Application of sesquiterpenoids derivative in preparation of anti-breast cancer drugs

Abstract

The invention discloses application of sesquiterpenoids derivatives in preparation of anti-breast cancer drugs. The structural formula of the derivatives is as shown in formula I in the specification. In-vitro experiments show that the sesquiterpenoids derivative can significantly inhibit proliferation of human breast cancer cells MDA-MB-231 and BT-549, and can activate GSDME protein through splitting decomposition, increase lactic dehydrogenase release and cell membrane damage and induce pyroptosis of the breast cancer cells. In-vivo experiments further prove that in a 4T1-Luc tumor-bearing mouse model, the sesquiterpenoids derivative can significantly inhibit tumor growth and reduce the tumor volume and weight. More importantly, when the sesquiterpenoids derivative and the PD-L1 monoclonal antibody Atezolizumab are combined for use, a synergistic effect is shown, the anti-tumor effect of the sesquiterpenoids derivative is remarkably superior to that of any single-drug treatment group, and infiltration of CD4 + and CD8 + T cells in a tumor microenvironment can be remarkably promoted. All treatment schemes have no obvious influence on the weight of the mouse, and the safety is good. The invention provides a new means for the treatment of breast cancer, especially the combined treatment strategy with the immune checkpoint inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of sesquiterpene derivatives in the preparation of anti-breast cancer drugs. Background Technology

[0002] Breast cancer is a prevalent malignant tumor in women worldwide, characterized by its complex pathogenesis and rapid disease progression. Triple-negative breast cancer lacks three key therapeutic targets: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), rendering endocrine therapy and anti-HER2 targeted therapy almost ineffective, and currently relying mainly on chemotherapy. However, chemotherapy is accompanied by significant side effects and drug resistance, and patients have a 20% risk of recurrence and metastasis within 5 years, highlighting the limitations of current treatment methods. Although studies have shown that patients with triple-negative breast cancer with high levels of lymphocyte infiltration have a better prognosis, suggesting that they may benefit from immune checkpoint inhibitors, and immunotherapies such as PD-1 / PD-L1 antibodies have been approved for clinical use, immunotherapy for triple-negative breast cancer still faces severe challenges. Due to tumor heterogeneity, immune escape mechanisms, and the complexity of the tumor microenvironment, only 10%–20% of patients respond well to existing immunotherapies. Therefore, the development of novel and highly effective drugs for triple-negative breast cancer is urgent, and how to overcome the current bottlenecks in immunotherapy has become a key scientific problem that urgently needs to be solved.

[0003] Pyroptosis is a programmed cell death mechanism in inflammation, characterized by morphological changes such as continuous cell enlargement until the cell membrane ruptures, leading to the release of cellular contents and triggering a strong inflammatory response. Pyroptosis is closely associated with diseases such as tumors, infectious diseases, and metabolic disorders. GSDME can be cleaved by Caspase-3, leading to the transition from apoptosis to pyroptosis. The pro-inflammatory and immunogenic contents released by tumor cell death induced by pyroptosis can alter the tumor immune microenvironment, increase the level of tumor-infiltrating lymphocytes, and ultimately enhance the body's anti-tumor immune response. Therefore, the discovery of drugs that induce tumor cell pyroptosis and improve tumor immune responses has significant scientific value and application prospects.

[0004] Sesquiterpenes are natural terpenoid compounds containing 15 carbon atoms and three isoprene units. They possess various skeletal structures, including chain and cyclic forms, and exhibit unique chemical structures and diverse biological activities. Natural sesquiterpenoid compounds can be used as raw materials to prepare sesquiterpenoid derivatives. CN 112970769 A discloses the application of sesquiterpenoid derivatives in the preparation of drugs for controlling wheat scab. Currently, there are no literature reports on the research and application of such sesquiterpenoid derivatives in inducing pyroptosis in breast cancer tumor cells and enhancing anti-tumor immune responses. Summary of the Invention

[0005] The purpose of this invention is to provide new uses for the above-mentioned sesquiterpene derivatives in the pharmaceutical field.

