CN113018357A

CN113018357A - Application of tea polyphenol and palbociclib combination in preparation of preparation for treating breast cancer

Info

Publication number: CN113018357A
Application number: CN202110182651.7A
Authority: CN
Inventors: 董一麟
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-06-25

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a combination of tea polyphenol and palbociclib in preparation of a preparation for treating breast cancer. The tea polyphenol extracted from tea leaves and the target drug palbociclib are combined, and in vitro experiments prove that the tea polyphenol and the target drug palbociclib have obvious synergistic effect when killing breast cancer cells. The new application of the combined medicine is beneficial to reducing the drug resistance of targeted medicines, improving the anti-tumor effect and providing scientific basis for researching and developing new medicines.

Description

Application of tea polyphenol and palbociclib combination in preparation of preparation for treating breast cancer

Technical Field

The invention provides a combined medicament for treating breast cancer, which contains unit preparations with different specifications and is used for simultaneously or respectively administering tea polyphenol and palbociclib. The combined medicine can effectively treat breast cancer, has obviously better effect than the single use of the palbociclib or the tea polyphenol, shows that the palbociclib and the tea polyphenol have synergistic effect after being matched and used under the specific proportion, and has good clinical application prospect.

Background

The breast is composed of skin fibrous tissue, breast gland and fat, and the malignant tumor occurring in the epithelial tissue of the breast gland is called breast cancer, which seriously threatens the health of women, and about 99 percent of breast cancer patients are women, and only 1 percent of breast cancer patients are men. Hundreds of thousands of women die of breast cancer every year in China, the morbidity and the mortality of the women are still continuously increased, and the breast cancer becomes a killer threatening the health of Chinese women, so that the search for a breast cancer treatment method is reluctant.

At present, researchers develop a series of drugs for treating breast cancer, such as tamoxifen, pyrroltinib, herceptin and the like, which are targeted drugs aiming at a single target point to develop a single pharmacophore, but in the actual treatment process, part of patients still have no response to the drugs, or the drugs generate drug resistance after a period of treatment. The breast cancer is a complex disease caused by multiple factors, and the traditional method for treating the cancer by using a single target has a remarkable effect in the early treatment stage, but the drug resistance is easy to appear along with the prolonging of the treatment time. In order to solve the problems, the mode of combined medication is usually adopted clinically, and the synergistic effect of two or more medicaments is utilized to increase the curative effect and reverse the drug resistance.

Palbociclib (Palbociclib), a novel antineoplastic drug developed by feverer usa, is the first cyclin-dependent kinase (CDK) inhibitor worldwide, and has achieved surprising efficacy in the treatment of Hormone Receptor (HR) positive, human epidermal growth factor receptor 2(HER2) negative breast cancers. Palbociclib selectively inhibits cyclin dependent kinases 4 and 6(CDK4/6), restores cell cycle control, blocks tumor cell proliferation, and is free of the side effects of traditional chemotherapy with minimal intestinal response.

Tea Polyphenol (TP) is a secondary metabolite of Tea, has multiple functions of resisting tumor, oxidation and aging, and is widely researched in the fields of food, medicine and the like. In the process of drug development, researchers find that the combination of tea polyphenol and analogues thereof and various anti-cancer drugs has a synergistic effect, and assist small-molecule targeted drugs in inhibiting the proliferation and metastasis of cancer cells. Tea polyphenol is polyhydroxy phenolic compound separated and purified from tea, can generate differentiation blocking effect on cancer cells in early stage of DNA synthesis of the cancer cells, and does not influence normal cell growth. Meanwhile, the tea polyphenol also has the function of resisting tumor and angiogenesis, and plays a role in resisting cancer by cutting off the way of nutrition sources for the survival of tumors and cutting off the migration channel for tumor amplification. In addition, the research shows that the tea polyphenol has good radiotherapy sensitization effect, the action concentration is far lower than the level required by cytotoxicity effect, and the tea polyphenol is an ideal combined medicament in clinic.

At present, no report about the combination of TP and Palbociclib for treating breast cancer is found.

Disclosure of Invention

The invention aims to provide application of TP and Palbociclib in treating breast cancer, relates to a combined medicine with a synergistic anti-tumor effect, and particularly relates to the combined application of TP and Palbociclib in the lowest effective dose, so that the breast cancer can be treated by high curative effect, less drug resistance and less toxic and side effects.

The invention is realized by the following technical scheme:

use of a combination of tea polyphenols and palbociclib for the preparation of a formulation for the treatment of breast cancer.

The application of the combination of tea polyphenol and palbociclib in preparing a breast cancer clinical preparation for adjuvant therapy of toxicity reduction and synergy.

Use according to

claim

1 or 2, characterized in that said breast cancer is ER-positive human breast cancer, HER 2-positive human breast cancer or triple-negative breast cancer.

Use according to

claim

1 or 2, wherein the tea polyphenol and palbociclib produce a synergistic effect.

Use according to claim 3, wherein the tea polyphenol and palbociclib produce a synergistic effect.

A pharmaceutical composition for treating breast cancer, which comprises tea polyphenol and palbociclib.

