Use of a combination of compounds in the manufacture of a medicament for the treatment of breast cancer

Abstract

The application discloses application of a compound combination in preparation of a drug for treating breast cancer, and the compound combination comprises an exportin XPO1 inhibitor and a CDK4 / 6 inhibitor. The application discloses a new drug administration mode of KPT-330 (selinexor) and a CDK4 / 6 inhibitor in treatment of breast cancer, and the medical name of KPT-330 is selinexor, which is a specific small molecule inhibitor of nuclear export protein 1 (XPO1). The CDK4 / 6 inhibitor is a kind of small molecule compound structural formula, which directly inhibits the formation of a CDK4 / 6-Cyclin D complex, reduces the phosphorylation of RB1, inhibits the release of an E2F family, and plays a role in blocking cells in the G1 phase. The application first proposes the concept of combination of selinexor and the CDK4 / 6 inhibitor in treatment of breast cancer, and is helpful to provide a suitable mode for treating breast cancer patients in the clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antitumor drugs, in particular to the application of a compound combination in the preparation of a drug for treating breast cancer. BACKGROUND

[0002] At present, cancer treatment is still one of the difficult problems to be solved worldwide. At present, multiple drug combination strategy is adopted in cancer treatment, but drug resistance is easily produced in treatment, leading to treatment failure. At present, breast cancer seriously threatens women's health. At present, CDK4 / 6 inhibitors have good effect in ER+HER2- breast cancer. At present, CDK4 / 6 inhibitors have poor effect in breast cancer. At present, KPT-330 can increase the activity of many antitumor drugs.

[0003] KPT-330 is known as selinexor in clinic, which has good antitumor activity in diffuse large B-cell lymphoma and multiple myeloma, but has poor antitumor activity in solid tumors.

[0004] CDK4 / 6 inhibitors are important first-line / two-line drugs for treating breast cancer in recent years, but only ER+HER2- breast cancer patients can benefit from them, and there is no obvious effect on the treatment of triple-negative breast cancer patients. SUMMARY

[0005] In view of the deficiency of the existing clinical drug treatment scheme for breast cancer patients, the present application proposes a new important scheme of drug combination for treating breast cancer.

[0006] To solve the above technical problems, the technical scheme of the present application is as follows: the application of a compound combination in the preparation of a drug for treating breast cancer, wherein the compound combination comprises an exportin XPO1 inhibitor and a CDK4 / 6 inhibitor.

[0007] As a preferred embodiment, the exportin XPO1 inhibitor is selinexor or a pharmaceutically acceptable salt thereof.

[0008] As a preferred embodiment, the CDK4 / 6 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

[0009] As a preferred embodiment, the breast cancer is triple-negative breast cancer; as a further preferred embodiment, the breast cancer is basal-like breast cancer in triple-negative breast cancer.

[0010] As a preferred embodiment, the mass ratio of the exportin XPO1 inhibitor and the CDK4 / 6 inhibitor is 1:10-1:50.

[0011] As a preferred embodiment, the drug use amount of the exportin XPO1 inhibitor is 0.5mg / kg-10mg / kg.

[0012] The application has the characteristics as follows: the application discloses a new drug use mode of KPT-330 (selinexor) combined with a CDK4 / 6 inhibitor for treating breast cancer, the medical name of KPT-330 is selinexor, which is a specific small molecule inhibitor of exportin 1 (XPO1). The CDK4 / 6 inhibitor is a small molecule compound structure formula, which directly inhibits the formation of the CDK4 / 6-Cyclin D complex, reduces the phosphorylation of RB1, inhibits the release of the E2F family, and blocks the cell in the G1 phase to play a role. The application first proposes the concept of using selinexor and the CDK4 / 6 inhibitor in combination for treating breast cancer, which helps to provide a suitable treatment mode for breast cancer patients in the clinic.

[0013] In triple-negative breast cancer, RB1 often exists in the form of phosphorylation, and a phosphorylation inhibitor of RB1 has appeared in the clinic, that is, a CDK4 / 6 inhibitor. In theory, the response effect of the CDK4 / 6 inhibitor in triple-negative breast cancer should be good, but the fact is not so. The application speculates that after using the CDK4 / 6 inhibitor, the non-phosphorylated RB1 is transported out of the nucleus by XPO1, and the RB1 loses its anticancer function in the nucleus. Therefore, the XPO1 inhibitor and the CDK4 / 6 inhibitor are used in combination for treating triple-negative breast cancer, on the one hand, the phosphorylation of RB1 is inhibited, and on the other hand, the nuclear localization of RB1 is increased, and finally the anti-tumor activity of RB1 is increased. The XPO1 inhibitor is selinexor, and the CDK4 / 6 inhibitor is palbociclib, and the combination of the two greatly enhances the treatment effect of triple-negative breast cancer.

