A pharmaceutical composition for treating breast cancer and its application

Abstract

The present invention provides a pharmaceutical composition for treating breast cancer and its application, belonging to the field of biomedicine technology. A pharmaceutical composition for treating breast cancer, comprising eranine and cisplatin in a mass ratio of (5~100): (1~1.250). The application of the pharmaceutical composition for treating breast cancer in the preparation of a drug for preventing and / or treating triple-negative breast cancer. The pharmaceutical composition can reduce the cell activity of triple-negative breast cancer cells, inhibit the growth of triple-negative breast cancer tumors, and induce ferroptosis in triple-negative breast cancer cells. It can be seen that the technical solution provided by the present invention provides a new means for the treatment or drug preparation of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition for treating breast cancer and application thereof. Background Art

[0002] Cancer is a fatal disease that threatens human life worldwide and imposes a serious economic and disease burden on society. Breast cancer is the cancer with the highest morbidity and mortality rate among women worldwide, and is also the leading cause of cancer-related death in women. The incidence has been increasing year by year in recent years. Triple-negative breast cancer accounts for about 15% to 20% of all breast cancers, with a mortality rate of 40% within 5 years after diagnosis. The conventional standard treatment of this type of breast cancer is ineffective, and distant metastasis is prone to occur. The prognosis is worse than other types of breast cancer, and the recurrence rate and mortality rate are higher than other types of breast cancer. At present, triple-negative breast cancer lacks effective therapeutic targets, and chemotherapy is the main treatment method. However, the high recurrence rate after surgery and adjuvant chemotherapy leads to cancer treatment failure, which is still an unresolved medical challenge. Therefore, it is necessary and urgent to develop new drugs to treat triple-negative breast cancer.

[0003] Erianin is a small molecule compound isolated from the traditional Chinese medicine Dendrobium chrysotoxum. In addition, Erianin is also an important bioactive component of Dendrobium nobile and Dendrobium chrysotoxum, and belongs to the bibenzyl class of compounds. It has a wide range of pharmacological activities, including anti-tumor, antibacterial and antiviral effects. In recent years, a large number of literatures have reported that Erianin has a broad-spectrum anti-tumor effect, including inhibiting the proliferation of more than 30 malignant tumor cells such as lung cancer, liver cancer, breast cancer, cervical cancer, gastric cancer, osteosarcoma, leukemia, colorectal cancer, etc., and has good broad-spectrum anti-tumor activity. Regarding the anti-tumor activity of breast cancer, Erianin can induce cell cycle arrest of Luminal A breast cancer, but there is no information on whether Erianin has anti-tumor activity against triple-negative breast cancer.

[0004] Cisplatin (DDP), also known as cis-dichlorodiammineplatinum, is a platinum-containing anticancer drug. It is an orange-yellow or yellow crystalline powder, slightly soluble in water and easily soluble in dimethylformamide. It can gradually convert into trans and hydrolyze in aqueous solution. Cisplatin is currently a commonly used metal platinum complex. The platinum atom in the molecule is of great significance to its anti-tumor effect. However, only the cis form is meaningful, and the trans form is ineffective. Cisplatin can cross-link with DNA chains and show cytotoxic effects. After dissolution, it can pass through the charged cell membrane without the need for carrier transport in the body. Cisplatin can bind to the bases of DNA in the cell nucleus to form three forms of cross-linking, causing DNA damage, disrupting DNA replication and transcription, and also inhibiting RNA and protein synthesis at high concentrations. Cisplatin has the advantages of a wide anti-cancer spectrum, effective in hypoxic cells, and strong action. It has been widely used to treat testicular cancer, ovarian cancer, uterine cancer, bladder cancer, neck cancer, prostate cancer, brain cancer, etc., with significant efficacy. However, currently cisplatin is only used in combination with other types of chemotherapy drugs to treat breast cancer, and no reports have been found on the combined use of cisplatin and erythrin to treat triple-negative breast cancer. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a pharmaceutical composition for treating breast cancer, which combines cisplatin and Erianin to induce ferroptosis of triple-negative breast cancer cells, thereby achieving the efficacy of inhibiting the growth of triple-negative breast cancer tumors.

[0006] The present invention provides a pharmaceutical composition for treating breast cancer, comprising Erianin and cisplatin;

[0007] The mass ratio of Erianin to cisplatin is (5-100): (1-1.250).