[0006] This invention provides the application of the above-mentioned sesquiterpene derivative (denoted as HCl-3) in the preparation of anti-breast cancer drugs, and the structural formula of the sesquiterpene derivative is shown below:

[0007]

[0008] This invention also provides the application of the above-mentioned sesquiterpene derivatives in combination with the PD-L1 monoclonal antibody Atezolizumab in the preparation of anti-breast cancer drugs.

[0009] The synthetic methods for the above-mentioned sesquiterpene derivatives are described in CN 112970769 A.

[0010] In the above applications, the breast cancer cells used include human breast cancer cells MDA-MB-231 and BT-549, and mouse breast cancer cells 4T1-Luc. The efficacy of anti-breast cancer drugs includes at least one of the following:

[0011] (1) It can effectively inhibit the proliferation of human breast cancer cells MDA-MB-231 and BT-549 in vitro, and induce pyroptosis of breast cancer cells by activating the GSDME-dependent pyroptosis pathway.

[0012] (2) It can significantly inhibit the growth of mouse breast cancer cells 4T1-Luc in vivo, reduce tumor volume and weight. When used in combination with the PD-L1 monoclonal antibody Atezolizumab, it shows a synergistic anti-tumor effect and can significantly promote CD4 in the tumor microenvironment. + With CD8 + T-cell infiltration enhances the anti-tumor immune response. None of the treatment regimens significantly affected mouse body weight, indicating good safety.

[0013] In the above applications, the sesquiterpene derivatives and the PD-L1 monoclonal antibody Atezolizumab can be prepared into anti-breast cancer drugs according to conventional pharmaceutical preparations and pharmaceutically acceptable carriers using conventional preparation processes for various formulations. These drugs can be any of the following: oral liquid, injection, tablet, capsule, granule, or drop pill.

[0014] The present invention further provides a pharmaceutical composition for treating breast cancer, comprising a therapeutically effective amount of the above-mentioned sesquiterpene derivative and the PD-L1 monoclonal antibody Atezolizumab.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention provides a novel application of sesquiterpene derivatives in the preparation of anti-breast cancer drugs. This compound, by specifically activating the GSDME-dependent pyroptosis pathway, not only directly inhibits the proliferation of human breast cancer cells MDA-MB-231 and BT-549 in a concentration-dependent manner and induces pyroptosis, but more importantly, releases a large amount of inflammatory factors and tumor antigens through this unique cell death mechanism, transforming the immunosuppressive "cold" tumor microenvironment into an immune-activated "hot" tumor microenvironment, creating favorable conditions for immunotherapy. In the 4T1-Luc mouse breast cancer model, monotherapy with this sesquiterpene derivative significantly inhibited tumor growth and reduced tumor volume and weight. When used in combination with the traditional PD-L1 inhibitor atezolizumab, it not only exhibited a significant synergistic anti-breast cancer effect but also effectively promoted CD4+ in breast cancer tissue. + and CD8 + T-cell infiltration and activation enhance the body's immune response against breast cancer. Importantly, this sesquiterpene derivative had no significant effect on mouse body weight throughout the treatment process, demonstrating good safety profile. This invention elucidates the anti-breast cancer effect of this sesquiterpene derivative for the first time from the dual mechanism of inducing tumor cell pyroptosis and synergistic immunotherapy. It provides a lead compound with a clear mechanism of action and a solid experimental foundation for developing novel "pyroptosis inducer + immune checkpoint inhibitor" combination therapy regimens, possessing significant clinical application value and development prospects in the field of breast cancer treatment. Attached Figure Description

[0017] Figure 1 These are images showing the morphology of human breast cancer cells MDA-MB-231 and BT-549 after treatment with different concentrations of HCl-3.