[0014] Compared with the prior art, the application has the following beneficial effects: the combination of selinexor and palbociclib plays a synergistic role and greatly enhances the treatment effect of triple-negative breast cancer. The application updates the treatment scheme for breast cancer, so that the breast cancer in the clinic has better drug selectivity. The treatment of breast cancer patients has more biological treatment options in addition to radiotherapy, chemotherapy and surgical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Results of KPT-330 combined with Palbociclib for treating breast cancer cells: A-D. KPT-330 combined with Palbociclib has a good effect on inhibiting the proliferation of tumor cells in breast cancer cell lines (HCC1806 and SUM149PT), and blocks the breast cancer cells in the G0 / G1 phase of the cell cycle;

[0016] Figure 2 A-B: KPT-330 combined with Palbociclib shows a synergistic effect in the treatment of breast cancer cell lines (HCC1806 and SUM149PT).

[0017] Figure 3 A-B: KPT-330 combined with Palbociclib also has good anti-tumor activity in animals;

[0018] Figure 4 Toxicity evaluation results of KPT-330 combined with Palbociclib in treating breast cancer nude mice transplanted tumors: the toxicity of the combination drug was evaluated using mouse body weight, liver function and cell morphology in metabolic organs, and the results showed that the combination drug had low toxicity. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific examples, but the present application is not limited to the following technical solutions.

[0020] The human triple-negative breast cancer cells HCC1806 and SUM149PT described in the following examples and the conventional cancer cells used above are all from the Kunming Institute of Zoology, Chinese Academy of Sciences.

[0021] Unless otherwise specified, the reagents in the examples of the present application can be purchased through commercial channels. RPMI1640 medium, DMEM medium, fetal bovine serum were purchased from Biological Industries; phosphate buffer (PBS) and 0.25% trypsin (containing EDTA) were purchased from Gibco company.

[0022] The cell strains were cultured in DMEM or RPMI1640 medium containing 10% fetal bovine serum at 37°C, 5% CO2 and 90% humidity.

[0023] The structure of KPT-330 (selinexor) used in the following examples is shown in formula I, and the structure of palbociclib (palbociclib) is shown in formula II: KPT-330 and palbociclib are both purchased from Tao Shu Biology.

[0024]

[0025] Example 1 Anti-tumor activity detection of KPT-330 and Palbociclib

[0026] CCK-8 assay was used to detect the anti-tumor activity of the combination drug. CCK-8 kit absorbance detection method is mainly used to detect cell proliferation. CCK-8 contains water-soluble tetrazolium salt WST-8, which is reduced to highly water-soluble yellow formazan product under the action of electron carrier 1-methoxy-5-methyl phenazine methosulfate in cells. CCK-8 method is a high-sensitivity, non-radioactive colorimetric detection method for determining the number of viable cells in cell proliferation or toxicity experiments, which can replace the traditional MTT method. Absorption peak is generated at 450 nm wavelength, and the absorbance value is linearly positively correlated with the cell amount, so it can be used for quantitative detection of cell number.

[0027] KPT-330 (Formula I) was dissolved in DMSO (dimethyl sulfoxide), and then 0.1% DMSO solution of each compound concentration of 800 nM was prepared with cell culture medium. Palbociclib (Formula II) was dissolved in DMSO, and then 0.1% DMSO solution of each compound concentration of 5 μM was prepared with culture medium.

[0028] Breast cancer cells SUM149PT and HCC1806 (6 x 10 3 cells / well) were inoculated in 96-well plates, respectively, and cultured at 37°C in a 5% CO2 incubator for 24 h, then KPT-330 and Palbociclib dissolved in DMSO were added to each well of cell culture, respectively; the control group was only added with the same volume of DMSO to the cell culture, and incubated in the incubator for 48 h. Then 10 μL of CCK-8 reagent was added, and the absorbance value at 450 nm was measured after incubation at 37°C in a 5% CO2 incubator for 1.5 hours. The percentage of absorbance value of the experimental group relative to the absorbance value of the control group represents the survival rate of cells or the proliferation level of cells, that is, the control group is set as 100%. Figure 1 ).

[0029] The results are shown in Figure 1 KPT-330 and Palbociclib can inhibit the protein expression of XPO1 in breast cancer cell lines, and can inhibit the phosphorylation of RB1, and the combination of the two drugs can significantly inhibit the proliferation ability of triple-negative breast cancer cells. The combination of the two drugs can block the cell cycle of tumor cells in the G0 / G1 phase, which is the main factor for the synergistic effect of the two drugs to play an important function.

[0030] Example 2 synergistic experiment results

[0031] First, the IC50 values of the two target drugs were determined by CCK-8 experiments, and then concentration gradients of the target drugs were set near the IC50 values. Cells were plated in three 96-well plates, and after adhesion, drug treatment was performed. The two drug concentrations were distributed from low to high on the X and Y axes of each 96-well plate, and then the drugs were added in the set concentrations in turn. After 48 hours, the cells were fixed with 10% TCA, and then SRB experiments were performed to obtain the target absorbance values. After normalization, an Excel table was created according to the Martix template of the Synergy Finder web application 2.0 (https: / / synergyfinder.fimm.fi / ), the data was input, and then uploaded to the website. According to the website's guidance, the operation was performed, and finally the synergy scores of the two drugs were obtained. A score greater than 10 indicates that the two drugs have a synergistic effect, a score less than -10 indicates that the two drugs are antagonistic, and a score between -10 and 10 indicates that the drugs have an additive effect. Figure 2 The results showed that the synergy scores of the two drugs were 17.22 and 23.96, respectively, indicating that KPT-330 and Palbociclib showed good synergy.