[0008] Preferably, the mass ratio of Erianin to cisplatin is (10-80):1.

[0009] Preferably, the mass ratio of Erianin to cisplatin is 25:1.

[0010] The present invention provides use of the pharmaceutical composition for treating breast cancer in preparing a drug for preventing and / or treating triple-negative breast cancer.

[0011] Preferably, the treatment of triple-negative breast cancer is manifested in at least one of the following aspects: reducing the cell activity of triple-negative breast cancer cells, inhibiting the growth of triple-negative breast cancer tumors, and inducing ferroptosis in triple-negative breast cancer cells.

[0012] Preferably, the inducing ferroptosis in triple-negative breast cancer cells comprises increasing the content of reactive oxygen species, malondialdehyde and ferrous iron in the triple-negative breast cancer cells and / or reducing the expression of ferroptosis-related proteins.

[0013] Preferably, the dosage form of the drug includes an injectable preparation that is administered via at least one of intravenous, arterial or intracavitary administration.

[0014] Preferably, the injectable preparation includes a lyophilized preparation and / or an aqueous injection preparation.

[0015] The present invention provides the pharmaceutical composition for treating breast cancer, and use of Erianin or cisplatin in preparing an in vitro triple-negative breast cancer cell inhibitor.

[0016] The present invention provides a method for inhibiting the growth of triple-negative breast cancer cells in vitro, wherein the triple-negative breast cancer cells are cultured in an environment containing Erianin and / or cisplatin;

[0017] The concentration of Erianin is not less than 50 μg / mL;

[0018] The concentration of cisplatin is not less than 2.5 μg / mL.

[0019] The present invention provides a pharmaceutical composition for treating breast cancer, comprising Erianin and cisplatin; the mass ratio of Erianin to cisplatin is (5-100): (1-1.250). The embodiment of the present invention records that the CCK-8 method is used to detect the effect of Erianin and cisplatin on the cell activity of triple-negative breast cancer cells. The results show that Erianin and cisplatin reduce the survival rate of MDA-MB-231 cells in a dose-dependent manner, and the combined efficacy of the two significantly improves the inhibitory effect on triple-negative breast cancer cells due to single administration. At the same time, the embodiment of the present invention carried out a drug administration experiment on triple-negative breast cancer model mice. The results showed that after Erianin, cisplatin and Erianin plus cisplatin combined drug treatment, the tumor growth of breast cancer model mice was significantly reduced, and the combined drug treatment group had a more significant inhibitory effect than the single drug treatment group. At the same time, the results of the toxicity experiment showed that the drug composition formed by Erianin and cisplatin was administered to nude mice, and there was no significant difference compared with the solvent control group, indicating that the drug composition has good drug safety. It can be seen that the pharmaceutical composition provided by the present invention can not only effectively inhibit the activity of triple-negative breast cancer cells and inhibit the growth of tumors in the body, but is also safe and non-toxic, providing a new idea for the treatment of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of CCK8 detection of cell activity of MDA-MB-231 cells after treatment with Erianin, cisplatin and combined drug administration, among which A is the detection result of Erianin treatment with different concentrations, B is the detection result of cisplatin treatment with different concentrations; C is the detection result of Erianin combined with cisplatin treatment with different concentrations, and D is the detection result of Erianin combined with different concentrations of cisplatin treatment;

[0021] Figure 2MDA (left), F e2+ (middle) and ROS (right) level result graphs;

[0022] Figure 3 The graph (left) and bar graph (right) show the effects of Erianin and cisplatin on the protein expression levels of GPX4 and SLC7A11 in MDA-MB-231 cells by WB.

[0023] Figure 4 The mean tumor volume of mice in each group (mean ± SD). DETAILED DESCRIPTION

[0024] The present invention provides a pharmaceutical composition for treating breast cancer, comprising Erianin and cisplatin; the mass ratio of Erianin to cisplatin is (5-100): (1-1.250).

[0025] In the present invention, the molecular formula of Erianin is C 18 H 22 O5, CAS No. 95041-90-0, molecular formula is shown in Formula I. The present invention has no special restrictions on the source of the Erianin, and it can be obtained by using the commercial purchase channels of Erianin known in the art. In the embodiment of the present invention, the Erianin is purchased from MCE Company.