[0018] Figure 2 The effect of different concentrations of HCl-3 on the proliferation of MDA-MB-231 and BT-549 cells.

[0019] Figure 3 The results are obtained by PI staining after MDA-MB-231 and BT-549 cells were treated with different concentrations of HCl-3 for 24 h.

[0020] Figure 4 The results show the effects of different concentrations of HCl-3 on LDH in MDA-MB-231 and BT-549 cells.

[0021] Figure 5 The results show the effects of different concentrations of HCl-3 on GSDME expression in MDA-MB-231 and BT-549 cells.

[0022] Figure 6This is a graph showing the effects of HCL-3, Atezolizumab, and the combination of HCL-3 and Atezolizumab on tumors in tumor-bearing mice. In the graph, A is a representative in vivo imaging image of tumor-bearing mice in different groups, B is the change in tumor size in different groups, C is the change in tumor weight in different groups, and D is the tumor growth curve in different groups.

[0023] Figure 7 HCL-3, Atezolizumab, and the combination of HCL-3 and Atezolizumab are used to target CD4 in tumors. + and CD8 + Results of T cell ratio detection.

[0024] Figure 8 This is a graph showing the effects of HCL-3, Atezolizumab, and the combination of HCL-3 and Atezolizumab on the body weight of tumor-bearing mice. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] Application of HCl-3 and HCl-3 + Atezolizumab in the preparation of anti-breast cancer drugs

[0028] 1. Cell morphology observation

[0029] MDA-MB-231 and BT-549 cells were respectively divided into groups of 3.0 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and incubated overnight at 37°C. After cell attachment, MDA-MB-231 cells were treated with different concentrations (final concentrations of 0.5, 1, and 2 μM) of HCl, and BT-549 cells were treated with different concentrations (final concentrations of 1.25, 2.5, and 5 μM) of HCl, for 24 h in each group. 0.1% DMSO was used as a control. Morphological changes in each group were observed using an inverted microscope (magnification: 200x, scale bar: 100 μm).

[0030] like Figure 1 As shown, with the increase of HCl-3 concentration, the cell density of the drug-treated group decreased significantly in a concentration-dependent manner compared with the control group, and cell blistering and rupture increased with increasing concentration, leading to pyroptosis.

[0031] 2. MTT assay for cell proliferation

[0032] Healthy MDA-MB-231 and BT-549 cells were collected and seeded at 6000 cells / well in 96-well plates. After cell adhesion and growth, different concentrations (final concentrations of 0.5, 1, 1.25, 2, 2.5, and 5 μM) of HCl-3 were added for treatment, with 0.1% DMSO as a control. After 0 h, 24 h, 48 h, 72 h, and 96 h of drug treatment, 10 μL of MTT solution was added to each well, and the plates were incubated for 4 h. After centrifugation, the blue-purple crystals at the bottom of the plate were retained, and 160 μL of DMSO was added to each well. After complete dissolution, the optical density (OD) value of each well was measured at a wavelength of 490 nm using a microplate reader.

[0033] MTT assay results showed that, compared with the control group, HCL-3 significantly inhibited the proliferation of breast cancer cells MDA-MB-231 and BT-549 in a concentration-dependent manner, with an IC50 concentration of 100%. 50 The values ​​were 1.19±0.21 μM and 2.2±0.06 μM, respectively. Figure 2 As shown.

[0034] 3. PI staining to detect the degree of cell damage

[0035] MDA-MB-231 and BT-549 cells were respectively divided into groups of 3.0 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and incubated overnight at 37°C. After cell attachment, MDA-MB-231 cells were treated with different concentrations (final concentrations of 0.5, 1, and 2 μM) of HCl-3, and BT-549 cells were treated with different concentrations (final concentrations of 1.25, 2.5, and 5 μM) of HCl-3 for 24 h. Cells were washed three times with serum-free medium, and 500 μL of PI solution prepared with serum-free medium (10 μL of PI staining solution was added to 500 μL of serum-free medium to achieve a final PI staining concentration of 20 μg / mL) was added. After incubation at 37°C for 20 min, cells were washed three times with serum-free medium and photographed under an inverted fluorescence microscope at 200x magnification and a scale bar of 100 μm.