The pharmaceutical composition of claim 6, wherein the molar ratio of tea polyphenol to palbociclib is 8:1, 5:1 or 4: 1.

The invention has the following advantages and positive effects:

the invention has the positive effects that TP and Palbociclib are used together, and the low-dose composition of the two medicines has a synergistic effect in treating malignant tumors, particularly breast cancer. The two medicines are combined for use, so that the curative effect can be enhanced, the toxic and side effects can be reduced, and a new idea is provided for treating drug-resistant malignant tumors.

The invention adopts TP combined Palbociclib to test breast cancer cells, and in vitro experiments prove that the TP combined Palbociclib has synergistic effect, thereby being beneficial to reducing the toxic and side effect of chemotherapy drugs, improving the effect of inhibiting tumors and providing scientific basis for developing new drugs.

Drawings

FIG. 1 shows that the two drugs are used singly to inhibit the proliferation of MCF-7 cells at a concentration of 0.05-500. mu.M;

FIG. 2 shows the inhibition of MCF-7 cell proliferation when used alone and in combination with a fixed amount of TP Palbociclib (8: 1);

FIG. 3 CI values for MCF-7 cells for two doses alone and with a fixed value in combination with TP Palbociclib (8: 1);

FIG. 4 shows that MDA-MB-453 cell proliferation inhibition effect is achieved when two medicines are used singly at a concentration of 0.05-500. mu.M;

FIG. 5 shows inhibition of MDA-MB-453 cell proliferation when the two drugs are administered alone and in combination at a ratio of TP to Palbociclib (5: 1);

FIG. 6 is a graph showing the CI value of TP Palbociclib (5:1) on MDA-MB-453 cells;

FIG. 7 shows that MDA-MB-231 cells are inhibited from proliferating by using two single drugs at a concentration of 0.05-500. mu.M;

FIG. 8 shows inhibition of MDA-MB-231 cell proliferation when the two drugs are administered alone and in combination at a ratio of TP to Palbociclib (4: 1);

FIG. 9 CI values for MDA-MB-231 cells at the association ratio TP: Palbociclib (4: 1).

Detailed Description

The invention aims to provide a high-activity anti-tumor medicine composition and application thereof in preparing a medicine for treating breast cancer.

The anti-tumor medicine composition contains TP and Palbociclib.

The structural formulas of TP and Palbociclib are shown as I, II

The biomaterials, drugs and experimental methods used in the specific examples were as follows:

the breast cancer cell strain comprises ER positive human breast cancer cells MCF-7, HER2 positive human breast cancer cells MDA-MB-453 and triple negative breast cancer cells MDA-MB-231, wherein the cells are purchased from Shanghai cell banks of Chinese academy of sciences.

The medicines described in the invention are TP standard and Palbociclib standard, and the medicines are purchased from an Allantin chemical reagent net. Dissolving water and dimethyl sulfoxide respectively to prepare a solution with TP concentration of 10mmol/L and Palbociclib concentration of 10mmol/L, and storing the stock solution at-20 ℃. Diluted to the appropriate concentrations as listed in the table at the time of use.

MTT assay cell proliferation: the cells were cultured in DMEM cell culture medium containing 1% penicillin-streptomycin solution (double antibody) and 10% fetal bovine serum at 37 deg.C under 5% CO₂Culturing in an incubator. After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (concentration 2X 10) in a volume of 100. mu.L per well⁴cell/ml) at 37 ℃ with 5% CO₂Culturing in incubator for 24 hr, adding medicines with different concentration gradients, standing at 37 deg.C and 5% CO₂Culturing in a constant temperature incubator for 48 h. 20. mu.L of 5mg/mL MTT solution (prepared in PBS, 0.22 μm filter sterilized) was added to each well and placed at 37 ℃ in 5% CO₂And (5) continuously incubating for 4h in the constant-temperature incubator, and terminating the culture. The culture supernatant was carefully removed from the wells, 100. mu.L of DMSO was added to each well, the mixture was left at 37 ℃ for 10min, and the purple crystals were dissolved sufficiently, and the absorbance (OD) of each well was measured with a microplate reader (550nm, 570 nm).

Cell survival (%) × (experimental OD-blank OD)/(control OD-blank OD) × 100%.

IC₅₀: also known as the half-effective inhibitory concentration, i.e., the concentration of the drug at which cell viability is 50%. Solving a linear regression equation according to the MTT result and calculating IC₅₀The value is obtained.

Evaluation of drug interaction: the joint action of TP and Palbociclib is calculated by a CI value method and is calculated by using computer software CompuSyn, wherein CI <1 indicates that the two medicines have a synergistic action after being combined; CI is 1, which shows that the two medicines have additive effect after being combined; CI is greater than 1, and shows that the two drugs have antagonism after being combined.

The results of experiments on the proliferation inhibition of MCF-7, MDA-MB-453 and MDA-MB-231 cells by TP and Palbociclib monomers are shown in Table 1 below, and the IC50 values of TP and Palbociclib monomers in the three cells are shown.

