Use of clozapine and its combination with a chemotherapeutic agent for the preparation of a medicament for the treatment of triple negative breast cancer or breast cancer stem cells

Abstract

This invention discloses the application of clozapine and its combination with chemotherapeutic drugs in the preparation of drugs for treating triple-negative breast cancer or breast cancer stem cells, belonging to the field of pharmaceutical technology. This invention experimentally verifies that clozapine can effectively inhibit the growth of breast cancer stem cells and has a strong growth-inhibiting effect on triple-negative breast cancer cell lines, but has no growth-inhibiting effect on other types of breast cancer cell lines and normal tissue cells, thus it can be used as a treatment drug for triple-negative breast cancer. Simultaneously, the combination of clozapine and chemotherapeutic drugs can significantly inhibit the growth of triple-negative breast cancer cells, and the inhibitory effect is significantly better than that of chemotherapeutic drugs alone. Clozapine alone and the combination of clozapine and chemotherapeutic drugs significantly inhibited the growth of triple-negative breast cancer tumors in mouse models, successfully solving the problem of drug resistance in triple-negative breast cancer caused by the inability of existing basic treatment drugs (chemotherapeutic drugs) to inhibit breast cancer stem cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of clozapine in the preparation of drugs for the treatment of triple-negative breast cancer and breast cancer stem cell therapy, and also relating to the application of clozapine and chemotherapy drugs in the preparation of drugs for the treatment of triple-negative breast cancer. Background Technology

[0002] Breast cancer is one of the most common cancers that threaten women's health. Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It accounts for about 15% to 20% of all breast cancers. It is characterized by high malignancy, high risk of recurrence and metastasis, and ineffectiveness of endocrine therapy and anti-HER2 targeted therapy. Clinical treatment is based on chemotherapy, combined with a multidisciplinary comprehensive plan including surgery, radiotherapy, immunotherapy, and novel targeted therapy.

[0003] Chemotherapy is the foundational treatment for triple-negative breast cancer, but approximately 30% of early and mid-stage triple-negative breast cancers are insensitive to chemotherapy, and almost all advanced triple-negative breast cancers develop chemotherapy resistance. The main reason is that chemotherapy drugs primarily target mature triple-negative breast cancer cells rather than breast cancer stem cells (BCSCs). BCSCs possess chemotherapy resistance, mediating chemotherapy resistance in triple-negative breast cancer and being a key reason for chemotherapy failure.

[0004] Breast cancer stem cells (BCSCs) are a small group of tumor cells in breast cancer tissue that possess self-renewal capacity, multi-lineage differentiation potential, and strong tumorigenicity. They are also the "root cells" of tumor occurrence, development, recurrence, metastasis, and drug resistance. Triple-negative breast cancer (TNBC) has the most prominent BCSC characteristics among all breast cancer subtypes, which is also the key molecular mechanism that makes it a clinically refractory subtype. Therefore, novel therapeutic strategies targeting breast cancer stem cells (BCSCs) are completely different from the molecular design and activity testing of existing therapeutic drugs and hold the promise of overcoming chemotherapy resistance in TNBC caused by BCSCs, representing a new hope for the future treatment of TNBC.

[0005] Clozapine is an atypical antipsychotic and an important drug in the clinical treatment of schizophrenia, especially for treatment-resistant schizophrenia. As of January 2026, a search of PubMed, UEmbase, and the Chinese Biomedical Literature Database (CMBdisc) revealed no reports of studies using clozapine as a breast cancer stem cell killer or a treatment for triple-negative breast cancer. Summary of the Invention

[0006] Therefore, this invention aims to provide the application of clozapine, a drug currently used clinically to treat schizophrenia, as a therapeutic agent for triple-negative breast cancer and breast cancer stem cells, and its combination with chemotherapeutic drugs in the preparation of therapeutic agents for triple-negative breast cancer. Extensive in vitro and in vivo experiments have demonstrated that clozapine can specifically inhibit the growth of triple-negative breast cancer cells and breast cancer stem cells, but has no growth-inhibiting effect on other types of breast cancer cell lines and normal tissue cells. Simultaneously, the combination of clozapine and chemotherapeutic drugs can significantly inhibit the growth of triple-negative breast cancer cells, and the inhibitory effect is significantly better than that of chemotherapeutic drugs alone. Furthermore, clozapine alone and the combination of clozapine and chemotherapeutic drugs significantly inhibited the growth of triple-negative breast cancer masses in mouse models, proving that it can serve as a novel therapeutic agent for triple-negative breast cancer, successfully solving the problem of drug resistance in triple-negative breast cancer caused by the inability of existing basic therapeutic agents (chemotherapeutic drugs) to inhibit breast cancer stem cells.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides the use of clozapine in the preparation of a medicament for treating triple-negative breast cancer or breast cancer stem cells.