[0032] Example 3 Animal Experiment Results

[0033] Construction of a Nude Mouse Subcutaneous Tumor Model

[0034] Logarithmic growth phase HCC1806 cells were digested, collected, and counted. According to 1x10 6 After centrifugation to remove the supernatant, the cells were resuspended with pre-cooled 1xPBS, and then Matrigel was added. The mixture was thoroughly mixed with a pre-cooled gun head, and then injected into the abdominal subcutaneous tissue of the mouse with an insulin injection needle at a volume of 75 μL per point. After one week, the subcutaneous tumor formation of the mice was observed. When the tumor volume reached 50-70 mm 3 The mice were randomly divided into groups of 4, and different drug treatment groups were set up. According to the formula of 100 μL per mouse, 10% (DMSO or drug) drug mixture + 90% corn oil, drug injection solution was prepared. Intragastric administration was performed every 2 days, and the body weight of the mice was recorded. At the same time, the tumor of the mouse was measured with a vernier caliper, and the tumor volume was calculated by the formula V = 0.5xlengthxwidth2. A total of 7 drug treatments were performed. After drug treatment, the mice were sacrificed by cervical dislocation. The tumor tissue was separated using surgical instruments, weighed, photographed, and the mouse serum was taken for subsequent liver and kidney function tests. The drugs used in this example were KPT-330 and Palbociclib, respectively, and combined administration.

[0035] Figure 3In vivo inhibition results of compound I and palbociclib respectively or in combination on human breast cancer mouse xenograft tumor; Figure 3 A is the proliferation result of measuring the tumor volume of nude mice once every three days; Figure 3 B-C are the photographing and weighing results of the tumor after the nude mice are sacrificed. Figure 4 A-B are the results of mouse subcutaneous tumor model construction and body weight measurement, and administration at the same time; C is the liver function test of the orbital blood before the mice are sacrificed after administration; D is the state evaluation of parenchymal cells in organs. The above results show that the toxicity of the combined administration method is smaller.

[0036] Tumor volume was measured every 3 days after the start of administration treatment, and the results were expressed by a broken line graph, as shown in Figure 3 A can be seen that compared with the control group, after administration of the drug, the tumor volume of the mice is lower than that of the control group; Figure 3 C is the tumor weight of the control group (DMSO) and the experimental group (Palbociclib, KPT-330 and Palbociclib+KPT-330), and the tumor weight of the experimental group is lower than that of the control group, especially the tumor weight after Palbociclib+KPT-330 combined administration treatment is the lowest; Figure 4 B is the change of the body weight of the mice after administration, and there is basically no difference in the body weight of the mice between the experimental group and the control group; Figure 4 C is after administration is completed, the orbital blood of the mice is taken after anesthesia, and the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum are detected to evaluate the hepatotoxicity of administration treatment; Figure 4 D is after administration is completed, the organs of the mice are taken for HE staining to evaluate the toxic effect of the drug on the organs. **P<0.01, ns represents no difference; from Figure 4 B-D can be seen that combined administration treatment does not cause the toxic effect of the drug on the organs of the nude mice.

[0037] In summary, KPT-330 combined with Palbociclib has good synergistic antitumor effect in breast cancer, can obviously inhibit the growth of triple-negative breast cancer cells in vivo and in vitro, and the combined administration does not affect the body weight of the nude mice and has smaller drug toxicity.

[0038] It should be noted that the foregoing examples have been provided merely for the purposes of explanation and are in no way to be construed as limiting. The present application is susceptible to modifications in the concepts as herein illustrated and it is understood that the examples are to be considered merely illustrative of the principles of the application and not a limitation thereof. Modifications in the methods and materials as described herein can be resorted to by those skilled in the art, which though not explicitly described herein, fall within the scope and spirit of the application. While the present application has been described with reference to the specific methods, materials and examples shown, the application is not to be limited to the particulars thereof. It is recognized that variations in the methods and materials can be resorted to by those skilled in the art, which though not specifically described herein, fall within the scope and spirit of the application.

Claims

1. The use of a compound composition in the preparation of a drug for treating basal triple-negative breast cancer, characterized in that, The compound composition includes an XPO1 inhibitor and a CDK4 / 6 inhibitor, wherein the XPO1 inhibitor and the CDK4 / 6 inhibitor, in combination, enhance the anti-tumor effect by synergistically inhibiting RB1 nuclear export and phosphorylation. The XPO1 inhibitor is celiniso or a pharmaceutically acceptable salt thereof; The CDK4 / 6 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

2. The application according to claim 1, characterized in that, The mass ratio of the XPO1 inhibitor to the CDK4 / 6 inhibitor is 1:10 to 1:

50.

3. The application according to claim 1, characterized in that, The combined drug therapy caused tumor cell cycle arrest in the G0 / G1 phase.

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