.2±5.4
DOX	44.6±7.1^#
		CA（0.25μM）+DOX	50.8±6.3^*
CA（0.5μM）+DOX	49.3±2.7^*
		CA（1μM）+DOX	53.8±8.1^*
CA（1.5μM）+DOX	60.8±6.2^*
		CA（3μM）+DOX	57.2±7.3^*
CA（3μM）	91.1±6.0^#

In comparison with the Control group,^# P<0.05; compared with the DOX group, the method has the advantages that,^* P<0.05。

example 2 Effect of caffeic acid on myocardial cell injury caused by Adriamycin

The cardiomyocytes grouped in example 1 were incubated at 37 ℃ with 5% CO₂After incubation in an incubator for 24h, cell supernatants of each group are extracted, and the Lactate Dehydrogenase (LDH) content in each group is detected by a full-automatic biochemical analyzer. FIG. 1 is a graph comparing the results of myocardial cell injury experiments in various groups; as shown in FIG. 1, the LDH content in the supernatant of DOX group was 36.87. + -. 1.86U/mL, which is much higher than that of Control group (6.92. + -. 0.37U/mL), while the LDH content in each of CA + DOX group was lower than that of DOX group, andexhibit some dose dependence; the LDH content of the CA group is close to that of the Control group, compared with that of the Control group,^* P<0.05; compared with the DOX group, the method has the advantages that,^# P<0.05. therefore, the adriamycin causes damage or death of the myocardial cells, the caffeic acid has no influence on the damage of the myocardial cells, and the caffeic acid and the adriamycin can inhibit the damage of the myocardial cells after being used together in a certain range.

Example 3 Effect of caffeic acid on Adriamycin-induced apoptosis of myocardial cells

The cardiomyocytes grouped in example 1 were incubated at 37 ℃ with 5% CO₂After incubation in an incubator for 24h, OD values reflecting nucleosome content (nucleosome can reflect apoptosis) in the lysate were detected in the ElISA manner. Note: after cell lysis, high speed centrifugation is required, and nucleosomes are contained in supernatant fluid of the cells.

FIG. 2 is a comparison of OD values of nucleosome contents in lysates of various groups of myocardial cells; as shown in FIG. 2, the OD value of the supernatant of DOX group is 0.88 + -0.013, which is much higher than that of Control group (0.38 + -0.013), while the OD value in each CA + DOX group is lower than that of DOX group and shows a certain dose dependence; OD values of the CA group and the Control group are close to each other, compared with the Control group,^* P<0.05; compared with the DOX group, the method has the advantages that,^# P<0.05. therefore, the adriamycin causes the apoptosis of the myocardial cells, compared with the control group, the caffeic acid has no influence on the myocardial cells, and the caffeic acid in a certain concentration range can inhibit the apoptosis of the myocardial cells after being combined with the adriamycin.

Example 4 Effect of caffeic acid on myocardial cell mitochondrial injury caused by Adriamycin

Taking SD suckling mice on one day of birth, taking the heart tip part after being sterilized and killed, and obtaining the myocardial cells by conventional digestion of pancreatin. Suspending the cardiomyocytes in DMEM medium containing 10% fetal calf serum at 37 deg.C with 5% CO₂The resulting cardiomyocytes were purified after 2h of culture. According to 2.5X 10⁵one/mL of the cells was inoculated in a 24-well plate and cultured for another 48 h. The cardiomyocytes were randomly divided into 3 groups, namely a normal control group, an adriamycin group (DOX), a caffeic acid group (CA) and 5 groups of adriamycin + caffeic acid mixed groups (DOX + CA) with different concentrations. Dissolving adriamycin with normal saline to obtain a final concentration of 10 μ M; dissolution of caffeic acidThe final concentration of the solution was 30. mu.M in preheated double distilled water, and the solution was diluted to the desired concentration in each group. Each set of detailed information is as follows:

① Control group (Control group) 1000. mu.L DMEM

② doxorubicin group (1. mu.M) 900. mu.L DMEM + 100. mu.L DOX

③ caffeic acid (1.5 μ M) + doxorubicin group (1 μ M) 800 μ L DMEM +100 μ L CA +100 μ L DOX.

The grouped myocardial cells were 37 ℃ and 5% CO₂After incubation in the incubator for 24h, the cells were disrupted with PBS buffer as a homogenization medium, and the supernatant was centrifuged. Performing enzymatic reaction, determining phosphorus, adjusting color of double distilled water to zero at 660nm with spectrophotometer with light path of 1cm, measuring absorbance value, and calculating mitochondria Na by phosphorus standard curve⁺- K⁺-ATPase and Ca²⁺-ATPase activity.

FIG. 3 shows Na in each group⁺- K⁺-a comparison of ATPase activity; FIG. 4 shows Ca in each group²⁺-a comparison of ATPase activity; as shown in FIG. 3, mitochondrial Na of the DOX group⁺- K⁺ATPase activity 0.63. + -. 0.14U/mL, much lower than that of Control (1.69. + -. 0.18U/mL), and mitochondrial Na in CA + DOX⁺- K⁺The ATPase activity is 1.06 +/-0.21U/mL, the activity is higher than that of the DOX group, and compared with that of the Control group,^* P<0.05; compared with the DOX group, the method has the advantages that,^# P<0.05. as shown in FIG. 4, group DOX mitochondrial Ca²⁺The ATPase activity is 1.99 +/-0.44U/mL, the activity is far lower than that of the Control group 3.83 +/-0.28U/mL, and the CA + DOX group mitochondrial Na⁺- K⁺-atpase activity 2.95 ± 0.13U/mL, higher than that of DOX group; in comparison with the Control group,^* P<0.05; compared with the DOX group, the method has the advantages that,^# P<0.05。

it will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention, and that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention.

Claims

1. A pharmaceutical composition characterized by: including caffeic acid and doxorubicin.

2. The pharmaceutical composition according to claim 1, wherein the molar ratio of caffeic acid to doxorubicin is 0.25-3: 1.

3. The pharmaceutical composition of claim 1, wherein the molar ratio of caffeic acid to doxorubicin is 1.5: 1.

4. A medicament for treating drug-induced cardiomyopathy, which comprises the pharmaceutical composition as claimed in any one of claims 1 to 3 and pharmaceutically acceptable auxiliary materials.

5. The medicament of claim 4, wherein the drug-induced cardiomyopathy is a myocardial injury disease caused by adriamycin.

6. Use of a pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of a drug-induced cardiomyopathy.

7. The use of claim 6, wherein the pharmaceutical cardiomyopathy is a cardiomyopathy induced by doxorubicin.