Application of compound composition in preparation of medicine for treating breast cancer

Abstract

The invention discloses an application of a compound combination in preparation of a medicine for treating breast cancer. The compound combination comprises a nuclear transport protein XPO1 inhibitor and a CDK4 / 6 inhibitor. The invention discloses a novel medication mode for treating breast cancer by combined use of KPT-330 and a CDK4 / 6 inhibitor, the medical name of the KPT-330 is the Sanisole, and the KPT-330 is a specific small-molecule inhibitor of nuclear export protein 1 (Exportin 1, XPO1). The CDK4 / 6 inhibitor has a small molecular compound structural formula, can directly inhibit formation of a CDK4 / 6-Cyclin D complex, reduce phosphorylation of RB1, inhibit release of an E2F family, and retard cells in a G1 phase to play a role. According to the invention, the concept of treating the breast cancer through combined use of the Selanisole and the CDK4 / 6 inhibitor is provided for the first time, and a proper way for treating a breast cancer patient is favorably provided for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of anti-tumor drugs, and in particular to the application of a compound combination in the preparation of drugs for treating breast cancer. Background Art

[0002] At present, cancer treatment is still one of the most difficult challenges to overcome worldwide. Currently, cancer treatment adopts a multi-drug combination strategy, but it is easy to develop drug resistance during treatment, leading to treatment failure. Breast cancer currently poses a serious threat to women's health. Currently, CDK4 / 6 inhibitors have a good effect in ER+HER2- breast cancer. Currently, CDK4 / 6 inhibitors have poor efficacy in breast cancer. Currently, KPT-330 can increase the activity of many anti-tumor drugs.

[0003] KPT-330, known as selinexor in the clinic, has good anti-tumor activity in diffuse large B-cell neoplasms and multiple myeloma, but poor anti-tumor activity in solid tumors.

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

[0005] In view of the insufficiency of existing clinical drug treatments for breast cancer patients, the present invention proposes an important new drug combination treatment for breast cancer.

[0006] In order to solve the above technical problems, the technical solution of the present invention is as follows: use of a compound combination in the preparation of a drug for treating breast cancer, wherein the compound combination comprises a nuclear export protein XPO1 inhibitor and a CDK4 / 6 inhibitor.

[0007] Preferably, the nuclear export protein XPO1 inhibitor is selinexor or a pharmaceutically acceptable salt thereof.

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

[0009] Preferably, the breast cancer is triple-negative breast cancer; further preferably, the breast cancer is basal breast cancer among triple-negative breast cancer.

[0010] Preferably, the mass ratio of the nuclear export protein XPO1 inhibitor to the CDK4 / 6 inhibitor is 1:10 to 1:50.

[0011] Preferably, the dosage of the nuclear export protein XPO1 inhibitor is 0.5 mg / kg to 10 mg / kg.

[0012] The characteristics of the present invention are as follows: The present invention discloses a new medication method for treating breast cancer by combining KPT-330 (Selinexor) with a CDK4 / 6 inhibitor. The medical name of KPT-330 is Selinexor, which is a specific small molecule inhibitor of nuclear export protein 1 (Exportin1, XPO1). CDK4 / 6 inhibitors are a class of small molecule compound structures that directly inhibit the formation of the CDK4 / 6-Cyclin D complex, reduce the phosphorylation of RB1, inhibit the release of the E2F family, and block cells in the G1 phase to exert their effects. The present invention proposes for the first time the concept of combining Selinexor with a CDK4 / 6 inhibitor to treat breast cancer, which helps to provide a suitable method for treating breast cancer patients in the clinic.

[0013] In triple-negative breast cancer, RB1 often exists in a phosphorylated form, and RB1 phosphorylation inhibitors, namely CDK4 / 6 inhibitors, have appeared in the clinic. Theoretically, the response effect of CDK4 / 6 inhibitors in triple-negative breast cancer should be better, but this is not the case. The present invention speculates that after using CDK4 / 6 inhibitors, non-phosphorylated RB1 is transported out of the nucleus by XPO1, and RB1 loses its tumor suppressor function without being in the nucleus. Therefore, XPO1 inhibitors and CDK4 / 6 inhibitors are used to treat triple-negative breast cancer in combination, which, on the one hand, inhibits the phosphorylation of RB1, and on the other hand, increases the nuclear localization of RB1, and ultimately increases the anti-tumor activity of RB1. The XPO1 inhibitor is selinexor, and the CDK4 / 6 inhibitor is palbociclib. The combined use of the two greatly enhances the therapeutic effect of triple-negative breast cancer.