[0009] Based on the above technical solution, clozapine can specifically inhibit the growth of triple-negative breast cancer cells without affecting the growth of other types of breast cancer and normal breast cells.

[0010] Based on the above technical solution, the triple-negative breast cancer cell lines further include BT549, MDA-MB-231, MDA-MB-468 and HCC1806 cells.

[0011] Based on the above technical solution, clozapine further significantly inhibited the growth of triple-negative breast cancer masses in a tumor-bearing mouse model.

[0012] Based on the above technical solution, the concentration of clozapine is further 1~50 μM.

[0013] Based on the above technical solution, clozapine can further specifically inhibit the growth of breast cancer stem cells.

[0014] Based on the above technical solution, clozapine further demonstrates that its inhibitory effect on breast cancer stem cells is significantly superior to that of chemotherapy drugs for triple-negative breast cancer.

[0015] Based on the above technical solution, the drug further includes pharmaceutically acceptable excipients.

[0016] Based on the above technical solutions, the dosage forms of the drugs further include oral liquids, granules, tablets, capsules, drop pills, injections, nano-formulations, and targeted formulations.

[0017] In a second aspect, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for treating triple-negative breast cancer or breast cancer stem cells, wherein the active ingredient of the pharmaceutical composition comprises clozapine and a chemotherapy drug.

[0018] Based on the above technical solution, the chemotherapy drugs further include antimetabolites, anthracyclines, and taxanes.

[0019] Based on the above technical solution, the chemotherapy drugs further include capecitabine, 5-fluorouracil (5-FU), tetiracetam, doxorubicin, epirubicin, pirarubicin, paclitaxel, and docetaxel.

[0020] Based on the above technical solution, the mass ratio of clozapine to chemotherapy drugs is further 1:1 to 300:1.

[0021] Based on the above technical solution, the triple-negative breast cancer cell lines further include BT549, MDA-MB-231, MDA-MB-468 and HCC1806 cells.

[0022] Based on the above technical solution, the concentration of clozapine is further 5~50 μM.

[0023] Based on the above technical solution, the drug further includes pharmaceutically acceptable excipients.

[0024] Based on the above technical solutions, the dosage forms of the drugs further include oral liquids, granules, tablets, capsules, drop pills, injections, nano-formulations, and targeted formulations.

[0025] Based on the above technical solution, furthermore, compared with chemotherapy drugs alone, the combination of clozapine and chemotherapy drugs significantly improves the ability to inhibit the growth of triple-negative breast cancer cells.

[0026] Based on the above technical solution, the combination of clozapine and chemotherapy drugs can significantly inhibit the growth of triple-negative breast cancer tumors in mouse models, and the inhibitory effect is significantly better than that of chemotherapy drugs alone.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a new strategy for treating triple-negative breast cancer and breast cancer stem cells, and inspires new anti-tumor therapies that improve the efficacy of triple-negative breast cancer treatment and reduce tumor recurrence. It is of great significance for improving the quality of life and prolonging the survival time of triple-negative breast cancer patients. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0029] Figure 1 Microscopic images of triple-negative breast cancer cells (A) BT549, (B) MDA-MB-231, (C) MDA-MB-468 and (D) HCC1806 after flow cytometry sorting and serum-free culture.

[0030] Figure 2 The results of the experiment on the activity of clozapine and its combination with the chemotherapeutic drug paclitaxel in inhibiting the growth of triple-negative breast cancer tumors in a mouse model bearing tumors include: (A) the tumor volume change curves of each group of mice during the administration process; and (B) the in vitro images of each group of tumors after the administration process. Detailed Implementation

[0031] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the raw materials and reagents used can be purchased from chemical or biological reagent companies or prepared from disclosed methods.