[0026] Formula I

[0027] In the present invention, the molecular formula of cisplatin is C l2 H4N2Pt, CAS No. 15663-27-1, structural formula as shown in Formula II; the present invention has no special restrictions on the source of the cisplatin, and it can be obtained by using the commercial purchase channels of cisplatin known in the art. In the embodiment of the present invention, the cisplatin is purchased from MCE.

[0028] Formula II

[0029] In the present invention, the mass ratio of Erianin to cisplatin is preferably (10-80):1, more preferably (20-40):1, and most preferably 25:1.

[0030] In one embodiment of the present invention, the effect of the combination of Erianin and cisplatin on the cell activity of triple-negative breast cancer cells (MDA-MB-231) was detected at the cellular level. The results showed that the IC 50 At around 100 μg / mL, the IC of cisplatin 50Around 5 μg / mL. The combined use of Erianin and cisplatin significantly improves the inhibitory effect on tumor cells compared with single drugs. When the concentration of Erianin is 25-100μg / mL and cisplatin is 5μg / mL or the concentration of Erianin is 100μg / mL and cisplatin is 1.25μg / mL, 2.5μg / mL and 5μg / mL, the cell survival rate is below 70%, which is significantly lower than that of the blank control and DMSO control group. At the same time, when the concentration of Erianin is 200-400μg / mL and the concentration of cisplatin is 10-20μg / mL, the cell survival rate is below 40%, and the most preferred value is below 20%.

[0031] The present invention provides use of the pharmaceutical composition for treating breast cancer in preparing a drug for preventing and / or treating triple-negative breast cancer.

[0032] In the present invention, the treatment of triple-negative breast cancer is preferably manifested in at least one of the following aspects: reducing the cell activity of triple-negative breast cancer cells, inhibiting the growth of triple-negative breast cancer tumors, and inducing ferroptosis in triple-negative breast cancer cells. The induction of ferroptosis in triple-negative breast cancer cells preferably includes increasing the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and ferrous iron (Fe 2+ ) content and / or reduce the expression of ferroptosis-related proteins.

[0033] In the present invention, the dosage form of the drug preferably includes an injection preparation that is administered through at least one of intravenous, arterial or intracavitary administration. The injection preparation preferably includes a lyophilized preparation and / or a water injection preparation. The injection preparation preferably includes a pharmaceutically acceptable injection oil and a surfactant. The injection oil is preferably selected from synthetic or natural fatty acids, fatty acid triglycerides, vitamin E, ethyl oleate or a combination thereof, and more preferably medium chain triglycerides, oleic acid or a combination thereof. The present invention has no special restrictions on the type of the surfactant, and the type of surfactant known in the art can be used. The mass ratio of the injection oil and the surfactant is preferably (1~2): (1~10), and more preferably 1: (3~5). When the drug is made into a lyophilized preparation, it is preferably further included. The lyophilized scaffold agent is selected from mannitol, glucose, fructose, lactose, sucrose, glycine, lysine, histidine or a combination thereof. The weight ratio of the lyophilized scaffold agent is 10%~90% of the total mass of the drug, more preferably 30%~80%, further preferably 40%~60%, and most preferably 50%. When the drug is made into an aqueous injection preparation, it is preferred to also contain a pharmaceutically acceptable solvent and an osmotic pressure regulator. The solvent is preferably water, ethanol, glycerol, propylene glycol, polyethylene glycol or a mixture thereof, more preferably water. The osmotic pressure regulator is preferably selected from glucose, sodium chloride, glycerol, mannitol, lactose or a combination thereof. The osmotic pressure regulator is preferably 0.1% to 10% (weight to volume ratio, w / v) of the total mass of the drug. The 10% means that 10 g of solute is dissolved in 100 ml of solvent.

[0034] In view of the fact that Erianin and cisplatin can inhibit the growth of triple-negative breast cancer cells in vitro, the present invention also provides the use of the pharmaceutical composition for treating breast cancer in the preparation of an in vitro triple-negative breast cancer cell inhibitor.

[0035] The present invention provides a method for inhibiting the growth of triple-negative breast cancer cells in vitro, wherein the triple-negative breast cancer cells are cultured in an environment containing Erianin and / or cisplatin;

[0036] The concentration of Erianin is not less than 50 μg / mL;

[0037] The concentration of cisplatin is not less than 2.5 μg / mL.