[0036] Figure 3 The results showed that, compared with the control group, the drug-treated group had more PI-positive cells, indicating that HCL-3 increases the degree of cell membrane damage in a concentration-dependent manner.

[0037] 4. LDH release experiment

[0038] To detect the ability of HCl-3 to induce the release of lactate dehydrogenase (LDH, a typical marker of pyroptosis), MDA-MB-231 and BT-549 cells were divided into two groups of 8 × 10⁻⁶ cells each. 3Cells were seeded per well in 96-well plates and incubated overnight at 37°C. After cell attachment, MDA-MB-231 cells were treated with different concentrations (final concentrations of 0.5, 1, and 2 μM) of HCl, and BT-549 cells were treated with different concentrations (final concentrations of 1.25, 2.5, and 5 μM) of HCl, for 24 h. LDH release from the cells was measured using an LDH assay kit.

[0039] Figure 4 The results showed that the drug-treated group released more LDH from cells compared with the control group, indicating that HCl-3 increases LDH release in a concentration-dependent manner.

[0040] 5. Detection of GSDME expression

[0041] GSDME expression was detected by Western blot. MDA-MB-231 and BT-549 cells were injected with 1.0 × 10⁻⁶ cells per cell line. 6 Cells were evenly seeded at a density of cells / well in 10 cm diameter culture dishes and incubated overnight. MDA-MB-231 cells were treated with different concentrations (final concentrations of 0.5, 1, and 2 μM) of HCl-3, and BT-549 cells were treated with different concentrations (final concentrations of 1.25, 2.5, and 5 μM) of HCl-3 for 24 h. Cells were scraped off and collected, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cell pellet was washed with 1×PBS and transferred to a 1.5 mL centrifuge tube, centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. An appropriate amount of IP lysis buffer was added, and benzyl sulfonyl fluoride was added at a ratio of 100:1. The cell pellet was resuspended and homogenized, and then placed on ice for lysis for 1 h. After centrifugation at 12000 rpm for 15 min, the supernatant was the protein sample. Protein quantification was performed using the BCA protein quantification kit. Samples were loaded, followed by SDS gel electrophoresis, membrane transfer, blocking, incubation with primary antibody at 4°C overnight, collection of primary antibody, washing three times with washing buffer, incubation with secondary antibody, and finally protein band development.

[0042] Figure 5 Western blot results showed that as HCl-3 concentration increased, the total amount of GSDME protein decreased, while the amount of cleaved GSDME increased, indicating that HCl-3 cleaved and activated GSDME, inducing pyroptosis in breast cancer cells.

[0043] 6. In vivo experiments in mice

[0044] Female BALB / c mice aged 4–6 weeks were housed in sterile cages with free access to food and water. 4T1-Luc cells were cultured in DMEM medium containing 10% fetal bovine serum. Cells were harvested at stable condition and approximately 80% confluence after passage, and cultured at 5.0 × 10⁻⁶ cells / mL.5 4T1-Luc cells were seeded at a density of 10 cells / mouse into the mammary fat pads of mice. After modeling, mouse body weight and tumor size were measured the following day. Tumor volume was expressed as Volume (mm²). 3 ) = 1 / 2 × a × b 2 Calculations were performed, where a and b represent the long and short diameters of the tumor, respectively. After successful model establishment, mice were divided into four groups (control group, HCL-3 group, Atezolizumab group, and HCL-3+Atezolizumab group, 10 mice in each group). HCL-3 was administered at a dose of 0.02 g / kg, and Atezolizumab at a dose of 0.02 g / kg, injected intraperitoneally every other day. The control group received the same volume of saline intraperitoneally each time. Tumor volume and mouse weight were measured. Once the tumor reached a certain size, D-Luciferin sodium was injected, and the bioluminescence intensity of the tumor was detected using a mouse in vivo imaging system. After anesthesia and euthanasia, tumor samples were collected, and tumor weight was recorded. Subsequently, a portion of the tumor was ground, stained with CD4 and CD8 antibodies, and CD4 was analyzed by flow cytometry. + and CD8 + The proportion of T cells.