[0014] Compared with the prior art, the present invention has the following beneficial effects: the combined use of selinexor and palbociclib exerts a synergistic effect, greatly enhancing the therapeutic effect of triple-negative breast cancer. The present invention updates the treatment regimen for breast cancer, making breast cancer in clinical practice have better drug selectivity. In addition to radiotherapy, chemotherapy and surgical treatment, the treatment of breast cancer patients has an additional biological treatment option. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Results of KPT-330 combined with Palbociclib in the treatment of breast cancer cells: AD. KPT-330 combined with Palbociclib has a good inhibitory effect on tumor cell proliferation in breast cancer cell lines (HCC1806 and SUM149PT) by blocking breast cancer cells in the G0 / G1 phase of the cell cycle;

[0016] Figure 2 AB: KPT-330 combined with Palbociclib showed synergistic effects in the treatment of breast cancer cell lines (HCC1806 and SUM149PT);

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

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

[0019] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

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

[0021] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased from commercial sources: RPMI1640 culture medium, DMEM culture medium, and fetal bovine serum were purchased from Biological Industries; phosphate buffered saline (PBS) and 0.25% trypsin (containing EDTA) were purchased from Gibco.

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

[0023] The structural formula of KPT-330 (selinexor) used in the following examples is shown in Formula I, and the structural formula of palbociclib (palbociclib) is shown in Formula II: KPT-330 and palbociclib were both purchased from Taoshu Biotechnology.

[0024]

[0025] Example 1 Antitumor Activity Detection of Antitumor Activity of KPT-330 and Palbociclib

[0026] The CCK-8 experiment is used to detect the anti-tumor activity of the combination drug. The absorbance detection method of the CCK-8 kit is mainly used to detect cell proliferation. CCK-8 contains water-soluble tetrazolium salt WST-8, which is reduced to a highly water-soluble yellow formazan product by dehydrogenase in the cell under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate. The CCK-8 method is a highly sensitive, non-radioactive colorimetric detection method for determining the number of live cells in cell proliferation or toxicity experiments, which can replace the traditional MTT method. The absorption peak is generated at a wavelength of 450nm, 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 prepared into 0.1% DMSO solution with a concentration of 800 nM for each compound using cell culture medium. Palbociclib (Formula II) was dissolved in DMSO, and then prepared into 0.1% DMSO solution with a concentration of 5 μM for each compound using culture medium.

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

[0029] The results are as follows Figure 1 As shown, KPT-330 and Palbociclib can inhibit the protein expression of XPO1 in breast cancer cell lines and inhibit the phosphorylation of RB1. 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 role.

[0030] Example 2 Collaborative Experimental Results

[0031] First, the IC50 values ​​of the two target drugs were found out through CCK-8 experiment, and then the concentration gradients of the target drugs were set near the IC50 values. The cells were plated on 3 96-well plates at the same time, and the drugs were treated after they adhered to the wall. The concentrations of the two drugs were distributed from low to high on the X-axis and Y-axis of each 96-well plate, and then the drugs were treated according to the set concentrations. After 48 hours, the cells were fixed with 10% TCA, and then the SRB experiment was performed. After the target absorbance value was obtained and normalized, a new Excell table was created according to the Martix template of the Synergy Finder webapplication 2.0 (https: / / synergyfinder.fimm.fi / ) website, and the data was entered and then uploaded to the website. The synergistic scores of the two drugs were finally obtained according to the guidance of the website. >10 points indicate that the two drugs have a synergistic effect, <-10 indicates that the two drugs are antagonistic, and between -10 and 10 points are additive effects of the drugs. Figure 2 The results showed that the synergistic scores of the two drugs were 17.22 and 23.96, respectively, so KPT-330 and Palbociclib showed good synergistic effects.