TABLE 1 IC50 values of TP and Palbociclib on breast cancer cells

Example 1

The results of experiments on the proliferation inhibition effect of TP and Palbociclib at different concentrations, which were diluted with water and dimethyl sulfoxide, are shown in FIGS. 1-3 and tables 2-4.

TABLE 2 cell viability of TP to MCF-7

TP concentration (μ M)	Cell survival rate (%)
		50	78.21±1.83
300	15.86±3.82
		400	4.77±0.45

TABLE 3 cell viability of Palbociclib on MCF-7

TABLE 4 cell viability and CI values for MCF-7 with combinations of TP and Palbociclib

The combination has a synergistic effect, an antagonistic effect and an additive effect. The specific effect is generally judged by adopting a combination index CI, wherein CI is less than 1, which indicates that the two medicines have a synergistic effect after being combined; CI is 1, which shows that the two medicines have additive effect after being combined; CI is greater than 1, and shows that the two drugs have antagonism after being combined. From the above results, it can be seen that when TP and Palbociclib were administered simultaneously, the CI value reached <1 at a dose ratio of 8:1, at which time, the combination of the two had a better synergistic effect on MCF-7 cells.

Example 2

The results of experiments on the proliferation inhibition effect of TP and Palbociclib at different concentrations, which were diluted with water and dimethyl sulfoxide, are shown in FIGS. 4-6 and tables 5-7.

TABLE 5 cell viability of TP to MDA-MB-453

TP concentration (μ M)	CellsSurvival rate (%)
		37.5	81.67±1.73
150	52.69±2.65
		200	43.76±4.11
300	30.63±2.77
		400	14.75±4.24

TABLE 6 cell viability of Palbociclib against MDA-MB-453

Palbociclib concentration (. mu.M)	Cell survival rate (%)
		7.5	88.35±2.86
30	52.98±1.15
		40	43.77±2.89
60	30.72±2.30
		80	17.85±1.53

TABLE 7 cell viability and CI values for MDA-MB-453 with TP in combination with Palbociclib

There are 3 effects after drug combination: synergistic, antagonistic and additive effects. The specific effect is generally judged by adopting a combination index CI, wherein CI is less than 1, which indicates that the two medicines have a synergistic effect after being combined; CI is 1, which shows that the two medicines have additive effect after being combined; CI is greater than 1, and shows that the two drugs have antagonism after being combined. From the above results, it can be seen that when TP and Palbociclib were administered simultaneously, the CI value reached <1 at a dose ratio of 5:1, and at this time, the combination of TP and Palbociclib had a better synergistic effect on MDA-MB-453 cells.

Example 3

The results of experiments on the proliferation inhibition effect of TP and Palbociclib at different concentrations, which were diluted with water and dimethyl sulfoxide, are shown in FIGS. 7-9 and tables 8-10.

TABLE 8 cell viability of TP to MDA-MB-231

TP concentration (μ M)	Cell survival rate (%)
		20	87.78±3.52
30	80.37±2.80
		40	75.81±3.61
60	70.66±1.77
		80	64.09±2.96
120	58.74±3.03
		160	53.48±2.62
240	41.25±3.15
		320	33.29±2.44
480	18.59±3.17

TABLE 9 cell viability of Palbociclib against MDA-MB-231

Palbociclib concentration (. mu.M)	Cell survival rate (%)
		5	91.07±4.61
7.5	83.69±1.82
		10	75.74±2.30
15	71.76±1.76
		20	66.98±3.23
30	60.72±4.92
		40	54.86±5.05
60	42.78±2.61
		80	30.83±3.37
120	17.66±2.18

TABLE 10 cell viability and CI values for MDA-MB-231 with combination of TP and Palbociclib

There are 3 combined effects after drug combination: synergistic, antagonistic and additive effects. The specific effect is generally judged by adopting a combination index CI, wherein CI is less than 1, which indicates that the two medicines have a synergistic effect after being combined; CI is 1, which shows that the two medicines have additive effect after being combined; CI is greater than 1, and shows that the two drugs have antagonism after being combined.

From the above results, it can be seen that when TP and Palbociclib were administered simultaneously, the CI value reached <1 at a 4:1 dose ratio, at which time, the combination of both had a better synergistic effect on MDA-MB-231 cells.

Claims

1. Use of a combination of tea polyphenols and palbociclib for the preparation of a formulation for the treatment of breast cancer.

2. The application of the combination of tea polyphenol and palbociclib in preparing a breast cancer clinical preparation for adjuvant therapy of toxicity reduction and synergy.

3. Use according to claim 1 or 2, characterized in that said breast cancer is ER-positive human breast cancer, HER 2-positive human breast cancer or triple-negative breast cancer.

4. Use according to claim 1 or 2, wherein the tea polyphenol and palbociclib produce a synergistic effect.

5. Use according to claim 3, wherein the tea polyphenol and palbociclib produce a synergistic effect.

6. A pharmaceutical composition for treating breast cancer, which comprises tea polyphenol and palbociclib.

7. The pharmaceutical composition of claim 6, wherein the molar ratio of tea polyphenols to palbociclib is: ER positive human breast cancer 8:1, HER2 positive human breast cancer 5:1, triple negative breast cancer 4: 1.