[0038] In the present invention, the environment containing Erianin and / or cisplatin is preferably a triple-negative breast cancer cell culture medium containing Erianin and / or cisplatin. The growth temperature of the triple-negative breast cancer cells is preferably 36-38°C, more preferably 37°C. The growth conditions of the triple-negative breast cancer cells are preferably 5% CO2 conditions. The concentration of Erianin is preferably 50 μg / mL-100 μg / mL, more preferably 100 μg / mL. The concentration of cisplatin is preferably 2.5 μg / mL-5 μg / mL, more preferably 5μg / mL.

[0039] The pharmaceutical composition for treating breast cancer and its application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following examples, human triple-negative breast cancer cells MDA-MB-231 are used as an example to verify the anticancer effect of Erianin and cisplatin in combination. The MDA-MB-231 cell line is from ATCC (American Type Culture Collection, Manassas, VA, United States). MDA-MB-231 is cultured in DMEM (Gibco, Grand Island, NY, USA) medium containing 10% fetal bovine serum and double antibiotics at 37°C and conventional culture conduction (5% CO2, 95% air).

[0041] Example 1

[0042] Breast cancer cell viability assay.

[0043] CCK-8 (Cat: MA0218) (meilunbio, Dalian, China) proliferation assay was used to detect the cell viability of MDA-MB-231 cells after treatment with different drugs. Approximately 5 × 10 3 cells / well. Then different concentrations of Erianin (25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL and 400μg / mL), cisplatin (1.25μg / mL, 2.5μg / mL, 5μg / mL, 10μg / mL, 20μg / mL) and different concentrations of Erianin and cisplatin were used for 24 h. After 24 h of treatment, 10 μl / 100 μl of CCK-8 solution was added and placed at 37℃ for 2 h. The test wavelength was 450 nm. The data are expressed as mean ± standard deviation, and the data analysis was performed by t test.

[0044] The results are as follows Figure 1As shown in Figure 2, cell counting kit-8 (CCK-8) assay showed that Erianin and cisplatin reduced the survival rate of MDA-MB-231 cells in a dose-dependent manner. The results showed that the IC 50 At around 100 μg / mL, the IC of cisplatin 50 At around 5 μg / mL, the combined use of the two drugs significantly improved the inhibitory effect on tumor cells compared with using either drug alone.

[0045] Example 2

[0046] ROS, MDA, Fe 2＋ Determination of.

[0047] MDA-MB-231 cells were treated with Erianin (100 μg / mL), cisplatin (5 μg / mL), and Erianin (100 μg / mL) + cisplatin (5 μg / mL) for 24 h. The levels of ROS, MDA, and ferrous iron in cell lysates were evaluated using H2DCF-DA (Cat: S0033) (Beyotime Biotechnology, Shanghai, China), lipid peroxidation assay kit (ab118970, Abcam), and iron assay kit (MAK025, Sigma-Aldrich), respectively. The specific procedures were performed according to the instructions of the above kits. Data are expressed as mean ± standard deviation, and data analysis was performed using t test.

[0048] It is well known that lipid peroxidation and ferrous iron accumulation are important phenomena of ferroptosis. Figure 2 The results showed that after 24 h of treatment with Erianin, cisplatin and Erianin + cisplatin, the intracellular ROS level increased, significantly upregulated the cellular MDA level, and triggered ferrous iron accumulation.

[0049] Example 3

[0050] Western Blot analysis experiment.

[0051] MDA-MB-231 cells were treated with Erianin (100 μg / mL), cisplatin (5 μg / mL), and Erianin (100 μg / mL) + cisplatin (5 μg / mL) for 24 h. Protein lysates were prepared using RIPA lysis buffer (Beyotime) and quantified using a BCA protein assay kit (Beyotime). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The blots were then blocked with 5% skim milk and incubated with primary antibodies overnight at 4°C, followed by secondary antibodies. Signals were detected using an ECL substrate (Beyotime). The primary antibodies used for western blot were as follows: anti-GPX4 (1:1000, ab125066, Abcam), anti-SLC7A11 (1:1000, ab175186, Abcam). Data are presented as mean ± standard deviation, and data analysis was performed using t-test.