[0032] Example 1: Induction, culture, and spheroidization experiments of breast cancer stem cells BT549, MDA-MB-231, MDA-MB-468, and HCC1806 cells were digested with trypsin and mechanically pipetted to obtain single-cell suspensions. Cell clumps were removed by filtration, and fluorescently labeled CD44, CD24, and ALDH1 antibodies (Invitrogen) were added. The suspensions were incubated at 4°C in the dark for 30–60 min, and unbound antibodies were washed away. The target cell population (CD44+ / CD24- / ALDH1+) was then separated using a flow cytometry sorter.

[0033] The sorted cells were collected at a density of 200-500 cells / cm³. 2Cells were seeded at a density of 1.2% poly(2-hydroxyethyl methacrylate) and 95% ethanol-coated plates (Sigma-Aldrich) and cultured in serum-free medium (SFM) containing a DMEM / Ham nutrient mixture F-12 (1:1), supplemented with 20 ng / mL epidermal growth factor (EGF, Invitrogen), 10 ng / mL human basic fibroblast growth factor (hFGFb, Invitrogen), 1% penicillin-streptomycin solution (Cytiva), and 2% human leukocyte antigen B27 (Gibco) for 7–21 days. After the suspension cells formed spheroids, the SFM was replaced every 3–5 days. Spherical cells were collected and enzymatically dissociated (treated with 0.05% trypsin containing 0.53mM EDTA-4Na for 10–20 minutes at 37°C); cells were then stained with Hoechst 33342 (Solarbio) and resorted via FACS to determine the percentage of spheroidal (SP) cells. Target cells were counted by seeding 1000 target cells in SFM supplemented with EGF, hFGFb, penicillin antibody, and B27. Spheroidal cells were collected and enzymatically dissociated, harvested, counted, and re-seeded with 1000 cells for the second and third rounds of screening. For each round, tumor cell spheroids were counted on day 7. The percentage of tumor cell SP (%MFU) was calculated by dividing the number of SP cells formed (size ≥50μm) by the number of cells seeded and multiplying by 1000.

[0034] The results are as follows Figure 1 As shown, after the above treatment, cell clusters with a size ≥50μm were formed, indicating that breast cancer stem cells induced by human breast cancer cell lines BT549, MDA-MB-231, MDA-MB-468, and HCC1806 had been initially formed. The obtained breast cancer stem cells were named BT549-SC, MDA-MB-231-SC, MDA-MB-468-SC, and HCC1806-SC, respectively.

[0035] Example 2: Clozapine and chemotherapy drugs inhibit the proliferation of triple-negative breast cancer cells and breast cancer stem cells as single agents. The half-maximal inhibitory concentration (IC50) of a drug on cell growth 50 The result was calculated based on cell viability assay results. Various cell lines in the logarithmic growth phase were collected at a rate of 2 × 10⁻⁶. 5Cells were seeded at a concentration of 100 μL / mL in 96-well plates, with four replicates per group. After overnight culture (24 h) according to the culture conditions for each cell type, different concentrations of clozapine and chemotherapy drugs were added for 24 h. Cell viability was then assessed using the standard MTT assay (Sigma). The concentration of the compound at which cell viability decreased by half compared to the control group without treatment was calculated; this was the half-maximal inhibitory concentration (IC50) of the drug on cell growth. 50 The specific results are shown in Table 1.

[0036] Table 1. Half-maximal inhibitory concentrations (IC50) of clozapine and chemotherapy drugs on cell growth 50 (μM)

[0037] The results in Table 1 show that the chemotherapy drugs paclitaxel, doxorubicin, and capecitabine have significantly lower ability to inhibit the proliferation of breast cancer stem cells (BT549-SC, MDA-MB-231-SC, MDA-MB-468-SC, and HCC1806-SC) than they have inhibited the proliferation of mature triple-negative breast cancer cells (BT549, MDA-MB-231, MDA-MB-468, and HCC1806). This indicates that breast cancer stem cells are the cause of resistance to chemotherapy drugs such as paclitaxel, doxorubicin, and capecitabine in the treatment of triple-negative breast cancer. Conversely, clozapine's ability to inhibit the proliferation of breast cancer stem cells (BT549-SC, MDA-MB-231-SC, MDA-MB-468-SC, and HCC1806-SC) was significantly higher than its ability to inhibit the proliferation of mature triple-negative breast cancer cells (BT549, MDA-MB-231, MDA-MB-468, and HCC1806), indicating that clozapine specifically inhibits breast cancer stem cells and has a completely different therapeutic effect and mechanism from chemotherapy drugs. The results also showed that clozapine specifically inhibits the proliferation of triple-negative breast cancer cells and breast cancer stem cells, without affecting the proliferation of other types of breast cancer cells (SKBR3, MCF-7) and normal breast tissue cells (MCF10A).