[0045] like Figure 6 As shown in Figure A, compared with the control group, the bioluminescence intensity at the tumor site was significantly reduced in the HCL-3 group, the Atezolizumab group, and the HCL-3+ Atezolizumab group, and the HCL-3+ Atezolizumab group showed better results than the HCL-3 or Atezolizumab monotherapy groups. Figure 6 As shown in B and 6C, the tumor volume and weight in the HCL-3 group, the Atezolizumab group, and the HCL-3+ Atezolizumab group were significantly lower than those in the control group, with the HCL-3+ Atezolizumab group exhibiting the smallest tumor volume and weight. Figure 6 As shown in Figure D, the tumor volume measurement results in mice showed that, compared with the control group, the tumor growth rate of the HCL-3 group, the Atezolizumab group, and the HCL-3+ Atezolizumab group was significantly lower than that of the control group, and the tumor growth rate of the HCL-3+ Atezolizumab group was the lowest. Figure 7 The flow cytometry analysis results showed that, compared with the control group, the HCL-3 group, the Atezolizumab group, and the HCL-3+ Atezolizumab group had significantly higher levels of CD4+ in tumors. + and CD8 + The proportion of T cells was significantly higher in the HCL-3+ Atezolizumab group than in the control group, and CD4+ levels were higher in tumors. +and CD8 + The proportion of T cells was the highest. Figure 8 The results of mouse weight measurement showed that, compared with the control group, the HCL-3 group, the Atezolizumab group, and the HCL-3+ Atezolizumab group had no significant effect on mouse weight.

Claims

1. The use of sesquiterpene derivatives in the preparation of anti-breast cancer drugs, wherein the structural formula of the sesquiterpene derivatives is shown below: 。 2. The sesquiterpene derivative according to claim 1 in the preparation of an anti-breast cancer drug, characterized in that: The cancer cells of the breast cancer include any one or more of the following: human breast cancer cells MDA-MB-231, human breast cancer cells BT-549, and mouse breast cancer cells 4T1-Luc.

3. The sesquiterpene derivative according to claim 1 or 2 in the preparation of an anti-breast cancer drug, characterized in that: The dosage form of the drug is any one of oral liquid, injection, tablet, capsule, granule, or drop pill.

4. The use of sesquiterpene derivatives in combination with the PD-L1 monoclonal antibody Atezolizumab in the preparation of anti-breast cancer drugs, wherein the structural formula of the sesquiterpene derivatives is shown below: 。 5. The use of the sesquiterpene derivative according to claim 4 in combination with the PD-L1 monoclonal antibody Atezolizumab in the preparation of an anti-breast cancer drug, characterized in that: The cancer cells of the breast cancer include any one or more of the following: human breast cancer cells MDA-MB-231, human breast cancer cells BT-549, and mouse breast cancer cells 4T1-Luc.

6. The use of the sesquiterpene derivative according to claim 4 or 5 in combination with the PD-L1 monoclonal antibody Atezolizumab in the preparation of an anti-breast cancer drug, characterized in that: The dosage form of the drug is any one of oral liquid, injection, tablet, capsule, granule, or drop pill.

7. A pharmaceutical composition for treating breast cancer, comprising a therapeutically effective amount of a sesquiterpene derivative and a PD-L1 monoclonal antibody, Atezolizumab, wherein the sesquiterpene derivative has the following structural formula: 。

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