[0052] The results are as follows Figure 3 As shown. Western blotting was used to detect the effects of Erianin and cisplatin on ferroptosis-related proteins. Compared with the control group, glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (xCT / SLC7A11) in MDA-MB-231 cells were significantly decreased after Erianin and cisplatin treatment. Taken together, these results indicate that ferroptosis plays a major role in Erianin and cisplatin-induced breast cancer cell death.

[0053] Example 4

[0054] Effects of Erianin and Cisplatin alone and in combination on breast cancer model mice.

[0055] Nude mice (6 weeks old, 6 mice per group) were ordered from the SLAC Laboratory Animal Center in Shanghai, China. All animal experiments were performed with the approval of the Animal Experiment Ethics Committee of the First Hospital of Jilin University; Certificate No. KT202402380. Approved by the Laboratory Animal Center. To study the antitumor effects of Erianin and cisplatin in vivo, mice were randomly divided into five groups: control group, positive control group, Erianin (100 mg / kg), DDP (4 mg / kg), and Erianin (100 mg / kg) + DDP (4 mg / kg). 5 × 10 cells were injected subcutaneously with 0.2 ml of cell suspension into the second nipple fat pad on the right side of nude mice. 7 / ml MDA-MB-231 cell suspension. After the model was successfully established, different doses of drugs were injected into the tail vein of the mice every day according to the grouping situation, and the drugs were administered continuously for 4 weeks. The positive control group was given docetaxel, and the control group was given an equal volume of saline every day. The tumor size was measured every 4 days, and the tumor volume was calculated as: 0.5 × length (L) × width (W) 2Tumors were collected and weighed on day 28, and mice in each group were weighed separately. Data are expressed as mean ± standard deviation, and data analysis was performed using t test.

[0056] The experimental results are as follows Figure 4 The results showed that compared with the control group without medication, the tumor growth of breast cancer model mice was significantly reduced after treatment with Erianin, cisplatin and Erianin plus cisplatin, and the inhibitory effect of the combined drug treatment group was more significant than that of the single drug treatment group. There was no statistically significant difference in the weight of mice in the control group and the treatment group.

[0057] Example 5

[0058] Acute toxicity test of Erianin + cisplatin combination.

[0059] Nude mice (6 weeks old, 20 mice in each group) were randomly divided into two groups, namely, the Erianin + cisplatin group and the solvent control group, and the drugs were administered after fasting (without water deprivation) for 12 hours. The Erianin + cisplatin group was intragastrically administered with Erianin (100 mg / kg) and cisplatin (4 mg / kg) once; the solvent control group was given an equal amount of distilled water. After administration, the mice were observed for two consecutive weeks, and the behavior, activity, body weight, food intake, feces and death of the mice were recorded. After two weeks, they were killed for autopsy.

[0060] After two weeks of administration of the Erianin + cisplatin combination, the nude mice had smooth fur, normal behavior, weight gain, food intake, and feces, with no significant difference compared with the solvent control group; and no death or other abnormal conditions occurred within two weeks. The two groups (Erianin + cisplatin combination group and solvent control group) were killed at the same time and autopsied. No abnormal changes were observed in the main organs (heart, liver, spleen, lung, and kidney) by naked eye observation. There was no significant difference between the Erianin + cisplatin combination group and the solvent control group. The results showed that there was no obvious toxic reaction in the 20 nude mice after administration, and no death occurred within two weeks.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for treating triple-negative breast cancer, wherein the pharmaceutical composition comprises Erianin and cisplatin; The mass ratio of Erianin to cisplatin is (20-25):

1.

2. The use according to claim 1, characterized in that: The treatment of triple-negative breast cancer is manifested in at least one of the following aspects: reducing the cell activity of triple-negative breast cancer cells, inhibiting the growth of triple-negative breast cancer tumors, and inducing ferroptosis in triple-negative breast cancer cells.

3. The application according to claim 2, characterized in that: The inducing ferroptosis in triple-negative breast cancer cells comprises at least one of the following: increasing the content of reactive oxygen, malondialdehyde and ferrous iron in the triple-negative breast cancer cells and reducing the expression of ferroptosis-related proteins.

4. The use according to claim 1, characterized in that: The dosage form of the drug includes an injectable preparation administered via at least one of intravenous, arterial or intracavitary administration.

5. The application according to claim 4, characterized in that: The injectable preparation includes a freeze-dried preparation and / or an aqueous injection preparation.

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