[0032] Example 3 Animal Experiment Results

[0033] Construction of subcutaneous tumor model in nude mice

[0034] HCC1806 cells in the logarithmic growth phase were digested, collected and counted. 1×10 6 After centrifugation of the cells to remove the supernatant, resuspend the cells in pre-cooled 1× PBS, then add Matrigel, mix thoroughly with a pre-cooled pipette tip, and inject into the subcutaneous tissue of the mouse abdomen with an insulin injection needle at a volume of 75 μL per point. One week after inoculation, observe the subcutaneous tumor formation of the mice. When the tumor volume grows to 50-70 mm 3 The mice were randomly divided into groups of 4 mice each, and different drug treatment groups were set up. The drug injection was prepared according to the formula of 100 μL per mouse, 10% (DMSO or drug) drug mixture + 90% corn oil. The drug was administered by gavage once every 2 days, and the weight of the mice was weighed and recorded. At the same time, the mouse tumor was measured with a vernier caliper, and the tumor volume was calculated by the formula V=0.5×length×width2. A total of 7 drug administration treatments were performed. After the drug treatment, the mice were killed by cervical dislocation. The tumor tissue was separated using surgical instruments, weighed and photographed, and mouse serum was taken for subsequent liver and kidney function tests. The drugs described in this embodiment are KPT-330 and Palbociclib, respectively and in combination.

[0035] Figure 3The results are as follows: ... Figure 3 A is the proliferation result of the nude mouse tumor volume measured every three days; Figure 3 BC is the photographic and weighing results of the tumor after the nude mice were killed. Figure 4 AB are the results of mouse subcutaneous tumor model construction and body weight measurement, and the drug was administered at the same time; C is the liver function test of blood drawn from the eye socket before the mouse was killed after administration; D is the status assessment of parenchymal cells in the organ. The above results show that the combined administration method has less toxicity.

[0036] The tumor volume was measured every 3 days after the start of treatment and is shown in a line graph. Figure 3 A shows that compared with the control group, the tumor volume of mice after drug administration 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). The tumor weight of mice in the experimental group was lower than that in the control group, especially after the combined administration of Palbociclib+KPT-330, the tumor weight was the lowest; Figure 4 B shows the weight changes of mice after administration, and there is basically no difference in the weight of mice in the experimental group and the control group; Figure 4 C: After the administration, the mice were anesthetized, and orbital blood was collected to detect the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum to evaluate the hepatotoxicity of the drug treatment; Figure 4 D: After the drug administration, each organ of the mouse was taken for HE staining to evaluate the toxic effects of the drug on the organ. **P<0.01, ns means no difference; Figure 4 It can be seen from BD that combined drug treatment does not cause toxic effects of drugs on the organs of nude mice.

[0037] In summary, KPT-330 combined with Palbociclib has a good synergistic anti-tumor effect in breast cancer, and can significantly inhibit the growth of triple-negative breast cancer cells in vitro and in vivo. At the same time, in vivo animal experiments have shown that combined administration does not affect the body weight of nude mice and has less drug toxicity.

[0038] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention extends to all other methods and applications with the same functions.

Claims

1. Use of a compound combination in the preparation of a drug for treating breast cancer, characterized in that: The compound combination comprises a nuclear export protein XPO1 inhibitor and a CDK4 / 6 inhibitor.

2. Use of the compound combination according to claim 1 in preparing a drug for treating breast cancer, characterized in that: The nuclear export protein XPO1 inhibitor is selinexor or a pharmaceutically acceptable salt thereof.

3. Use of the compound combination according to claim 1 in preparing a drug for treating breast cancer, characterized in that: The CDK4 / 6 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

4. The use of the compound combination according to claim 1 in the preparation of a drug for treating breast cancer, characterized in that: The breast cancer is triple-negative breast cancer.

5. Use of the compound combination according to claim 4 in the preparation of a drug for treating breast cancer, characterized in that: The breast cancer is basal breast cancer among triple-negative breast cancer.

6. Use of the compound combination according to claim 1 in preparing a drug for treating breast cancer, characterized in that: The mass ratio of the nuclear export protein XPO1 inhibitor to the CDK4 / 6 inhibitor is 1:10 to 1:

50.

7. Use of the compound combination according to claim 1 in preparing a drug for treating breast cancer, characterized in that: The dosage of the nuclear export protein XPO1 inhibitor is 0.5 mg / kg to 10 mg / kg.

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