[0038] Example 3: The drug composition of clozapine and chemotherapy drugs inhibits the proliferative activity of triple-negative breast cancer cells. This experiment examined the half-maximal inhibitory concentration (IC50) of chemotherapy drugs in inhibiting the proliferation of triple-negative breast cancer cells when 10 μM or 20 μM clozapine and different concentrations of chemotherapy drugs were added, respectively. 50 ), compared with the IC when chemotherapy drugs are added as a single agent 50 The half-maximal inhibitory concentration (IC50) of a drug on cell growth. 50 The detection method is the same as in Example 2, and the specific results are shown in Table 2.

[0039] Table 2. Half-maximal inhibitory concentration (IC50) of chemotherapeutic drugs on cell growth in clozapine and chemotherapeutic drug combinations. 50 (μM)

[0040] The results in Table 2 show that when chemotherapy drugs are combined with clozapine, their ability to inhibit the proliferation of triple-negative breast cancer cells is significantly better than that of chemotherapy drugs alone. This demonstrates that the combination of clozapine and chemotherapy drugs can serve as a new and more effective treatment for triple-negative breast cancer.

[0041] Example 4: The inhibitory activity of clozapine monotherapy and its combination with chemotherapeutic drugs on triple-negative breast cancer tumor growth in tumor-bearing mouse models. Three groups of 6-week-old nude mice (n=5 per group) were randomly selected and subcutaneously transplanted with triple-negative breast cancer cells (MDA-MB-231). Group A served as the control, Group B was injected with clozapine 10 mg / kg every 3 days, and Group C was injected with a combination of clozapine 10 mg / kg and paclitaxel 5 mg / kg every 3 days. The specific experimental results are shown in Table 3.

[0042] Table 3. Construction and drug administration regimens of three tumor-bearing mouse models

[0043] Mouse test results as follows Figure 2 As shown in the data, after 21 days of treatment with both clozapine monotherapy and clozapine combined with the chemotherapy drug paclitaxel, the tumor size in the treatment groups was significantly smaller than that in the control group. The tumor inhibition rate in the combination therapy group exceeded 60% (calculated by tumor volume). These results demonstrate that clozapine and its combination with chemotherapy drugs can serve as novel and more effective treatments for triple-negative breast cancer.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of clozapine in the preparation of drugs for the treatment of triple-negative breast cancer or breast cancer stem cells.

2. The application according to claim 1, characterized in that, The clozapine described herein can specifically inhibit the growth of triple-negative breast cancer cells; the clozapine described herein can specifically inhibit the growth of breast cancer stem cells.

3. The application according to claim 1, characterized in that, The triple-negative breast cancer cell lines include BT549, MDA-MB-231, MDA-MB-468, and HCC1806 cells.

4. The application according to claim 1, characterized in that, The drug includes pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that, The dosage forms of the drugs include oral liquids, granules, tablets, capsules, drop pills, injections, nano-formulations, and targeted formulations.

6. The use of a pharmaceutical composition in the preparation of a medicament for treating triple-negative breast cancer or breast cancer stem cells, characterized in that, The active ingredient of the pharmaceutical composition consists of clozapine and a chemotherapy drug.

7. The application according to claim 6, characterized in that, The chemotherapy drugs mentioned include antimetabolites, anthracyclines, and taxanes.

8. The application according to claim 7, characterized in that, The chemotherapy drugs include capecitabine, 5-fluorouracil (5-FU), tetiazem, doxorubicin, epirubicin, pirarubicin, paclitaxel, and docetaxel; the mass ratio of clozapine to the chemotherapy drugs is 1:1 to 300:

1.

9. The application according to claim 6, characterized in that, The drug includes pharmaceutically acceptable excipients.

10. The application according to claim 6, characterized in that, The dosage forms of the drugs include oral liquids, granules, tablets, capsules, drop pills, injections, nano-formulations, and targeted formulations.

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